Manufacturing process of annexin V

Abstract

To provide a process for recovery and / or purification of a protein comprising annexin A5 (AnxA5) sequence recombinantly expressed in an endotoxin-producing host cell with a cell wall.SOLUTION: The process comprises the release of the intracellular protein from the host cell, where the release step of the intracellular AnxA5 protein is conducted in the presence of a homogenization buffer comprising a nonionic detergent. Preferably the process does not include any centrifugation steps for recovery and / or purification of the AnxA5 protein after the release from the host cell, and / or the AnxA5 protein remains in solution throughout the entire process except for temporally binding to any chromatographic resins.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] This application relates to a process for producing a protein containing the sequence of annexin A5 (AnxA5). More specifically, the process is for the recovery and / or purification of the AnxA5 protein from recombinant host cells, particularly bacterial host cells. The process described herein is highly efficient and cost-effective and can be used on a commercial scale (e.g., in recombinant host cell cultures with a culture volume of about 1000 L or more) to rapidly and conveniently produce pharmaceutical-grade AnxA5 protein products. [Background technology]

[0002] Any listing or discussion of previously published literature in this specification should not necessarily be interpreted as an endorsement that such literature is part of the latest technology or common general knowledge.

[0003] Atherothrombosis, formed from underlying atherosclerotic plaques, is the primary pathogenic mechanism underlying the majority of clinically apparent ischemic cardiovascular diseases, including acute coronary artery disease, cerebrovascular, and peripheral artery occlusion. As discussed in Cederholm and Frostegard, 2007, Drug News Perspect., 20(5):321-6, annexin A5 (formerly known as annexin V), a member of the annexin superfamily, is a protein with potent and unique antithrombotic properties. The antithrombotic effect exerted by annexin A5 is thought to be primarily mediated by the mechanical shielding of phospholipids, particularly phosphatidylserine, thereby reducing their availability to coagulation reactions. However, other interesting properties of annexin A5 that potentially contribute to its antithrombotic function have been reported, including the downregulation of tissue factor expressed on its surface, or its interaction with additional homeostatic ligands such as sulfatides and heparin, as well as the upregulation of urokinase-type plasminogen activator. The biological importance of annexin A5 as a member of the endogenous antithrombotic system in the body for large vascular structures and placental microcirculation has also been suggested.

[0004] In fact, annexin A5 is known to have broad utility in providing direct therapeutic effects in pharmaceuticals. Examples of annexin A5's uses include the following: For the prevention of atherosclerosis and / or plaque rupture as described in WO2005 / 099744 (the contents of which are incorporated herein by reference), For the treatment of vascular dysfunction, reduction of ischemic pain and / or treatment of vascular disease, as described in WO2009 / 077764 (the contents thereof are incorporated herein by reference), For the prevention or treatment of restenosis as described in WO2009 / 103977 (the details of which are incorporated herein by reference), For use in inhibiting the activity of cardiolipin oxidized (oxCL), as described in WO2010 / 069605, and for treating, preventing and / or reducing the risk of developing cardiovascular disease, autoimmune disease or inflammatory conditions (the details of which are incorporated herein by reference), For the prevention and / or reduction of perioperative or postoperative complications following surgical interventions, such as complications following vascular surgery, particularly peripheral vascular surgery, as described in WO2012 / 136819 (the details of which are incorporated herein by reference).

[0005] Thus, annexin A5 represents a protein of therapeutic interest and potential. Therefore, there is an urgent need for an effective method for producing therapeutic-grade annexin A5 protein by an efficient and cost-effective process that can be scaled up to and conveniently adapted for commercial-scale production (for example, to collect annexin A5 protein from recombinant host cell cultures with a culture volume of approximately 1000 L or more).

[0006] A specific challenge in recombinantly expressing annexin A5 in standard bacterial host cells such as E. coli is contamination by host cell-derived components, particularly endotoxins. Endotoxins are lipopolysaccharides (LPS) composed of covalently linked lipids and polysaccharides consisting of an O antigen, an outer core, and an inner core. LPS are found in the outer membrane of Gram-negative bacteria and elicit a potent immune response in animals. Annexin A5 is characterized by its strong binding to biological membranes containing electrically charged phospholipids, and therefore has a particularly high affinity for endotoxins. This makes large-scale commercial production of annexin A5 from endotoxin-producing hosts more difficult.

[0007] Currently, there are no processes available that produce therapeutic-grade annexin A5 protein in an efficient and cost-effective manner that can be scaled up to commercial production (for example, to collect annexin A5 protein from recombinant host cell cultures with a culture volume of approximately 1000 L or more) and conveniently adapted to that, let alone processes that address endotoxin contamination.

[0008] In 1991, Kumar reported on the development of a process for the production and purification of Annexin A5 (Undergraduate Honors College Thesis titled "Expression, Purification, and Large-Scale Production of the Human Recombinant Annexin-V Protein," submitted to the Department of Chemical Engineering, College of Engineering, University of Arkansas (Fayetteville, AR), available online at https: / / uarkive.uark.edu / xmlui / handle / 10826 / 981). Kumar's process involved expressing annexin A5 in recombinant E. coli host cells in a 100 mL culture flask, pelletizing the cells, resuspending them in the presence of a homogenization / lysis buffer consisting of 50 mM Tris HCl and 10 mM CaCl2 at pH 7.2, and then degrading the cells via sonication to release the annexin A5 protein. The addition of CaCl2 induced calcium-dependent binding of annexin A5 to the cell membrane in the cell debris. The mixture was then subjected to a first purification centrifugation step of 20 minutes, after which the supernatant was discarded and the pellet containing the cell debris and the bound annexin A5 was recovered. Annexin A5 was released from the pellet using EDTA, followed by a second purification centrifugation step of 20 minutes and collection of annexin A5 in the supernatant. Subsequently, the buffer for annexin A5 was changed to Tris HCl at pH 8.0 after overnight dialysis, followed by further steps of anion exchange on a DEAE-Sepharose column and elution of annexin A5 using a salt gradient.

[0009] The applicant has found that Kumar's method has numerous limitations and drawbacks. First, it is only demonstrated on a small scale using 100 mL cultures and requires two separate centrifugation steps during the purification process. This prevents efficient scaling to commercial processes using high-volume cultures (e.g., 1000 L or more). As will be further discussed below, centrifugation of such high-volume fluids is extremely time-consuming and costly. Nevertheless, centrifugation is required as a preliminary capture step in Kumar's method, which relies on the calcium-inducible binding of annexin A5 to the membrane in cell debris. Second, the applicant has found that Kumar's method leads to high loss of annexin A5 protein, for example, by disposing of unbound soluble annexin in the supernatant of the product from the first purification centrifugation step. Third, it is noteworthy that Kumar's method is completely unable to remove endotoxins to levels suitable for therapeutic use, and no testing is performed regarding endotoxin levels in the final product. Thus, Kumar's method cannot be scaled up to commercial production in an efficient and time-effective manner, leading to high loss (i.e., low yield) of annexin A5 protein and resulting in low-grade protein purification unsuitable for therapeutic use.

[0010] In 2008, the Tait Research Laboratory, Department of Laboratory Medicine, University of Washington Medical Center, published a paper titled "Production of Recombinant Annexin V from plasmid pET12a-PAPI," which is available online at https: / / depts.washington.edu / labweb / Faculty / Tait / 108.pdf. The described method is very similar to the method proposed by Kumar. This method involves expressing annexin A5 in recombinant E. coli host cells in a 1 L culture, pelleting the cells, resuspending them in a homogenization / lysis buffer consisting of 50 mM Tris HCl and 10 mM CaCl2 at pH 7.2, and then degrading the cells via sonication to release the annexin A5 protein. The addition of CaCl2 induced calcium-dependent binding of annexin A5 to the cell membrane in the cell debris, and the mixture was then subjected to a first purification centrifugation step of 20 minutes, after which the supernatant was discarded and the pellet containing the cell debris and the bound annexin A5 was recovered. Annexin A5 was released from the pellet using EDTA, followed by a second purification centrifugation step of 20 minutes and collection of annexin A5 in the supernatant. Subsequently, a dialysis step was performed to change the buffer for annexin A5 to Tris HCl at pH 8.0, followed by further steps of anion exchange on a Mono Q column and elution of annexin A5 using a salt gradient. The applicant noted that many of the limitations and drawbacks in Kumar's method also apply to this method.

[0011] In 2014, a further method for purifying annexin A5 was proposed in Marder et al., 2014, BMC Biotechnology, 14:33, entitled "Production of recombinant human annexin V by fed-batch cultivation." Marder et al. reported that this method is a fed-batch method for large-scale production of recombinant human annexin V, and it is suggested that this method could expand the commercial utility of recombinant human annexin A5 to applications such as in in-vivo imaging studies.

[0012] Nevertheless, Marder et al.'s method is very similar to Kumar's 1991 method and the 2008 method by the Department of Laboratory Medicine at the University of Washington Medical Center. Marder et al. expressed annexin A5 in recombinant E. coli host cells in 1 L cultures held in 2 L tanks. As discussed (in the "Purification" section of Marder et al.'s "The Methods"), the collected cells were resuspended in the presence of a homogenization / lysis buffer (buffer A) consisting of 50 mM Tris HCl and 10 mM CaCl2 at pH 7.2, and the cells were then degraded via sonication to release the annexin A5 protein. The addition of CaCl2 induced annexin A5 binding to the cell membrane in cell debris in a calcium-dependent manner, and the mixture was then subjected to a first purification centrifugation step of 30 minutes, after which the supernatant was discarded and the pellet containing cell debris and bound annexin A5 was recovered. Annexin A5 was released from the pellet using EDTA, followed by a second purification centrifugation step of 30 minutes and collection of annexin A5 in the supernatant. Subsequently, a dialysis step was performed to change the buffer for annexin A5 to Tris HCl at pH 8.0, followed by a third purification centrifugation step of 20 minutes to remove residual precipitate, and then further steps of anion exchange on a Mono Q column and elution of annexin A5 using a salt gradient were performed. Again, the applicant noticed that many of the limitations and drawbacks in Kumar's method also apply to this method.

[0013] The 1997 method by Kumar, the 2008 method by the Department of Laboratory Medicine of the University of Washington Medical Center, and the 2014 method by Marder clearly show that the art has developed and established techniques for the commercial production and purification of annexin A5 products. However, the drawbacks in these methods have not been understood in the art without readily available alternatives.

[0014] All of these prior art methods for annexin A5 recovery have only been demonstrated in laboratory-scale processes and cannot be adopted for scale-up or take into account industry standards or equipment available on a larger scale. This process has inherent disadvantages that make it unsuitable for large-scale manufacturing. In particular, the very restrictive features of these prior art processes are the two or (in the case of the 2014 method by Marder et al.) three high G-force centrifugations required by the process whether annexin A5 is alternatively in solution or as a precipitate. Applying only two centrifugation steps to a 1000 L batch processing would reasonably be expected to result in a process that would take approximately 12 weeks with a 12-hour shift per day in any well-equipped biomanufacturing facility, which results in unacceptably high production costs. See Comparative Example 1.

[0015] Therefore, for a manufacturing process operated on a commercial scale (e.g., a recombinant host cell culture having a culture volume of about 1000 L or more), it is an object of the present invention to provide a methodological process for the efficient and cost-effective purification and recovery of annexin A5, and further to overcome the yield losses and low purity (including endotoxin contamination) suffered by prior art processes.

[0016] It is also an object of the present invention to provide a pharmaceutical-grade annexin A5 product produced by the method of the present invention. SUMMARY OF THE INVENTION

[0017] The applicant has made numerous developments and improvements to processes for the production of proteins containing the annexin A5 (AnxA5) sequence, and has devised several highly efficient purification steps that can be used independently and / or in combination to improve existing processes. Most preferably, the present process for the production of annexin A5 comprises all of the developed process steps.

[0018] In particular, the applicant's development offers the possibility of a highly efficient process for the recovery of AnxA5 protein, preferably via a method in which the AnxA5 protein remains in solution throughout the entire process (except when it is temporarily bound to a chromatographic resin). That is, the applicant's development provides a process for the recovery of AnxA5 protein that can preferably be carried out without requiring any purification centrifugation step to be applied to the AnxA5 protein after its release from the host cell. This has enormous industrial benefits, as high-G force centrifugation for collecting precipitates is difficult, time-consuming, and expensive to apply in large-scale biopharmaceutical manufacturing plants. Furthermore, this means that the entire process of the present invention can be carried out without relying on the ability of annexin A5 to bind to membranes (e.g., host cell membranes and / or liposomes), which can often result in annexin A5 being co-purified with undesirable contaminants such as endotoxins.

[0019] Therefore, this process can be applied to the processing of large volumes of host cell cultures (e.g., approximately 100 L, 500 L, 1,000 L, 5,000 L, 10,000 L, 50,000 L, 100,000 L or more) in a highly time-efficient manner, without bottlenecks caused by one or more purification centrifugation steps. For example, it may be preferable that the purification process be carried out in 5, 4, 3, or 2 weeks per 1,000 L of host cell culture to be processed, or less, most typically less than 1 week. Moreover, this process can remarkably provide improved yield and / or improved purity (e.g., improved endotoxin removal) compared to more time-consuming and less efficient prior art processes.

[0020] Accordingly, a first aspect of the present invention provides an improved process for protein release from host cells. More specifically, it provides a process for the recovery and / or purification of a recombinantly expressed intracellular protein comprising the sequence of annexin A5 (AnxA5) from an endotoxin-producing host cell with a cell wall, the process comprising releasing the intracellular protein from the host cell, characterized in that the step of releasing the intracellular AnxA5 protein is carried out in the presence of a homogenization buffer comprising a nonionic detergent. Preferably, the nonionic detergent is a polysorbate, more preferably a polysorbate selected from Tween 20 and Tween 80, most preferably Tween 80.

[0021] The applicant also discovered that, in contrast to conventional methods where yield loss continues to increase with the sequential addition of purification steps (because the product is lost at each step), the combination of anion exchange and heparin affinity chromatography has the remarkable advantage of achieving the high purity that can be obtained with heparin affinity chromatography alone, but with substantially increased yield (i.e., recovery increases from about 30-40% to about 70-90%) (further discussed in Example 2 below). This is the exact opposite of what would normally be expected from a combination of purification steps.

[0022] Therefore, a second aspect of the present invention provides a process for the recovery and / or purification of a protein containing the annexin A5 (AnxA5) sequence from a solution containing the AnxA5 protein and one or more impurities, the method being described below. In order to perform a first anion exchange step and thereby produce a first anion exchange product containing the released AnxA5 protein, a solution containing AnxA5 protein and one or more impurities is applied to an anion exchange resin. The method includes directly or indirectly subjecting a first anion exchange product to an affinity chromatography step to produce a first affinity chromatography product containing the released AnxA5 protein.

[0023] Preferably, according to the process of a second aspect of the present invention, the affinity chromatography step may include binding of the AnxA5 protein to immobilized heparin, optionally facilitated by the presence of calcium ions, and further optionally, the AnxA5 protein is eluted from the immobilized heparin using an elution buffer containing a calcium ion chelating agent such as EDTA.

[0024] In addition, as discussed in Example 3, the applicant found that Tween 80 has a particularly advantageous effect on the heparin affinity chromatography process (compared to other nonionic detergents, including other Tweens such as Tween 20). Incorporating Tween 80 into the buffer used in the heparin affinity chromatography process at, for example, around 0.1% (w / v), can assist in the elution of the AnxA5 protein at a single peak, reduce pressure, and prevent precipitation.

[0025] Accordingly, a third aspect of the present invention provides a process for the recovery and / or purification of a protein containing the sequence of annexin A5 (AnxA5) from a solution containing the AnxA5 protein and one or more impurities, the method comprising subjecting the solution containing the AnxA5 protein and one or more impurities (which may or may not be direct or indirect products of a first anion exchange chromatography capture step as considered herein) to a heparin affinity chromatography step in the presence of Tween 80 (preferably in the presence of about 0.1 w / v% Tween 80) thereby producing a first affinity chromatography product containing the released AnxA5 protein.

[0026] A fourth aspect of the present invention is based on the applicant's realization that calcium metal ion chelators (e.g., EDTA) can adversely affect the effectiveness of anion exchange processes. Free EDTA (or other chelators) can directly bind to anion exchange functional groups, thereby reducing the volume and even separation achieved by the anion exchange process. On the other hand, attempts to remove calcium metal ion chelators before the anion exchange process waste time and, consequently, increase costs. Therefore, prior art methods involving a slow dialysis process for buffer exchange are inefficient. Furthermore, when incorporating calcium metal ion chelators into the AnxA5 product during the anion exchange process, they can be important components for preventing calcium-mediated binding of the AnxA5 protein to impurities, including endotoxins. Therefore, it would be advantageous and effective to introduce additives that block or reduce the binding of calcium ion chelators to the anion exchange resin, allowing the anion exchange process to be carried out without the inconveniences and costs associated with dialysis and without hindering the beneficial effects of the calcium metal ion chelators during the anion exchange process.

[0027] The applicant has found that this can be achieved by incorporating one or more additional selected metal ions into the AnxA5 protein product before anion exchange, wherein the additional selected metal ions are selected such that the calcium metal ion chelator has a binding affinity for the selected metal ions that is greater than its binding affinity to the anion exchange resin but smaller than its binding affinity to calcium ions. The appropriate selection of additional metal ions will depend on the properties of the calcium ion chelator and the properties of the anion exchange resin. For example, when using EDTA as a calcium ion chelator, Mg 2+ Ions are generally suitable for achieving the objectives of the present invention and can be added to the AnxA5 protein product before the anion exchange step.

[0028] Therefore, a fourth aspect of the present invention is a process for recovering and / or purifying a protein comprising the sequence of annexin A5 (AnxA5) from a composition comprising the AnxA5 protein and a calcium metal ion chelating agent, The process is characterized by comprising providing the composition to an anion exchange resin in order to perform an anion exchange step, thereby recovering and / or purifying the AnxA5 protein from the composition. The anion exchange process is further characterized by being carried out in the presence of additional selected metal ions. This additional selected metal ion provides a process in which the calcium metal ion chelating agent is selected such that it has a binding affinity for the selected metal ion that is greater than its binding affinity for the anion exchange resin but less than its binding affinity for calcium ions.

[0029] A fifth aspect of the present invention provides a composition comprising the AnxA5 protein, which is a direct or indirect product (or can be directly or indirectly obtained) of a process according to any of the first, second, third, or fourth aspects of the present invention. Optionally, the composition is pharmaceutically acceptable and / or veterinarily acceptable.

[0030] A sixth aspect of the present invention also provides a composition of the fifth aspect of the present invention for use in pharmaceuticals. In other words, a sixth aspect of the present invention provides a method comprising administering a therapeutically effective amount of the composition of the fifth aspect of the present invention to a human or animal in need of treatment.

[0031] Any method or composition described herein is intended to be applicable in relation to any other method or composition described herein.

[0032] The use of the words "a" or "an" in conjunction with the term "comprising" in the claims and / or herein may mean "one," but it is also consistent with the meanings of "one or more," "at least one," and "more than one."

[0033] These and further aspects of the present invention will be better recognized and understood when considered in conjunction with the following description and accompanying drawings. However, it should be understood that the following description, while illustrating various aspects and embodiments of the present invention and numerous specific details thereof, is given as examples and not as a limitation. Within the scope of the present invention, many substitutions, modifications, additions and / or rearrangements may be made without departing from its spirit, and the present invention includes all such substitutions, modifications, additions and / or rearrangements. [Brief explanation of the drawing]

[0034] [Figure 1-1] The sequence of sequence number 1, which is the sequence of human annexin A5, is shown. [Figure 1-2] The sequence of sequence number 1, which is the sequence of human annexin A5, is shown. [Figure 2] A schematic flowchart of the complete manufacturing process for Annexin A5 is shown. [Figure 3] A process flowchart for an example of AX capture chromatography is provided. [Figure 4] A process flowchart for an example of intermediate affinity chromatography is shown. [Figure 5] A process flowchart for an example AX refining chromatography step is shown. [Figure 6] This shows a process flowchart for ultrafiltration / dialysis and formulation, which is an example of Annexin A5. [Figure 7A] The results of Example 3, which demonstrates the effect of Tween80 on the heparin affinity chromatographic purification of annexin A5, are shown in Figure 7A, which shows the results for Test 1 (without Tween80), and Figure 7B, which shows the results for Test 2 (with Tween80). [Figure 7B] The results of Example 3, which demonstrates the effect of Tween80 on the heparin affinity chromatographic purification of annexin A5, are shown in Figure 7A, which shows the results for Test 1 (without Tween80), and Figure 7B, which shows the results for Test 2 (with Tween80). [Modes for carrying out the invention]

[0035] A. Annexin A5 protein The present invention relates to a method for purifying and / or recovering a protein containing the sequence of annexin A5 (AnxA5), as well as to products and formulations containing the AnxA5 protein produced in this manner.

[0036] In one embodiment of the present invention, the purified and / or recovered AnxA5 protein may contain, be essentially, or consist of a protein having the sequence of human annexin A5 (Sequence ID 1 shown in Figure 1), with or without the N-terminal methionine.

[0037] In another embodiment, the purified and / or recovered AnxA5 protein may contain, be essentially, or consist of a variant or mutant of the protein having the sequence of human annexin A5 (SEQ ID NO: 1 shown in Figure 1), with or without the N-terminal methionine. For example, the variant or mutant may differ from SEQ ID NO: 1 at any one or more positions, e.g., at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160 or more positions, or up to that number of positions (with or without the N-terminal methionine).

[0038] Therefore, variants or mutants of annexin A5 may be proteins in which one or more amino acid insertions, deletions, or substitutions have been made, whether conserved or non-conserved. Preferably, this change results in a protein whose fundamental properties are not significantly altered, allowing it to function in an equivalent manner to annexin A5. In this context, "significantly" means that the properties of the variant may still differ from those of the original protein, but this difference is not uncommon, as would be expected of someone skilled in the art.

[0039] Preferably, the equipotential point (pI) of the variant or mutant is not altered compared to the unmodified protein, or is not altered by more than 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 pH units.

[0040] In preferred embodiments, the AnxA5 protein is capable of binding to phosphatidylserine on biological membranes at levels of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or about 100% of the levels indicated by human annexin A5 (SEQ ID NO: 1) under the same conditions. Suitable methods for measuring annexin A5 binding to phosphatidylserine on biological membranes are known in the art (Vermes et al. (1995) J Immunol Methods, 184(1): p.39-51).

[0041] "Conservative substitutions" are intended combinations such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr.

[0042] Variants and mutants can be prepared using protein engineering and site-directed mutagenesis methods well known in the art.

[0043] In further embodiments, the purified and / or recovered AnxA5 protein may be a dimer of a protein that contains, or is essentially, a protein comprising, a variant or mutant thereof, of human annexin A5 (Sequence ID 1 shown in Figure 1), with or without the N-terminal methionine, or a variant or mutant thereof as described above.

[0044] In further embodiments, the purified and / or recovered AnxA5 protein may be a fusion protein comprising, essentially, or consisting of: (a) one or more protein sequences comprising the sequence of a fusion partner, fused to a protein having the sequence of human annexin A5 (SEQ ID NO: 1 shown in Figure 1), with or without the N-terminal methionine, or a variant or variant thereof, or a dimer as described above; or (b) one or more protein sequences comprising, essentially, or consisting of a protein having the sequence of human annexin A5 (SEQ ID NO: 1 shown in Figure 1), with or without the N-terminal methionine, or a variant or variant thereof, or a dimer as described above. For example, without limitation, the fusion protein may have a general structure selected from the following: -In the case of fusion of two amino acid sequences, for example, H2N-(a)-(b)-COOH, or H2N-(b)-(a)-COOH, -In the case of a fusion of three amino acid sequences, for example, H2N-(a)-(b)-(a)-COOH, or H2N-(b)-(a)-(b)-COOH, or H2N-(a)-(b)-(a)-COOH, or H2N-(a)-(a)-(b)-COOH, or H2N-(b)-(a)-(a)-COOH, or -In the case of a fusion of four amino acid sequences, for example, H2N-(a)-(a)-(a)-(b)-COOH, or H2N-(a)-(a)-(b)-(a)-COOH, or H2N-(a)-(b)-(a)-(a)-COOH, or H2N-(a)-(a)-(b)-(b)-COOH, or H2N-(a)-(b)-(a)-(b)-COOH, or H2N-(b)-(a)-(a)-(b )-COOH, or H2N-(a)-(b)-(b)-(a)-COOH, or H2N-(b)-(a)-(b)-(a)-COOH, or H2N-(b)-(b)-(a)-(a)-COOH, or H2N-(b)-(b)-(b)-(a)-COOH, or H2N-(b)-(b)-(a)-(b)-COOH, or H2N-(a)-(b)-(b)-(b)-COOH, or H2N-(a)-(b)-(b)-(b)-COOH, or -In the case of a fusion of five amino acid sequences, for example, or H2N-(a)-(a)-(a)-(a)-(b)-COOH, or H2N-(a)-(a)-(a)-(b)-(a)-(a)-COOH, or H2N-(a)-(b)-(a)-(a)-(a)-COOH, or H2N-(b)-(a)-(a)-(a)-(a)-COOH, or H2N-(a)-(a)-(a)-(b)-(b )-COOH, or H2N-(a)-(b)-(a)-(a)-(b)-COOH, or H2N-(b)-(a)-(a)-(a)-(b)-COOH, or H2N-(a)-(a)-(b)-(b)-(a)-COOH, or H2N-(a)-(b)-(a)-(b)-(a)-COOH, or H2N-(b)-(a)-(a)-(b)-(a)-COOH, or H2N-(a)-(b)-(a)-(a)-(a)-COOH, or H2N-(b)-(a)-(b)-(a)-(a)-COOH, or H2 N-(b)-(b)-(a)-(a)-(a)-COOH, or H2N-(a)-(a)-(b)-(b)-(b)-COOH, or H2N-(a)-(b)-(a)-(b)-(b)-COOH, or H2N-(b)-(a)-(a)-(b)-(b)-COOH, or H2N-(a)-(b)-(b)-(a)-(b)-COOH, or H2N-(b)-(a)-(b)-(a)-(b)-COOH, or H2N-(a)-(b)-(b)-(b)-COOH, or H2N-(a)-(b)-(b) -(b)-(a)-COOH, or H2N-(b)-(a)-(b)-(b)-(a)-COOH, or H2N-(b)-(b)-(b)-(a)-(a)-COOH, or H2N-(a)-(b)-(b)-(b)-(b)-COOH, or H2N-(b)-(a)-(b)-(b)-(b)-COOH, or H2N-(b)-(b)-(a)-(b)-(b)-COOH, or H2N-(b)-(b)-(b)-(b)-(a)-COOH, Here, (a) and (b) are as defined above in this paragraph. In the case of multiple fusion partner proteins as defined by (a), the multiple fusion partners may be the same or different. Any fusion partner of interest may be used. For example, the fusion partner polypeptide sequence(s) may be suitable for conferring additional therapeutic properties (e.g., anticoagulation, cell inhibition and / or killing) to the molecule, which may extend the half-life of the molecule in the patient's circulatory system and / or confer further functionality to the molecule. In the case of the fusion protein as defined by (b), which includes the sequence of human annexin A5 (SEQ ID NO: 1 shown in Figure 1), or a variant or variant thereof as described above, or a plurality of protein sequences having dimers, with or without N-terminal methionine, those proteins may be the same or different.

[0045] In further embodiments of the present invention, the purified and / or recovered AnxA5 protein may be a protein comprising, essentially, or consisting thereof, a sequence of annexin A5 or a functional variant or variant thereof selected from the following. a) Human annexin A5 (SEQ ID NO: 1) containing or not containing N-terminal methionine, b) Mammalian orthologs of human annexin A5, c) Allele or gene variant of a) or b), d) A protein that is 50%, 60%, 70%, 75%, for example, 80%, 85%, 90%, or more preferably 95% or 99% identical to any of a), b), or c), e) a) b) c) or d) dimer, A fusion protein containing one or more fusion partners fused to any of f)a), b), c), d), or e).

[0046] In certain embodiments, the AnxA5 protein is a functional variant or variant of annexin A5 that is 50%, 60%, 70%, 75%, for example, 80%, 85%, 90%, or more preferably 95% or 99% identical to human annexin A5, SEQ ID NO: 1, with or without an N-terminal methionine.

[0047] The percentage of identity between two amino acid sequences is determined as follows: First, the amino acid sequences are compared, for example, to Sequence ID No. 1, using the BLAST 2 Sequences (Bl2seq) program from the standalone version of BLASTZ, which includes BLASTN version 2.0.14 and BLASTP version 2.0.14. This standalone version of BLASTZ is available from the National Center for Biotechnology Information website of the U.S. government at ncbi.nlm.nih.gov. Instructions on how to use the Bl2seq program can be found in the readme file included with BLASTZ. Bl2seq compares the two amino acid sequences using the BLASTP algorithm. To compare the two amino acid sequences, the Bl2seq options are set as follows: -i is set for the file containing the first amino acid sequence to be compared (e.g., C:\seq1.txt), -j is set for the file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt), -p is set for blastp, -o is set for any desired filename (e.g., C:\output.txt), and all other options remain at their default settings. For example, the following command can be used to generate an output file containing a comparison between two amino acid sequences: C:\Bl2seq -ic:\seq1.txt -jc:\seq2.txt -p blastp -oc:\output.txt. If the two compared sequences share homology, the specified output file will present their homology regions as aligned sequences. If the two compared sequences do not share homology, the specified output file will not present aligned sequences. Once aligned, the number of matches is determined by counting the number of positions where identical nucleotides or amino acid residues are presented in both sequences.

[0048] The identity percentage is determined by dividing the number of matches by the length of the sequence shown in the identified sequence, and then multiplying the resulting value by 100. For example, if a sequence is compared to the sequence shown in sequence number 1 (the length of the sequence shown in sequence number 1 is 320), and the number of matches is 288, then the sequence has an identity percentage of 90 with respect to the sequence shown in sequence number 1 (i.e., 288 ÷ 320 × 100 = 90).

[0049] The AnxA5 protein may be a dimer of annexin A5 (such as dianexin) or a functional variant or mutant thereof. Dianexin A5 is disclosed in WO02 / 067857.

[0050] Preferably, the AnxA5 protein does not contain a His tag, and its recovery and purification are not achieved using an affinity binding step to a His tag sequence.

[0051] His tags are polyhistidine amino acid motifs in proteins, typically consisting of at least six histidine (His) residues, and often (but not always) located at the N-terminus or C-terminus of the protein. Polyhistidine tags are often used for affinity purification of polyhistidine-tagged recombinant proteins expressed in Escherichia coli and other prokaryotic expression systems by incubation with affinity resins containing bound divalent nickel or cobalt ions, which are commercially available in various types. These resins are generally Sepharose / agarose functionalized with chelating agents such as iminodiacetic acid (Ni-IDA) and nitrilotriacetic acid (Ni-NTA) for nickel, and carboxymethyl aspartate (Co-CMA) for cobalt, to which polyhistidine tags bind with micromolar affinity. The resins are then typically washed with phosphate buffer to remove proteins that do not specifically interact with cobalt or nickel ions. In the case of Ni-based methods, the washing efficiency can be improved by adding 20 mM imidazole (proteins are usually eluted with 150-300 mM imidazole).

[0052] While the His tagging method is convenient for purification, the presence of non-natural polyhistidine motifs in therapeutic proteins, including the AnxA5 protein, is undesirable because it can lead to adverse patient reactions, such as immunological responses. Alternatively, attempting to remove the His tagged motif after protein production is cumbersome, time-consuming, and costly, and in fact, His-tagged protein preparations typically retain one or more heterologous histidine residues.

[0053] As a result, preferably, the AnxA5 protein does not contain the His tag, and its recovery and purification are not achieved using an affinity binding step to the His tag sequence.

[0054] The AnxA5 protein of the present invention may or may not be (preferably not) be a variant or mutant of a protein having a human annexin A5 sequence, modified to include one or more, for example, up to 20 RGD (arginine-glycine-aspartic acid) motifs, as disclosed in WO2010 / 140886 (the contents of which are incorporated herein by reference). As described in WO2010 / 140886, the addition of one or more RGD motifs can enhance phagocytosis by using an AnxA5 variant that binds to phosphatidylserine (PS) on apoptotic cells, and can activate phagocytic cells to engulf apoptotic cells instead of inhibiting phagocytosis.

[0055] B. Host cell culture The AnxA5 protein can be recombinantly expressed in host cell cultures. Preferred host cell cultures expressing the AnxA5 protein according to the present invention are cultures on a commercial scale for AnxA5 protein production, for example, about 100 L, about 200 L, about 300 L, about 400 L, about 500 L, about 600 L, about 700 L, about 800 L, about 900 L, about 1,000 L, about 2,000 L, about 3,000 L, about 4,000 L, about 5,0 These are cultures having a culture volume of 00L, approximately 6,000L, approximately 70,000L, approximately 80,000L, approximately 90,000L, approximately 10,000L, approximately 20,000L, approximately 30,000L, approximately 40,000L, approximately 50,000L, approximately 60,000L, approximately 70,000L, approximately 80,000L, approximately 90,000L, approximately 100,000L, or higher. In this context, the term "approximately" may mean ±50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1% of the given volume.

[0056] Methods for recombinant expression of target genes are well known in the field.

[0057] Typically, the nucleotide sequence encoding the AnxA5 protein is recombinantly expressed in a host cell culture. For example, the sequence encoding the AnxA5 protein may be introduced into host cells by transformation of the host cells with a plasmid or other vector containing the sequence encoding the AnxA5 protein, and optionally, the sequence encoding the AnxA5 protein may be incorporated into the host cell chromosome (or plastome) or maintained on a replicable extrachromosomal vector.

[0058] Therefore, host cells can be transformed with a polynucleotide vector construct containing a sequence encoding the AnxA5 protein.

[0059] The host cell can be either prokaryotic or eukaryotic.

[0060] Bacterial cells are preferred prokaryotic host cells in connection with the present invention. The bacterial host cells may be, for example, Gram-positive or Gram-negative (however, Gram-neutral and Gram-undeciduous bacteria may also be used). Examples of Gram-negative bacteria include, but are not limited to, Escherichia coli, Salmonella, Shigella, Pseudomonas, Neisseria, Haemophilus influenzae, Bordetella pertussis, and Vibrio cholera.

[0061] In at least in relation to the first aspect of the present invention, and optionally in relation to all aspects of the present invention, the host cell is an endotoxin-producing host cell with a cell wall, and is therefore typically a Gram-negative bacterium such as E. coli, e.g., E. coli strain DH5 available from Bethesda Research Laboratories Inc. (Bethesda, MD, USA) and RR1 (number ATCC 31343) available from the American Type Culture Collection (ATCC) of Rockville, MD, USA.

[0062] To avoid misinterpretation, the term "endotoxin-producing host cells with cell walls" may be interpreted to exclude yeasts such as Saccharomyces cerevisiae, and other eukaryotic cells.

[0063] Further particularly preferred endotoxin-producing strains of E. coli include the BL21(DE3) strain (e.g., widely available and described in Marder et al., 2014, BMC Biotechnology, 14:33). However, the applicant has found that annexin A5 expressed from BL21(DE3) exhibits an unexpectedly high level of undesirable post-translational gluconoylation, with approximately 40% of the annexin A5 protein being gluconoylated. This is significantly higher than the level of gluconoylation of most other proteins recombinantly expressed in BL21(DE3), which typically show levels of only about 5–10% gluconoylation. Therefore, it is even more preferable that the endotoxin-producing strain of E. coli is a BL21(DE3) strain that has been engineered to reduce the level of gluconoylation of the AnxA5 protein by overexpressing phosphogluconolactonase (PGL), as described, for example, Aon et al. (Appl. Env. Microbiol., 2008, 74(4):950-958 (the contents of which are incorporated herein by reference), thereby suppressing posttranslational gluconoylation of recombinantly expressed proteins, and thus suppressing the formation of gluconoylated variants of the AnxA5 protein to a level below 40%, for example, below 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, preferably substantially 0%.

[0064] Typically, the bacterial host cells for use in the present invention are walled bacterial host cells, and therefore preferably exclude cells lacking a cell wall and (in the case of Gram-negative bacteria) an outer membrane, such as spheroplasts (as described in Liu et al, 2006, J. Exp. Microbiol., 9:81-85 (the contents of which are incorporated herein by reference)). Spheroplasts, in the context of this application, are not endotoxin-producing host cells and lack a cell wall. Spheroplasts are completely unsuitable for commercial-scale production, particularly due to their susceptibility and fragility resulting from the absence of a cell wall, which severely limits their ability to grow productively in large-volume cultures.

[0065] Optionally, the host cells are endotoxin-producing host cells with cell walls that cannot be lysed by osmotic shock and / or freeze / thaw treatment.

[0066] A further option is a host cell culture in which the host cells have not been cultured, or have never been cultured, in the presence of an antibiotic to which the host cells are not resistant, and optionally in the presence of any antibiotic. Specific antibiotics to be avoided are, in one embodiment, those that result in spheroblast formation, such as ampicillin.

[0067] Eukaryotic host cells include yeast and mammalian cells, preferably vertebrate cells such as those derived from mouse, rat, monkey, or human fibroblast cell lines. Yeast host cells include YPH499, YPH500, and YPH501, which are commonly available from Stratagene Cloning Systems (La Jolla, CA 92037, USA). Preferred mammalian host cells include Chinese hamster ovary (CHO) cells, available from ATCC as CCL61, NIH Swiss mouse embryonic cells NIH / 3T3, available from ATCC as CRL1658, and monkey kidney-derived COS-1 cells, available from ATCC as CRL1650. Preferred insect cells are Sf9 cells, which can be transfected with baculovirus expression vectors.

[0068] Typical prokaryotic vector plasmids include pUC18, pUC19, pBR322, and pBR329 available from Biorad Laboratories (Richmond, CA, USA), pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 available from Pharmacia (Piscataway, NJ, USA), pBS vector, and Phagescript, Bluescript, pNH8A, pNH16A, pNH18A, and pNH46A available from Stratagene Cloning Systems (La Jolla, CA 92037, USA).

[0069] A typical mammalian cell vector plasmid is pSVL, available from Pharmacia (Piscataway, NJ, USA). This vector uses the SV40 late promoter to induce the expression of cloned genes, the highest expression levels of which are found in T antigen-producing cells such as COS-1 cells. An example of an inducible mammalian expression vector is pMSG, also available from Pharmacia (Piscataway, NJ, USA). This vector uses a glucocorticoid-inducible promoter of long terminal repeats of mouse mammary tumor virus to induce the expression of cloned genes.

[0070] Useful yeast plasmid vectors are pRS403-406 and pRS413-416, which are generally available from Stratagene Cloning Systems (La Jolla, CA 92037, USA). Plasmids pRS403, pRS404, pRS405, and pRS406 are yeast integration plasmids (YIps) that incorporate the yeast-selective markers HIS3, TRP1, LEU2, and URA3. Plasmid pRS413-416 is a yeast centromere plasmid (YCps).

[0071] An expression vector containing the AnxA5 protein coding sequence and, for example, a suitable transcriptional or translational regulatory sequence can be constructed using methods well known to those skilled in the art. One such method involves ligation via a homopolymer tail. A homopolymer polydA (or polydC) tail is attached to an exposed 3'OH group on the DNA fragment to be cloned by a terminal deoxynucleotidyltransferase. The fragment is then annealable to a polydT (or polydG) tail attached to the end of a linearized plasmid vector. The gap remaining after annealing can be filled by a DNA polymerase, and the free end can be ligated by a DNA ligase.

[0072] Another method involves ligation via adherent ends. Compatible adherent ends can be generated on DNA fragments and vectors by the action of suitable restriction enzymes. These ends rapidly anneal by complementary base pairing, and any remaining nicks can be joined by the action of DNA ligases.

[0073] Further methods utilize synthetic molecules called linkers and adapters. Blunt-end DNA fragments are produced by bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, which removes the protruding 3' end and fills the recessed 3' end. Synthetic linkers, which are pieces of blunt-end double-stranded DNA containing recognition sequences for specific restriction enzymes, can be ligated to blunt-end DNA fragments by T4 DNA ligase. They are then digested with appropriate restriction enzymes to create adherent ends and ligated into expression vectors with compatible ends. Adapters are also chemically synthesized DNA fragments that contain one blunt end for ligation but also possess one implemented adherent end.

[0074] Synthetic linkers containing a variety of restriction endonucleases are commercially available from several sources, including Biotechnologies Inc (New Haven, CN, USA).

[0075] A preferred method for modifying the DNA encoding the AnxA5 protein is to use a polymerase chain reaction, as disclosed by Saiki et al (1988) Science 239, 487-491. In this method, two specific oligonucleotide primers are adjacent to the DNA to be enzymatically amplified, and these oligonucleotide primers themselves are incorporated into the amplified DNA. These specific primers may contain restriction endonuclease recognition sites that can be used for cloning into expression vectors using methods known in the art.

[0076] Transformation of a suitable cell host with a DNA construct containing the sequence encoding the AnxA5 protein is typically carried out by a well-known method depending on the type of vector used.

[0077] Regarding the transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110 and Sambrook et al (2001) Molecular Cloning, A Laboratory Manual, 3 rd See Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Transformation of yeast cells is described in Sherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, NY. The method of Beggs (1978) Nature 275, 104-109 is also useful. For vertebrate cells, reagents useful for transfecting such cells, such as calcium phosphate and DEAE-dextran or liposomal formulations, are available from Stratagene Cloning Systems or Life Technologies Inc. (Gaithersburg, MD 20877, USA).

[0078] Electroporation is also useful for cell transformation and is well known in the art for the transformation of yeast cells, bacterial cells, and vertebrate cells.

[0079] For example, many bacterial species may be transformed by the method described in Luchansky et al (1988) Mol. Microbiol. 2, 637-646 (incorporated herein by reference). The maximum number of transformants is consistently recovered after electroporation of a DNA-cell mixture suspended in 2.5-fold PEB using 6250 V per cm in 25 μFD.

[0080] A method for transforming yeast by electroporation is disclosed in Becker & Guarente (1990) Methods Enzymol. 194, 182.

[0081] Physical methods may be used to introduce DNA into animal and plant cells. For example, microinjection involves using a very fine pipette to directly inject DNA molecules into the nucleus of the cell to be transformed. Another example is high-speed micro-injection, which involves bombarding cells with gold or tungsten particles, usually coated with DNA.

[0082] Successfully transformed cells, i.e., cells containing a DNA construct with a sequence encoding the AnxA5 protein, can be identified by known techniques. For example, one selection technique involves incorporating a DNA sequence (marker) encoding a selectable trait of the transformed cell into an expression vector. These markers include dihydrofolate reductase, G418, or neomycin resistance for eukaryotic cell cultures, and tetracycline, kanamycin, or ampicillin resistance genes for cultures in E. coli and other bacteria. Alternatively, the genes for such selectable traits may be on a separate vector used for co-transformation of the desired host cells.

[0083] While marker genes can be used to identify transformants, it is desirable to determine which cells contain recombinant DNA molecules and which contain self-ligated vector molecules. This can be achieved using cloning vectors, where the insertion of a DNA fragment disrupts the integrity of one of the genes present in that molecule; therefore, recombinants can be identified by the loss of function of that gene.

[0084] Another method for identifying successfully transformed cells involves growing cells resulting from the introduction of an expression construct containing a sequence encoding the AnxA5 protein to produce the polypeptide of the present invention. The cells can be harvested and lysed, and their DNA content can be examined for the presence of their DNA using methods such as those described by Southern (1975) J. Mol. Biol. 98, 503 or Berent et al (1985) Biotech. 3, 208. Alternatively, the presence of the protein in the supernatant can be detected using antibodies as described below.

[0085] In addition to directly assaying for the presence of recombinant DNA, successful transformation can be confirmed by well-known immunological methods if the recombinant DNA is capable of directing protein expression. For example, cells successfully transformed with an expression vector produce proteins exhibiting appropriate antigenicity. Samples of cells suspected of being transformed are collected and assayed for proteins using suitable antibodies.

[0086] Therefore, the transformed host cells themselves can be cultured to provide cultured transformed host cells that express the AnxA5 protein. The culture may be a monoclonal (clonally homogeneous) culture in a nutrient medium, or a culture derived from a monoclonal culture.

[0087] Cultures of transformed host cells expressing the AnxA5 protein are grown under suitable growth conditions typically selected to balance productivity with the time and cost associated with the culture phase until the desired cell density is achieved, and the cells are then typically harvested. The optimal time for cell harvesting can be determined empirically for any given culture.

[0088] Collection involves, for example, the collection of host cells from the culture medium (typically intact and typically retaining substantially all of the AnxA5 protein within the cells (e.g., 80%, 90%, 95%, or over 99% or 100%)). This can generally be achieved by centrifugation or filtration, which collects the cultured host cells in biomass form. In the case of centrifugation, the supernatant may be discarded, and the cell pellet may be transferred directly or indirectly to the cell culture homogenization stage (e.g., after storage by freezing).

[0089] C. Homogenization of cell cultures As discussed above, a first aspect of the present invention provides an improved process for protein release from host cells. More specifically, it provides a process for the recovery and / or purification of a recombinantly expressed intracellular protein comprising the sequence of annexin A5 (AnxA5) from a cell walled endotoxin-producing host cell, the process comprising releasing the intracellular protein from the host cell, characterized in that the step of releasing the intracellular AnxA5 protein is carried out in the presence of a homogenization buffer comprising a nonionic detergent.

[0090] Preferably, the nonionic detergent is a polysorbate, more preferably a polysorbate selected from Tween 20 and Tween 80, most preferably Tween 80. Alternatively, though less preferred, other nonionic detergents may be used, provided that they have a similar UV absorption rate λ to the protein absorption maximum, i.e., 275-280 nm. maxThe use of nonionic detergents containing λ is preferably avoided (in both the cell culture homogenization step and any other step of this process), because it may interfere with the ability to monitor the presence of proteins by UV absorption rate during the recovery process. In this context, "similarly" means λ max However, this could mean that the absorption maximum of the purified AnxA5 protein is within 10nm, 9nm, 8nm, 7nm, 6nm, 5nm, 4nm, 3nm, 2nm, or 1nm. Therefore, for example, nonionic detergents are λ max It may be preferable that Triton X-100 does not have a wavelength of 275 nm, and for this reason, it may be preferable that Triton X-100 is not used in the cell culture homogenization step and / or any other step of the process of the present invention.

[0091] It should be noted that, prior to cell homogenization to release the intracellular AnxA5 protein, a nonionic detergent may be included in the homogenization buffer to which the cells are added (for example, the homogenization buffer may be "pre-formed" with the present nonionic detergent), or the cells may be suspended in a homogenization buffer that does not contain a nonionic detergent, and then the nonionic detergent may be added to and mixed with the cells suspended in the homogenization buffer.

[0092] Preferably, the step of releasing intracellular AnxA5 protein is carried out in the presence of a homogenization buffer containing an amount of nonionic detergent effective in reducing (50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more) or preventing binding between annexin A5 and endotoxin. Endotoxin levels may be measured by methods well known and established in the art, for example, by the Limulus Amebocyte Lysate (LAL) test.

[0093] For example, the homogenization buffer may contain 0.01 to 10% (w / w) non-ionic detergent, such as 0.02 to 5% (w / w), 0.05 to 2% (w / w), or about 1% (w / w) non-ionic detergent. The term "about" in this context may include a meaning of ±0.5%, 0.4%, 0.3%, 0.2% or 0.1% (w / w).

[0094] Calcium ions or ionizable calcium compounds (such as CaCl2) are preferably not added or included in the homogenization buffer. Therefore, the free calcium ion concentration in the homogenization buffer at the time of release of intracellular AnxA5 protein from host cells is less than 10 mM, preferably less than 5 mM, 4 mM, 3 mM, 2 mM or 1 mM, more preferably less than 500 μM, 400 μM, 300 μM, 200 μM, 100 μM, 50 μM, 40 μM, 30 μM, 20 μM, 10 μM, 5 μM, 4 μM, 3 μM, 2 μM, 1 μm or substantially zero.

[0095] In one embodiment, the homogenization buffer may contain a calcium metal ion chelating agent. In light of an optional subsequent step involving enzymatic treatment and using such enzyme with Mg 2+ it may be preferable to select a calcium ion chelating agent that does not strongly bind to Mg 2+ such as ethylene glycol tetraacetic acid (EGTA). Alternatively, an optional subsequent step involving enzymatic treatment and using such enzyme with Mg 2+ may be replaced by another step that does not require Mg 2+ In that case, any calcium ion chelating agent such as EGTA or ethylenediaminetetraacetic acid (EDTA) may be included in the homogenization buffer. [[ID=D17]]

[0096] Optionally, the concentration of free calcium ions in the homogenization buffer and / or the amount of calcium metal ion chelating agent is in an amount effective in reducing (50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or more, etc.) or preventing the binding between annexin A5 and components of the host cell membrane and / or wall, compared to the level of binding that would be observed in the presence of a homogenization buffer consisting of 50 mM Tris HCl and 10 mM CaCl2 at pH 7.2.

[0097] For example, the homogenization buffer in the first aspect of the present invention may contain 0.01 to 500 mM, for example, 0.05 to 100 mM, 0.5 to 20 mM, 1 to 15 mM, 2 to 10 mM, or about 4 mM of a calcium metal ion chelating agent, preferably EDTA or EGTA.

[0098] As discussed above, the cell culture homogenization step in the first aspect of the present invention does not include a centrifugation step for the purification and separation of the released AnxA5 protein from host cell debris. Nevertheless, a simple test using centrifugation can be performed on aliquots of lysed cells to establish tolerance to free calcium ions and / or effective amounts of calcium metal ion chelating agents in the homogenization buffer. After cell homogenization, aliquots of lysed cells (e.g., 100 mL) are subjected to centrifugation (e.g., 38,900 g for 30 minutes), and the supernatant and pellet are separated. The amount of AnxA5 protein in the supernatant is determined to determine the level of "free" AnxA5 protein. The pellet is resuspended in 50 mM Tris HCl, 20 mM EDTA (pH 7.2) with stirring at 4°C for 30 minutes to release any bound AnxA5 protein. Then, it is centrifuged at 38,900 g at 4°C for 30 minutes to determine the amount of bound AnxA5 protein released into the supernatant, thereby determining the level of "bound" AnxA5 protein. In this context, the percentage of AnxA5 binding to the host cell membrane and / or wall components is given by (level of "bound" AnxA5 protein / (level of "bound" AnxA5 protein + level of "free" AnxA5 protein)) × 100.

[0099] Preferably, when determined by the method described above, the percentage of bound AnxA5 in the resulting biomass homogenate is less than 50%, 40%, 30%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or substantially 0%.

[0100] In this specification, the solution is referred to as a “homogenization buffer,” but it is not necessarily required to be a pH buffer. However, optionally, the homogenization buffer may further contain additional components including a buffer (e.g., Tris), and optionally, the pH may be adjusted as needed to, for example, a range of pH 6 to 8, more preferably pH 7 to 8.5, for example, about pH 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5. In one embodiment, pH 7.4 may be selected for use.

[0101] As discussed above, the subsequent process is Mg 2+ In several options, including enzymatic treatment with enzymes that require cofactors such as those mentioned above, it may also be desirable to include the cofactors in the homogenization buffer.

[0102] In one exemplary embodiment, the homogenization buffer for use in the first embodiment of the present invention comprises, or consists of, an aqueous solution of 50 mM Tris (pH 7.4), 1 mM MgCl2, and 1% (w / w) Tween 80.

[0103] The process of the first aspect of the present invention may include a step of mixing biomass from a host cell culture in a homogenization buffer at a concentration of biomass (wet weight) of about 1 g to 300 g per liter of homogenization buffer, for example, at a concentration of about 10 g to 200 g per liter of homogenization buffer. Examples of concentrations may be about 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, 90 g / L, 100 g / L, 110 g / L, 120 g / L, 130 g / L, 140 g / L, 150 g / L, 160 g / L, 170 g / L, 180 g / L, 190 g / L, or 200 g / L. A resuspend ratio of about 100 g of biomass per liter of homogenization buffer is particularly preferred. In this context, the term "approximately" is intended to include ±5 g / L, 4 g / L, 3 g / L, 2 g / L, or 1 g / L of the specified value.

[0104] Mixing is typically carried out at room temperature, i.e., typically between 18°C ​​and 28°C, for example, around 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, or 27°C. It may be preferable to control the temperature during the mixing process to maintain it at or around the temperature selected from the above enumerated items (e.g., ±5, 4, 3, 2, or 1°C), but dynamic temperature control at this stage is usually not required.

[0105] Optionally, the homogenization buffer may also contain, or be added to, one or more enzymes useful in enzymatic treatment, after mixing with biomass but before host cell homogenization. For example, it may be preferable to include or add one or more nuclease enzymes after homogenization to assist in the degradation of host cell nucleic acids (including DNA and / or RNA). This will reduce the viscosity of the homogenate subsequently produced, thereby assisting downstream processing steps. Any suitable enzyme may be used. The enzyme may be a nuclease, such as nuclease A, preferably nuclease A derived from Serratia marescens. One such example enzyme of interest is benzonase nuclease, an endonuclease derived from Serratia marescens, available from commercial sources including Merck / Novagen, Sigma Aldrich, etc., which can be used to degrade all forms of DNA and RNA while having no proteolytic activity. It is effective under a wide range of conditions and possesses high specific activity. This enzyme completely digests nucleic acids down to 5′-monophosphate-terminal oligonucleotides of 2–5 nucleotides in length (below the hybridization limit), which is ideal for the removal of nucleic acids from recombinant proteins and enables compliance with FDA guidelines regarding nucleic acid contamination. The Benzonase enzyme requires 1–2 mM Mg to activate. 2+It requires ionic and nonionic detergents, reducing agents, the protease inhibitor PMSF (1 mM), the chelating agent EDTA (1 mM), and urea to remain active (relative activity depends on specific conditions). While those skilled in the art would readily be able to determine the effective concentration of such nuclease enzyme, the applicant has identified that benzonase is effective when pre-diluted at least about 3.3 U per liter of host cell culture or about 1.85 U per liter of resuspended biomass. In this context, the term “about” may include ±90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the specified number of units.

[0106] The step of releasing intracellular AnxA5 protein from host cells in a homogenization buffer may involve any suitable method for cell homogenization or lysis. For example, this may include lysing, degrading, or homogenizing, sonicating, or pressurizing the host cells so that the cell wall and cell membrane barrier of the host cells are disrupted, thereby releasing the intracellular AnxA5 protein. In certain options of the first aspect of the present invention, this step does not involve the use of osmotic shock and / or freeze-thaw steps.

[0107] In a preferred embodiment of the first aspect of the present invention, the step of releasing intracellular AnxA5 protein from a host cell includes high-pressure homogenization, for example, one or more cycles of high-pressure homogenization at about 400 bar to about 2,500 bar, preferably three cycles of homogenization at about 600 bar, or two cycles of homogenization at about 800 bar. In this context, the term “about” may include ±500, 400, 300, 200, 100, 50, 40, 30, 20, or 10 bar of the defined value.

[0108] Optionally, for example, in a situation where no nuclease enzyme is added to decompose nucleic acids (for example, Mg 2+By incorporating chelating agents, etc., Mg in homogenization buffer 2+ If the concentration is too low, it may be beneficial to include several further high-pressure homogenizations (e.g., 2 to 4 times at pressures in the range of approximately 600 to 2500 bar) to degrade the nucleic acids and reduce the viscosity of the homogenate.

[0109] Therefore, the step of releasing intracellular AnxA5 protein after cell homogenization can produce a biomass homogenate containing the released AnxA5 protein. Preferably, the biomass homogenate is homogeneous, meaning that at least 80%, preferably at least 90%, more preferably at least 95%, 96%, 97%, 98%, 99%, or substantially 100% of the cellular biomass is degraded.

[0110] Depending on the cell homogenization technique applied, this technique may cause an increase in the temperature of the homogenate. It may be preferable to operate the cell homogenization technique in a manner that prevents an undesirable increase in temperature, and / or to apply temperature control to prevent an undesirable increase in temperature. However, if the homogenate contains an enzyme treatment agent such as benzonase, for example, it may be advantageous to use a temperature increase to approach, and preferably within, the optimal temperature range for the enzyme. In the case of benzonase, temperatures in the range of about 36-40°C may be particularly preferred.

[0111] Cell homogenization procedures involve metal ion cofactors (e.g., Mg 2+ If the process involves enzymatic treatment with an enzyme that requires ) and further, if the homogenization buffer excludes the calcium metal ion chelator, then in one option, the calcium ion chelator may be added after the completion of the enzymatic treatment. This may be done before or after the clarification step, as discussed below.

[0112] Following cell homogenization, biomass homogenates typically further contain one or more (typically all) impurities selected from the group consisting of host cell proteins, host cell wall components, host cell membrane components, host cell nucleic acids, and endotoxins.

[0113] The viscosity of the homogenate is preferably low enough to assist the downstream processing steps.

[0114] Clarification of D. homogenate In an optional and preferred embodiment, after the production of biomass homogenate, it is then treated in a clarification step.

[0115] Accordingly, in a further embodiment of the first aspect of the present invention, the process further includes a step of clarifying the biomass homogenate to produce a clarified product containing the released AnxA5 protein. This step is performed to reduce the nucleic acid content and, further, to obtain a solution with reduced particles, which may be applied to subsequent purification steps such as capture chromatography, which will be discussed further below.

[0116] Any suitable clarification step or combination of steps can be performed. For example, the clarification step may include (preferably following the nuclease treatment) passing the biomass homogenate containing the released AnxA5 protein through a filter such as a cellulose or polypropylene filter, and the filter effluent is the clarified product containing the released AnxA5 protein.

[0117] Preferably, the filter is a depth filter and / or preferably has a cutoff of less than 4 μm, for example, a cutoff of less than 3 μm, 2 μm, or 1 μm, most preferably in the range of 0.2 to 0.6 μm. For example, the homogenate may be clarified by filtration using a 0.6 to 0.2 μm cutoff depth filter, an example of which is commercially available, such as the cellulose-based Cuno 60 SP depth filter. Other depth filters found to offer good performance (i.e., good filtration with no product loss), though not as good as the preferred cellulose Cuno 60 SP depth filter with a 0.6-0.2 μm cutoff, include cellulose + kieselguhr filters with a 0.5 μm cutoff (e.g., PR12 UP available from Begerow), polypropylene filters with a 1.2 μm cutoff (e.g., Sartopure PP2 available from Sartorius), cellulose filters with a 0.1 μm cutoff (e.g., Sartoclear S9 available from Sartorius), polypropylene filters with a 0.65 μm cutoff (e.g., Sartopure PP2 available from Sartorius), and cellulose filters with a 0.2-0.5 μm cutoff (e.g., EK 1P or EKM-P, both available from Pall; these are good but slow). Further testing has shown that filters with larger cutoffs (e.g., above 4 μm) achieve only moderate particle removal, making them less desirable.

[0118] The applicant has also found that positively charged cellulose-based filters can further reduce the DNA content in the clarified homogenate and therefore represent a category of filters that are particularly preferred for use in the clarification process.

[0119] Furthermore, it was found that cellulose-based filters require a smaller filter area to provide effective clarification than the corresponding polypropylene filters. This may be another reason why cellulose-based filters are particularly preferred for use in clarification processes.

[0120] The selection of filter area may also depend on the degree and nature of the homogenization used. For example, the applicant suggests that if cell homogenization is performed by three homogenization cycles at approximately 600 bar, then approximately 60 cm³ per liter of homogenate is used. 2 A filter area of ​​this size is preferable, however, if cell homogenization is performed by two homogenization cycles at approximately 800 bar, then approximately 180 cm³ per liter of homogenate is required. 2 We found that the filter area is optimal. Therefore, the depth filter can be optionally set to 10-500 cm³ per liter of homogenate to be clarified. 2 For example, 30-400cm 2 / L, 40~250cm 2 / L, 50~200cm 2 / L or 60-180cm 2 / L, for example, 50-100cm 2 / L, or 60-80cm 2 / L; or 120~240cm 2 / L, or 150-210cm 2 It may be selected to have an area of ​​ / L.

[0121] After clarification, the clarified product may be further adjusted by adding one or more additional additives in preparation for subsequent steps. For example, it may be preferable to condition the clarified product by adding one or both of the following: (a) a nonionic detergent such as polysorbate and most preferably Tween 80, and (b) a calcium metal ion chelating agent such as EDTA (unless the clarified product already contains a sufficient level of chelating agent based on the incorporation of the chelating agent into the homogenization buffer and / or by the addition of the chelating agent after the enzymatic treatment step).

[0122] In one exemplary embodiment, the clarified product is diluted approximately 2-fold with a 1% nonionic detergent (most preferably Tween 80), and a calcium ion chelating agent (most preferably EDTA) is added to a final concentration of approximately 2 mM.

[0123] A further advantageous feature of the process of the present invention is the absence of any time-wasting dialysis steps on the clarified product prior to further chromatographic capture steps, such as anion exchange capture, as discussed below. Therefore, in embodiments of the present invention, the clarified product is not subjected to dialysis before chromatographic capture of the AnxA5 protein.

[0124] E. Anion exchange and capture In a first aspect of the present invention, the process may further include a step of performing a first anion exchange capture step, thereby providing the released AnxA5 protein to an anion exchange resin in order to produce a first anion exchange product containing the released AnxA5 protein.

[0125] Generally, protein capture from bacterial (e.g., E. coli) homogenates / lysates by anion exchange chromatography is not a preferred strategy because the binding of large amounts of host cell proteins (HCPs) and DNA to the capture resin negatively impacts the target product's binding capacity and also burdens the resin's performance. However, the applicant has determined that the cell homogenization and clarification procedure described above according to a first aspect of the present invention is effective in providing an AnxA5 protein product that can be effectively further purified using anion exchange chromatography.

[0126] Therefore, in one embodiment, the clarified product containing the released AnxA5 protein, produced by the homogenate / lysate clarification and / or nucleic acid degradation steps discussed above, is subjected to a first anion exchange capture step to produce a first anion exchange product containing the released AnxA5 protein.

[0127] Optionally, prior to the first anion exchange step, one or more parameters of the environment for the released AnxA5 protein are adjusted, selected from the group consisting of pH, conductivity, calcium ion chelating agent level, and nonionic detergent level. For example, the AnxA5 protein composition subjected to the anion exchange step may be formulated at a pH of approximately 6.9, optionally ±1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 (in one option, the preferred range is pH 6 to 8.5, more preferably pH 6.5 to 7.5, and most preferably pH 6.9). The applicant has found that low pH values ​​around this range (e.g., at pH 6) tend to result in the presence of detectable host cell proteins in the eluted product, while solubility is somewhat reduced at pH 8 and above. The AnxA5 protein composition subjected to the anion exchange step may optionally be adjusted to have a conductivity of approximately 2.8 mS / cm, ±1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mS / cm. After clarification, the clarified product may be further adjusted by the addition of one or more additional additives as preparation for subsequent steps. As already discussed above, prior to the first anion exchange step, it may be preferable to condition the AnxA5 protein composition subjected to the anion exchange step by adding either or both of (a) a nonionic detergent such as polysorbate and most preferably Tween 80, and (b) a calcium metal ion chelating agent such as EDTA (unless the clarified product already contains a sufficient level of chelating agent based on the incorporation of the chelating agent into the homogenization buffer and / or by the addition of the chelating agent after the enzymatic treatment step). In one exemplary embodiment, the AnxA5 protein composition subjected to the anion exchange process is diluted approximately 2-fold with a 1% nonionic detergent (most preferably Tween 80), and a calcium ion chelating agent (most preferably EDTA) is added to a final concentration of approximately 2 mM.

[0128] In this regard, the use of a nonionic detergent and / or a calcium metal ion chelating agent to condition the AnxA5 protein composition subjected to the first anion exchange capture step can be highly beneficial in enhancing the separation of the AnxA5 protein from host cell-derived impurities (including cell wall components, cell membrane components, endotoxins, nucleic acids, etc.) by the first anion exchange capture step. For example, the applicant has demonstrated highly efficient removal of endotoxins (reduction of approximately 99%) by the first capture anion exchange step when carried out in the presence of a nonionic detergent and a calcium metal ion chelating agent.

[0129] In relation to the second anion exchange refining step, as will be mainly discussed below, in the option where a calcium metal ion chelating agent (e.g., EDTA) is present in the AnxA5 protein composition subjected to the first anion exchange capture step, it may also be beneficial for the operation of the first anion exchange capture step to include one or more additional selected metal ions (not calcium), and these additional selected metal ions are selected such that the binding affinity of the calcium metal ion chelating agent to the selected metal ions is greater than the binding affinity of the calcium metal ion chelating agent to the anion exchange resin, but less than the binding affinity of the calcium ions to the selected metal ions. One example of a metal ion is Mg 2+ That is the case.

[0130] Typically, the anion exchange resin is equilibrated before contact with the AnxA5 product. Any suitable equilibration method may be used. For example, the anion exchange resin can be equilibrated with a buffer (e.g., 20 mM Tris pH 7.4), a nonionic detergent (e.g., 0.1% polysorbate, preferably Tween 80), and a salt (e.g., 25 mM NaCl). Any suitable equilibration volume may be used, and without limitation, the applicant has found that 3 column volumes (CV) are a suitable volume in exemplary embodiments.

[0131] Preferably, the first anion exchange capture step is performed in positive mode with respect to the AnxA5 protein, so that the AnxA5 protein is temporarily bound to the anion exchanger during the anion exchange step, a washing solution is typically passed through the column to remove impurities from the bound AnxA5 protein, and then an elution buffer is applied to the anion exchange resin to release the bound AnxA5 protein, thereby producing a first anion exchange product containing the released AnxA5 protein.

[0132] The applicant found that strong anion exchange resins provided an acceptable capacity for capturing the AnxA5 protein, while the performance of weak anion exchange resins was not very acceptable, and therefore strong anion exchange resins are preferred. Strong anion exchange resins are well known in the art and include resins containing quaternary ammonium functional groups, such as Type I resins having trialkylammonium chloride or ammonium hydroxide, or Type II resins having dialkyl2-hydroxyethylammonium chloride or ammonium hydroxide (e.g., Q Sepharose XL by GE Healthcare, Capto Q by GE Healthcare, Unosphere Q by Biorad, or Eshmuno Q by Merck). Weak anion exchange resins are unpreferred and include DEAE resins containing diethylaminoethyl functional groups. Q Sepharose XL resin (e.g., provided by GE Healthcare) may be the most preferred.

[0133] After loading onto the anion exchange resin, under positive mode, the AnxA5 protein can be temporarily bound to the resin and washed to reduce / remove impurities. Any suitable washing conditions can be used. For example, the washing solution may contain, essentially consist of, an aqueous solution of a buffer (e.g., 20 mM Tris pH 7.4), a nonionic detergent (e.g., 0.1% polysorbate, preferably Tween 80), and a salt (e.g., 25 mM NaCl). Any suitable washing volume may be used, and without limitation, the applicant has found that 10 column volumes (CV) is a suitable volume in an exemplary embodiment.

[0134] The bound AnxA5 protein is then released from the anion exchange resin using an elution buffer. Any suitable elution buffer may be used. For example, the elution buffer may contain, essentially, or consist of, a buffer (e.g., 20 mM Tris pH 7.4), a nonionic detergent (e.g., 0.01–1% (w / v), more preferably 0.1% (w / v) polysorbate, preferably Tween 80), and an aqueous solution of a salt at a higher concentration than the washing solution (e.g., 300 mM NaCl, optionally ±100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 mM). Any suitable elution volume may be used, and without limitation, the applicant has found that 9 column volumes (CV) is a suitable volume for elution in an exemplary embodiment.

[0135] Therefore, the AnxA5 protein is captured in the elution buffer, which provides the first anion exchange product.

[0136] The anion exchange resin can then be regenerated and purified. Suitable methods for regeneration and purification are known in the art, and one such suitable protocol is considered in the examples.

[0137] Typically, the first anion exchange product containing the released AnxA5 protein contains substantially more than 50% of the AnxA5 protein released from the host cell. Preferably, the first anion exchange product contains more than 60%, 70%, or 80% of the AnxA5 protein released from the host cell. In comparison with the process of Marder et al. (see above), it is clear that the prior art process exhibited a significant product loss of approximately 50% or more. For example, the process of Marder et al. (see above) involves an initial purification centrifugation step in which the supernatant is discarded and annexin A5 bound to the pellet is collected. The relative amounts of annexin A5 in the discarded supernatant and annexin A5 recovered from the pellet in the process of Marder et al. (see above) are shown in the second and third columns of Figure 3, respectively. From that figure, it is clear that the discarding of approximately half of the released annexin A5 in the process of Marder et al. (see above) leads to a low yield method.

[0138] For further comparison, the applicant has found that the yield of the example process provides a yield of approximately 5 g of AnxA5 protein per liter of culture at the end of the first anion exchange chromatography capture step, which is significantly higher than the yield of 0.983 g of purified annexin A5 protein per liter of culture reported by Marder et al. (see above) (see paragraph 2, "Conclusion").

[0139] Therefore, in the first aspect of the present invention, it is preferable that the first anion exchange product contains more than 1 g, more than 2 g, more than 3 g, more than 4 g, or about 5 g of AnxA5 protein per liter of culture.

[0140] Optionally, the first anion exchange product is subjected directly or indirectly to a filtration step (such as a sterile filtration step). For example, a 0.45-0.2 μM filtration step has been found to be preferable, without limitation.

[0141] F. Affinity chromatography The applicant also discovered that, in contrast to conventional methods where yield loss tends to increase with the sequential addition of purification steps (because the product is lost at each step), the combination of anion exchange and heparin affinity chromatography has the remarkable advantage of achieving the high purity that can be obtained with heparin affinity chromatography alone, but with a substantially increased yield (i.e., the recovery rate increases from about 30-40% to about 70-90%) (see, for example, Example 2 below). This significant increase in yield associated with the addition of purification steps is the opposite of what would normally be expected from a combination of purification steps.

[0142] Accordingly, a second aspect of the present invention provides a process for the recovery and / or purification of a protein containing the annexin A5 (AnxA5) sequence from a solution containing the AnxA5 protein and one or more impurities (which may or may not be products of the clarification step described above), the method being described To perform a first anion exchange step, thereby producing a first anion exchange product containing the AnxA5 protein, a solution containing the AnxA5 protein and one or more impurities (this solution may or may not be a direct or indirect product of the cell homogenization, clarification and / or first anion exchange chromatographic capture step discussed above) is applied to an anion exchange resin, The method includes directly or indirectly subjecting a first anion exchange product to an affinity chromatography step to produce a first affinity chromatography product containing the released AnxA5 protein.

[0143] Preferably, according to the process of a second aspect of the present invention, the affinity chromatography step may include binding of the AnxA5 protein to immobilized heparin, optionally facilitated by the presence of calcium ions, and further optionally, the AnxA5 protein is eluted from the immobilized heparin using an elution buffer containing a calcium ion chelating agent such as EDTA.

[0144] In addition, as discussed in Example 3, the applicant found that Tween 80 has a particularly advantageous effect on the heparin affinity chromatography process (compared to other nonionic detergents, including other Tweens such as Tween 20). Incorporating Tween 80 into the buffer used in the heparin affinity chromatography process at, for example, around 0.1% (w / v), can assist in the elution of the AnxA5 protein at a single peak, reduce pressure, and prevent precipitation.

[0145] Accordingly, a third aspect of the present invention provides a process for the recovery and / or purification of a protein containing the sequence of annexin A5 (AnxA5) from a solution containing the AnxA5 protein and one or more impurities, the method comprising subjecting the solution containing the AnxA5 protein and one or more impurities (which may or may not be a direct or indirect product of the cell homogenization, clarification and / or first anion exchange chromatography capture step discussed above) to a heparin affinity chromatography step in the presence of Tween 80 (preferably in the presence of 0.1% Tween 80) to produce a first affinity chromatography product containing the released AnxA5 protein.

[0146] Therefore, both the second and third aspects of the present invention can be operated independently of each other or in combination (i.e., the heparin affinity chromatography step in the second aspect of the present invention may include Tween 80 (e.g., around 0.1% (w / v)) in the buffer used in the heparin affinity chromatography step).

[0147] More generally, however, a first aspect of the present invention may include a step of subjecting the released AnxA5 protein to an affinity chromatography step to produce a first affinity chromatography product containing the released AnxA5 protein. For example, the AnxA5 protein in the first anion exchange product produced by the method described above may be particularly preferably subjected to the affinity chromatography step directly or indirectly (e.g., after sterile filtration and / or addition of further components).

[0148] Therefore, in one embodiment of the first aspect of the present invention (which may be combined with any or both of the features of the second and third aspects of the present invention), the process is (a) A step in which the biomass homogenate containing the released AnxA5 protein described above is clarified by the clarification process described above, thereby producing a clarified product containing the released AnxA5 protein, (b) The first anion exchange step described above is carried out, and the AnxA5 protein in the clarified product is subjected to an anion exchange resin in order to produce a first anion exchange product containing the AnxA5 protein. (c) The AnxA5 protein in the first anion exchange product is subjected (directly or indirectly) to an affinity chromatography step.

[0149] Preferably, the affinity chromatography step involves binding the AnxA5 protein to immobilized heparin, and optionally, the binding is facilitated by the presence of calcium ions.

[0150] Therefore, prior to the heparin affinity step, the AnxA5 product may be conditioned by the addition of one or more of the following: calcium ions (e.g., CaCl2), a nonionic detergent (preferably Tween 80), and optionally buffered (e.g., Tris buffer at pH 7.4). Without limitation, the applicant has demonstrated the beneficial effect of diluting the filtered anion exchange product about 8-fold with a dilution buffer containing 20 mM Tris (pH 7.4), 0.1% Tween 80, and 2 mM CaCl2.

[0151] Therefore, it may be preferable that the AnxA5 product be conditioned with polysorbate 80, and more preferably, the polysorbate 80 is in a final concentration of about 0.01% to a maximum of about 10% (w / v), for example, about 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, 0.16%, 0.17%, 0.18%, 0.19%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In this context, the term "approximately" refers to ±50%, 40%, 30%, 20%, 10%, or 5% of the given value.

[0152] Dilution with calcium allows annexin A5 to bind to immobilized heparin chromatography. This interaction is slower compared to ionic interactions. Contact time is important, and therefore this chromatography is preferably performed at 100 cm / h or less.

[0153] The conditions used in loading affinity chromatography columns (e.g., heparin affinity chromatography columns) allow the binding of the AnxA5 protein to heparin. In other words, affinity chromatography is typically performed in positive mode with respect to the AnxA5 protein.

[0154] Affinity chromatography columns (e.g., heparin affinity chromatography columns) can be loaded with a desired level of AnxA5 product. For example, loading may be carried out at approximately 5 g per liter of column resin volume, or at higher levels, such as approximately 10 g / L, 15 g / L, 20 g / L, 25 g / L, or 30 g / L or more. In this context, the term "approximately" refers to ±50%, 40%, 30%, 20%, 10%, or 5% of the specified value. In fact, the applicant has found that loading of approximately 20-30 g of AnxA5 product per liter of column resin volume provides very satisfactory results in terms of process efficiency and product purification and recovery. In relation to such high loading amounts, the applicant has found that the presence of polysorbate 80 in the loaded mixture is particularly beneficial in avoiding AnxA5 protein precipitation or insolubility. In the absence of polysorbate 80, precipitation and increased back pressure are observed, and the affinity chromatography process becomes less efficient. Please refer to Examples 3 and 4.

[0155] After loading with the AnxA5 protein, the column is typically washed one or more times to remove impurities. Any suitable washing protocol may be used. The applicant has found, without limitation, that suitable washing protocols include two-stage washing. For example, in the first washing stage, washing may be performed using a first washing buffer containing calcium (e.g., 20 mM Tris pH 7.4, 0.1% Tween 80, 2 mM CaCl2). The volume of the first washing stage may be varied depending on the desired results and the exact properties of the washing solution used. Without limitation, the applicant has found, for example, that the washing buffer described above can be successfully used for washing 15 CV. In the second washing stage, the washing buffer may contain calcium in a lower amount than in the first washing stage, or preferably, may not contain calcium (e.g., 20 mM Tris pH 7.4, 0.1% Tween 80). The volume of the second-stage wash may be varied depending on the desired results and the exact properties of the second-stage wash solution used. Without limitation, the applicant has found that, for example, the wash buffer described above can be successfully used in the case of washing 2CV.

[0156] Subsequently, the AnxA5 protein is eluted from the affinity chromatography column using an elution buffer, thereby providing a first affinity chromatography product containing the released AnxA5 protein. When using a heparin affinity chromatography column, it may be preferable to use an elution buffer containing a calcium metal ion chelator such as EDTA or EGTA.

[0157] For example, without limitation, the applicant has found that a suitable elution buffer is 20 mM Tris pH 7.4, 0.1% Tween 80, 10 mM EDTA, and 25 mM NaCl, which chelates calcium ions. The chelation reaction specifically elutes annexin A5, which can bind to heparin only in the presence of calcium.

[0158] To increase the concentration of the product, it may be preferable to reduce the flow rate to less than 100 cm / h during elution. For example, in the examples, without limitation, the applicant enabled concentrated elution by reducing the flow rate to 60 cm / h or less during elution. The complete elution peak may be collected, for example, starting with an increase in the UV signal at 0.05 AU and ending at a descending peak at 0.05 AU, which corresponds to approximately 7 CV. Preferably, the elution profile shows a single peak, which is most ideally a sharp peak.

[0159] Therefore, the first affinity chromatography product comprises the released AnxA5 protein and a calcium ion chelating agent such as EDTA or EGTA, which optionally is present in a concentration of about 0.1 mM to 500 mM, for example, in the range of about 1 mM to about 100 mM, more typically in the range of about 2 mM to about 50 mM, more preferably in the range of about 5 mM to about 15 mM, and most preferably about 10 mM. In this context, the term "about" may mean ±50%, 40%, 30%, 20%, 10%, 5%, or 1% of the given concentration(s).

[0160] The affinity chromatography step may be the most powerful purification step in this process scheme. The AnxA5 protein binds to calcium ions, and in this calcium-bound state, the product can form a highly specific binding to heparin. Typically, only correctly folded AnxA5 protein forms that have the ability to complex with calcium can bind to heparin. Thus, the affinity chromatography step may be useful in assisting the distinction between correctly folded and misfolded products. In addition, the intermediate step achieves a high removal factor when the highly specific interaction is combined with a specific elution mode by the chelation reaction of calcium with EDTA. Therefore, a strong reduction of endotoxins and HCP is observed, combined with a moderate reduction in DNA content. Endotoxins are typically already reduced (preferably by about 97%) during the preceding anion exchange capture step, but are further reduced (preferably by about 99%), which preferably brings the endotoxin level down to about 0.03% of the level in the clarified product before the first anion exchange capture step.

[0161] G. Anion exchange refining In further embodiments of the first, second, and / or third aspects of the present invention, the AnxA5 product (e.g., produced by an affinity chromatography step) may be further purified directly or indirectly by an anion exchange refining step.

[0162] If the AnxA5 product produced by the affinity chromatography step is further purified indirectly by an anion exchange refining step, the affinity chromatography step and the anion exchange refining step may be separated by adding one or more conditioning additives to the product of the affinity chromatography step.

[0163] Suitable additives may include, for example, diluents for further diluting the AnxA5 protein in the affinity chromatography product (e.g., water), buffers (e.g., Tris, e.g., at 35 mM and pH 8), nonionic detergents (e.g., polysorbate, more preferably Tween 80, at a concentration of about 0.1 w / v%, etc.), and / or one or more further additives based on a fourth aspect of the present invention as discussed below.

[0164] In other words, a fourth aspect of the present invention is based on the applicant's realization that calcium metal ion chelators (e.g., EDTA) can adversely affect the effectiveness of anion exchange steps. Free EDTA (or other chelators) can directly bind to anion exchange functional groups, thereby reducing the volume and even the separation achieved by the anion exchange step. This is particularly problematic when performing anion exchange on the product of a heparin affinity chromatography step, in which case the AnxA5 protein is bound to heparin in the presence of calcium ions, and then a calcium metal ion chelator (e.g., EDTA) is used to elute the bound AnxA5 protein. As a result, the eluted AnxA5 product from the heparin affinity chromatography step contains high levels of calcium ion chelator. It is generally desirable that the AnxA5 product be further purified in a further anion exchange step, but the calcium ion chelator is a problematic component during that further anion exchange step. On the other hand, attempts to remove the calcium metal ion chelator before the anion exchange step are time-consuming and thus costly. Therefore, for example, prior art methods involving a dialysis step for buffer exchange are slow and inefficient, thereby increasing production costs. Furthermore, incorporating calcium metal ion chelators into the AnxA5 product during the anion exchange step may be an important component in preventing calcium-mediated binding of the AnxA5 protein to impurities, including endotoxins. Thus, it would be advantageous and effective to introduce additives that block or reduce the binding of calcium ion chelators to the anion exchange resin, while still allowing the anion exchange step to be carried out without the inconveniences and costs associated with dialysis, and without hindering the beneficial effects of the calcium metal ion chelators during the anion exchange step.

[0165] The applicant has found that this can be achieved by incorporating one or more additional selected metal ions into the AnxA5 protein product before anion exchange, wherein the additional selected metal ions are selected such that the calcium metal ion chelator has a binding affinity for the selected metal ions that is greater than its binding affinity to the anion exchange resin but smaller than its binding affinity to calcium ions. The appropriate selection of additional metal ions will depend on the properties of the calcium ion chelator and the properties of the anion exchange resin. For example, when using EDTA as a calcium ion chelator, Mg 2+ Ions are generally suitable for achieving the objectives of the present invention and can be added to the AnxA5 protein product before the anion exchange step.

[0166] Therefore, a fourth aspect of the present invention is a process for recovering and / or purifying a protein comprising the sequence of annexin A5 (AnxA5) from a composition comprising the AnxA5 protein and a calcium metal ion chelating agent, The process is characterized by comprising providing the composition to an anion exchange resin in order to perform an anion exchange step, thereby recovering and / or purifying the AnxA5 protein from the composition. The anion exchange process is further characterized by being carried out in the presence of additional selected metal ions. This additional selected metal ion provides a process in which the calcium metal ion chelating agent is selected such that it has a binding affinity for the selected metal ion that is greater than its binding affinity for the anion exchange resin but less than its binding affinity for calcium ions.

[0167] Preferably, the additional selected metal ions are mixed with the composition containing the AnxA5 protein and calcium metal ion chelating agent before the composition is subjected to the anion exchange resin. The solution used in the subsequent anion exchange step (e.g., washing solution and / or elution buffer) may or may not also contain the additional selected metal ions. Therefore, in one embodiment of this aspect of the present invention, the step of carrying out the anion exchange step in the presence of additional selected metal ions means adding the additional selected metal ions to the composition containing the AnxA5 protein and calcium metal ion chelating agent before the composition is subjected to the anion exchange resin.

[0168] In one embodiment of a fourth aspect of the present invention, the calcium metal ion chelating agent is selected from EDTA or a salt thereof, EGTA or a salt thereof, and most preferably EDTA.

[0169] Calcium metal ion chelating agents may be present in the composition at concentrations exceeding approximately 0.1 mM to 500 mM, for example, in the range of approximately 1 mM to approximately 100 mM, more typically in the range of approximately 2 mM to approximately 50 mM, and / or at concentrations of approximately 0.1 mM, 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM or higher, or at least at those concentrations. In this context, the term "approximately" may include the meaning of ±0.5, 0.4, 0.3, 0.2, or 0.1 mM of the given value. In the context described above, the term “excess” may mean that there is a sufficient amount of calcium metal ion chelating agent to remove any divalent ions that could contribute to the binding of the AnxA5 protein to the immobilized heparin on the column in the preceding affinity chromatography step, thereby allowing the AnxA5 protein to be released from the column into solution and subsequently used directly or indirectly in the anion exchange refining step.

[0170] In one embodiment that exemplifies a fourth aspect of the present invention, the selected metal ion is Mg 2+ It is a divalent cation, such as an ion.

[0171] It is preferable that the selected metal ions be present in an amount effective in reducing (e.g., by about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more) or preventing the interaction between the calcium ion chelator and the anion exchange resin during the anion exchange process, when the composition is subjected to the anion exchange resin. For example, the selected metal ions may be present in an amount effective in reducing or preventing the interaction between the calcium ion chelator and the anion exchange resin during the anion exchange process, when the composition is loaded onto the anion exchange resin, and / or during one or more washing steps in which the AnxA5 protein binds to the anion exchange resin and impurities are removed by washing.

[0172] It is preferable that the selected metal ions are present in an amount effective in increasing the binding of the AnxA5 protein to the anion exchange resin in the presence of the calcium ion chelator during the anion exchange process, thereby reducing the loss of the AnxA5 protein in the pass-through fraction of the anion exchange process compared to the level of loss observed when the selected metal ions are not present during the anion exchange process (e.g., approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more).

[0173] The selected metal ions may preferably be present during the anion exchange process at concentrations of approximately 1 to 100 mM, for example, approximately 2 to 50 mM, approximately 5 to 25 mM, approximately 10 to 15 mM, or approximately 12.5 mM.

[0174] Furthermore, the calcium metal ion chelating agent is EDTA, and the selected metal ion is Mg 2+ It is an ion, more preferably Mg 2+The molar ratio of ions to EDTA is in the range of 0.5:1 to 2:1, most preferably at least 1:1 or greater than 1:1.

[0175] In relation to a fourth aspect of the present invention, in further embodiments, a composition comprising AnxA5 protein and a calcium metal ion chelating agent, which is subjected to an anion exchange resin, may be a direct or indirect product of a preceding process, which includes subjecting the AnxA5 protein to an affinity chromatography step, eluting the AnxA5 protein with a calcium ion chelating agent, thereby producing an affinity chromatography product which is a composition comprising AnxA5 protein and a calcium metal ion chelating agent. For example, the preceding affinity chromatography step may include binding of the AnxA5 protein to immobilized heparin, optionally facilitated by the presence of calcium ions, and further optionally, the AnxA5 protein is eluted from the immobilized heparin using an elution buffer containing a calcium ion chelating agent such as EDTA.

[0176] It is even more preferable that there is no dialysis step between the preceding affinity chromatography step and the anion exchange step, and / or that the calcium ion chelating agent is not removed from the product of the preceding affinity chromatography step, before the direct or indirect application of the product to the anion exchange step.

[0177] In connection with a fourth aspect of the present invention, in further embodiments, the selected metal ions are added to the composition before or during the anion exchange step.

[0178] In a preferred embodiment, the product of the affinity chromatography step according to any of the first, second, or third aspects of the present invention is treated in a further anion exchange step, such as an anion exchange step according to the fourth aspect of the present invention.

[0179] Optionally, prior to the (second) refining anion exchange step, one or more parameters of the environment of the released AnxA5 protein are adjusted, selected from the group consisting of the concentration of AnxA5 protein, pH, conductivity, level of calcium ion chelator and level of nonionic detergent, or level of selected additional metal ions (in a fourth aspect of the present invention).

[0180] Typically, prior to contact between the (second) anion exchange resin and the AnxA5 product in the refining process, the anion exchange resin is equilibrated. Any suitable equilibration method may be used. For example, the anion exchange resin can be equilibrated with a buffer (e.g., 20 mM Tris pH 7.4), a nonionic detergent (e.g., 0.1% polysorbate, preferably Tween 80), and a salt (e.g., 25 mM NaCl). Any suitable equilibration volume may be used, and without limitation, the applicant has found that 3 column volumes (CV) are a suitable volume in exemplary embodiments.

[0181] Preferably, the (second) anion exchange refining step is performed in positive mode with respect to the AnxA5 protein, so that the AnxA5 protein is temporarily bound to the anion exchanger during the anion exchange step, a washing solution is typically passed through the column to remove impurities from the bound AnxA5 protein, and then an elution buffer is applied to the anion exchange resin to release the bound AnxA5 protein, thereby producing a second anion exchange product containing the released AnxA5 protein.

[0182] Strong anion exchange resins are preferred. Strong anion exchange resins are well known in the art, and examples include resins containing quaternary ammonium functional groups, such as Type I resins having trialkylammonium chloride or ammonium hydroxide, or Type II resins having dialkyl2-hydroxyethylammonium chloride or ammonium hydroxide. Without limitation, an example of an anion exchange resin suitable for the second refining step is Source15 Q. Source 15Q anion exchange resin may be defined as a polymeric strong anion exchanger, containing a quaternary ammonium ligand and a median particle size (d) based on a cumulative volume distribution of approximately 15 μm. 50v It can be further characterized as having a polystyrene / divinylbenzene matrix and / or pressure / flow specifications of approximately 400 cm / h and 1000 kPa when evaluated as a FineLine 100 column with a bed height of 10 cm.

[0183] Without limitation, further examples of anion exchange resins suitable for the second refining process include Capto Q ImpRes. Capto Q ImpRes can be defined as a strong anion exchanger, comprising a quaternary amine ligand, a high-flow agarose matrix, and a median particle size of approximately 36-44 μm (d 50v ), about 0.15~0.18mmol Cl - It may be further characterized as having an ion capacity of at least 220 cm / h and a pressure / flow specification of approximately 300 kPa when evaluated as an ion capacity of at least 55 mg of BSA and at least 48 mg of β-lactoglobulin per ml of chromatography solvent, and / or as a 1 m diameter column with a bed height of 20 cm.

[0184] Without being constrained by theory, the applicant found that it may be particularly advantageous to use Capto Q ImpRes anion exchange resin in the (second) anion exchange refining step when the AnxA5 protein to be purified is derived from E. coli BL21(DE3) that has been further manipulated to overexpress PGL, such as Aon et al. (Appl. Env. Microbiol., 2008, 74(4):950-958 (the contents of which are incorporated herein by reference)), which eliminates or reduces the gluconylation of the AnxA5 protein expressed by bacteria. In this context, "low" means that the level of gluconylation is, in turn, derived from E. coli strain BL21(DE3) (e.g., widely available commercially, Marder et al., 2014, BMC). This can mean that the level of gluconoylation of the AnxA5 protein expressed in (as described in Biotechnology, 14:33) is below (e.g., less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%). For example, Capto Q for (second) anion exchange refining process. The level of gluconylated AnxA5 protein in the product applied to the ImpRes anion exchange resin can be within the range of 0.5–30%, 0.5–20%, 0.5–15%, or 0.5–10% of the total AnxA5 protein content in the applied product. The gluconylated variant of Anx5 can be measured and quantified, for example, by using ultra-high-performance liquid chromatography (UPLC) or high-performance liquid chromatography (HPLC) chromatographic equipment with appropriate anion exchange or reversed-phase columns. Various peaks can be further identified and characterized using mass spectrometry (MS).

[0185] The applicant found that the Capto Q ImpRes anion exchange resin has a high binding capacity, can withstand high flow rates without back pressure, can be packed at higher bed heights, and is less expensive, so the use of the Capto Q ImpRes anion exchange resin for the (second) anion exchange refining step results in a more efficient process (for example, compared to the use of the Source 15Q anion exchange resin for the (second) anion exchange refining step) when the AnxA5 protein to be purified is either not gluconoylated or has a low level of gluconoylation. The quality and purity of the final product are maintained regardless of whether Source 15Q or Capto Q ImpRes anion exchange resin is used. However, it is estimated that switching from Source 15Q anion exchange resin (with a particle size of 15 μm) to Capto Q ImpRes anion exchange resin (with a particle size of 40 μm) could provide a productivity increase of more than 5x by reducing the time required for the entire process operation (especially since the second anion exchange step used in refining is one of the most time-wasting steps), and could also reduce overall manufacturing costs by more than 50%. Avoiding resins with very small bead diameters (less than 30 μm) allows for higher flow rates, enabling chromatography columns to be packed with a higher resin bed height without causing unacceptable back pressure. This increases productivity because more resin can be packed into the column for a given floor area (column diameter), and therefore more proteins can be bound, while simultaneously enabling faster operation due to the higher flow rate.

[0186] After loading onto the (second) anion exchange resin for refining, under positive mode, the AnxA5 protein can be temporarily bound to the resin and washed to reduce / remove impurities. Any suitable washing conditions can be used. For example, the washing solution may consist of, essentially, or comprise an aqueous solution of a buffer (e.g., 20 mM Bis-Tris, pH 7) and a salt (e.g., 25 mM NaCl), and optionally, an aqueous solution of a nonionic buffer (e.g., polysorbate at a level of about 0.1 w / v, preferably Tween 80). Any suitable washing volume may be used, and without limitation, the applicant has found that 3 column volumes (CV) are a suitable volume in exemplary embodiments.

[0187] The bound AnxA5 protein is then released from the anion exchange resin using an elution buffer. Any suitable elution buffer may be used. For example, the elution buffer may consist of, or be essentially, an aqueous solution of a buffer (e.g., 20 mM Bis-Tris, pH 7), a salt at a higher concentration than the washing solution (e.g., 180 mM NaCl, optionally ±100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 mM) and optionally a nonionic buffer (e.g., polysorbate at a level of about 0.1 w / v, preferably Tween 80). Any suitable elution volume may be used, and without limitation, the applicant has found that in exemplary embodiments, a column volume (CV) with the concentration of the elution buffer increased linearly from 0 to 100% is suitable for elution.

[0188] In this way, the AnxA5 protein is released into the elution buffer, which provides a (second) refined anion exchange product. As discussed in Example 1, the process carried out using 0.1% Tween 80 increased the product yield after the intermediate process by approximately 30%.

[0189] The (second) anion exchange resin can then be regenerated and purified. Suitable methods for regeneration and purification are known in the art, and one such suitable protocol is considered in the examples.

[0190] The refining process is primarily carried out to reduce impurities associated with the product, such as the separation of various annexin A5 isoforms. In addition, the refining process achieves the highest removal coefficient in the process for residual DNA and strongly reduces HCP. Endotoxins, which are already at low levels after the intermediate process, are further reduced by approximately 99%, and this (when used in combination with preceding cell homogenization, nuclease treatment, clarification, capture anion exchange, filtration (such as a sterile filtration process), and heparin affinity processes) brings the endotoxin level down to approximately 0.0003% of the level in the clarified product before the first AX process.

[0191] Accordingly, further embodiments of the first, second, third and / or fourth aspects of the present invention provide a process for the recovery and / or purification of a recombinantly expressed protein comprising the sequence of intracellular annexin A5 (AnxA5) from a host cell with a cell wall, or from a culture in the first aspect of the present invention, preferably comprising the recovery and / or purification of the recombinantly expressed intracellular AnxA5 protein from a culture of host cells, the culture being at least about 100 L, about 200 L , having a volume of approximately 300L, approximately 400L, approximately 500L, approximately 600L, approximately 700L, approximately 800L, approximately 900L, approximately 1,000L, approximately 2,000L, approximately 3,000L, approximately 4,000L, approximately 5,000L, approximately 6,000L, approximately 7,000L, approximately 8,000L, approximately 9,000L, approximately 10,000L, approximately 20,000L, approximately 30,000L, approximately 40,000L, approximately 50,000L, approximately 60,000L, approximately 70,000L, approximately 80,000L, approximately 90,000L, approximately 100,000L or greater. (a) The process, according to a first aspect of the present invention, includes releasing intracellular proteins from host cells in the presence of a homogenization buffer containing a nonionic detergent, (b) Optionally, the discharge step is carried out according to one or more embodiments of the first aspect of the present invention described above in subsection C: (c) The process may further optionally include a step of clarifying the biomass homogenate by one or more of the embodiments described in Section D above, (d) The process further includes a step of directly or indirectly providing the released AnxA5 protein to an anion exchange resin in order to perform a first anion exchange step according to one or more of the embodiments described in subsection E, thereby producing a first anion exchange product containing the released AnxA5 protein, (e) The process further comprises subjecting the released AnxA5 protein directly or indirectly to an affinity chromatography step according to any of the first, second and / or third aspects of the present invention described above in subsection F, (f) The product of the affinity chromatography step is a composition containing AnxA5 protein and a calcium metal ion chelating agent. (g) The direct or indirect products of the affinity chromatography step, comprising the AnxA5 protein and a calcium metal ion chelating agent, are subjected to an anion exchange step according to any of the embodiments described above in this section (i.e., section G).

[0192] H. Product formulation A process according to any of the first, second, third, or fourth aspects of the present invention may preferably further include, at the end of the process, one or more further steps selected from the group consisting of concentration, buffer modification, conditioning, and filtration (such as a sterile filtration step), and optionally, a final step of storing the AnxA5 protein-containing product in a sterile container.

[0193] For example, one of the further steps used in product formulation may be ultrafiltration / diafiltration (UF / DF), and optionally, the product of the UF / DF step contains AnxA5 protein at concentrations of at least approximately 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 125 mg / mL or higher.

[0194] In another example, one of the further steps used in product formulation may be the addition of a nonionic surfactant, preferably polysorbate, and more preferably Tween 80. The nonionic surfactant may be added in an amount desired for the final product, for example, to a final concentration of about 0.05% (w / w) (e.g., ±0.05, 0.04, 0.03, 0.02, or 0.01 w / v%).

[0195] In another example, one of the further steps used in product formulation may be a filtration step (such as a sterile filtration step) using, for example, a 0.45-0.2 μm filter or a 0.22 μm filter.

[0196] As described above, the process described in the first, second, third, or fourth aspect of the present invention may be completed by a sterilization filtration step and a step of placing the sterilization filtered AnxA5 protein-containing product into a sterilization container.

[0197] The final concentration of AnxA5 protein in the filled container may be adjusted as needed. Without limitation, the applicant has shown a final concentration of 10 mg / mL as an example. Preferred concentrations may be, for example, 1–125 mg / mL, 2–100 mg / mL, 5–50 mg / mL, 7–30 mg / mL, or about 10–20 mg / mL.

[0198] Optionally, the process of the present invention may provide a final sterile AnxA5 protein product in a phosphate-free buffer (such as Bis-Tris or Tris buffer) at approximately pH 7.4 (e.g., ±0.5, 0.4, 0.3, 0.2, or 0.1 pH units) containing approximately 150 mM NaCl (e.g., ±50, 40, 300, 200, 100, or 50 μM), approximately 1 mM CaCl2 (e.g., ±500, 400, 300, 200, 100, or 50 μM), and approximately 0.05% (w / w) (e.g., ±0.05, 0.04, 0.03, 0.02, or 0.01 w / v%) of a polysorbate such as Tween 80 or other nonionic detergent. A pH of approximately 7.4 is a typical target pH for formulations intended for use in humans (particularly intravenous delivery), as it matches the pH of human blood and provides a stable AnxA5 protein with good solubility. Below pH 7, especially when the pH drops to around pH 6, the AnxA5 protein may lose solubility and begin to precipitate.

[0199] NaCl may be useful in maintaining the AnxA5 product in monomeric form during storage. Therefore, the process of the present invention may provide a final sterile AnxA5 protein product in which the present NaCl concentration maintains the AnxA5 protein in a predominantly monomeric form (i.e., more than about 50%, e.g., 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or substantially 100%). The monomeric level percentage can be readily determined using techniques well known in the art, such as gel permeation chromatography (GPC).

[0200] In one embodiment, the process of the present invention provides a therapeutically acceptable final sterile AnxA5 protein product in total yield of more than 1 g, more preferably at least about 1.5 g / L, and even more preferably in the range of about 2 to about 4 g / L per liter of host cell culture. In this context, the term “about” may include ±0.4, 0.3, 0.2, or 0.1 g / L of the specified value.

[0201] In another embodiment, the process of the present invention provides a therapeutically acceptable final sterile AnxA5 protein product with a total recovery rate of at least about 24% by weight (e.g., ±10, 9, 8, 7, 6, 5, 4, 3, 2, 1%) of the AnxA5 protein present in the host cell culture, or more. This can be determined, for example, by measuring the soluble AnxA5 protein in the initial homogenate (which can be captured and measured by centrifugating aliquots of the homogenate and testing the level of AnxA5 in the supernatant) and in the final purified product.

[0202] In addition, at any point in this process (if relevant, before filling sterile containers), and typically after the final purification step described above, the AnxA5 protein may be chemically modified. For example, the AnxA5 protein may be PEGylated. PEGylated annexin A5 is disclosed in WO02 / 067857. PEGylation is a method well known to those skilled in the art, in which a polypeptide or peptide mimetic compound (in this invention, the AnxA5 protein) is modified so that one or more polyethylene glycol (PEG) molecules are covalently bonded to the side chains of one or more amino acids or their derivatives. It is one of the most important molecular transformation structural chemistry techniques (MASC). Other MASC techniques may be used, which can improve the pharmaceutically active properties of a molecule, such as extending its in vivo half-life. The PEG-protein complex is first formed by activating the PEG moiety so that it reacts with and couples with the protein or peptide mimetic compound of the present invention. The PEG moieties differ considerably in molecular weight and conformation, with the earlier moieties (monofunctional PEG, i.e., mPEG) being linear with a molecular weight of 12 kDa or less, and the later moieties being of increased molecular weight. PEG2, a recent innovation in PEG technology, involves the coupling of 30 kDa (or less) mPEG to a lysine amino acid (although PEGation can be extended to the addition of PEG to other amino acids), which is then further reacted to form a branched structure that behaves like linear mPEG with a much larger molecular weight (Kozlowski et al., 2001). Methods that may be used to covalently bond PEG molecules to polypeptides are further described in Roberts et al. (2002) Adv Drug Deliv Rev, 54, 459-476, Bhadra et al. (2002) Pharmazie 57, 5-29, Kozlowski et al. (2001) J Control Release 72, 217-224, and Veronese (2001) Biomaterials 22, 405-417, as well as the references cited therein.The advantages of PEGylation include reduced renal clearance, which, for some products, can lead to more sustained adsorption and limited distribution after administration, resulting in constant and sustained plasma concentrations and therefore increased clinical efficacy (Harris et al. (2001) Clin Pharmacokinet 40, 539-551). Further advantages may include reduced immunogenicity of the therapeutic compound (Reddy (2001) Ann Pharmacother, 34, 915-923) and lower toxicity (Kozlowski et al. (2001) Biodrugs 15, 419-429). When the AnxA5 protein is chemically modified, it may be preferable to perform one or more additional purification steps, for example, to reduce or remove unreacted components and / or to select a homogeneous population of chemically modified AnxA5 protein for incorporation into the final product. Suitable techniques for purifying chemically modified proteins from the reaction process are known to those skilled in the art.

[0203] The final product may be a pharmaceutical or veterinary composition, or may be subsequently formulated to form one.

[0204] The final product may be presented in unit dosage forms. For example, a unit dosage form may contain approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg of AnxA5 protein (the term "approximately" refers to ±0.5, 0.4, 0.3, 0.2, or 0.1 mg), or more, for example, between 0.1 and 1000 mg, or between 1 and 100 mg.

[0205] Pharmaceutical or veterinary compositions may contain the AnxA5 protein mixed with a pharmaceutically or veterinarily acceptable adjuvant, diluent, or carrier, which are typically selected with respect to the intended route of administration and standard pharmacokinetics. The compositions may be in the form of immediate-release, delayed-release, or controlled-release applications. Preferably, the formulation is a unit dose containing a daily dose or unit, a daily partial dose, or an appropriate proportion thereof of the active ingredient.

[0206] The phrase "pharmaceutically or veterinarily acceptable" includes, where appropriate, references to compositions that, when administered to animals or humans, do not cause adverse reactions, allergic reactions, or other undesirable reactions. The preparation of such pharmaceutical or veterinary compositions is known to those skilled in the art in light of this disclosure, as exemplified in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Furthermore, with respect to administration to animals or humans, it will be understood that preparations should meet the sterility, pyrogenicity, overall safety, and purity requirements of the FDA Office of Biological Standards.

[0207] As used herein, “pharmaceutically or veterinarily acceptable carriers” include all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antimicrobial agents, antifungal agents), isotonic agents, salts, antiseptics, drugs, drug stabilizers, excipients, disintegrants, such similar materials, and combinations thereof, as would be known to those skilled in the art. Their use in therapeutic or pharmaceutical compositions is intended, except insofar as any conventional carrier is incompatible with the active ingredient.

[0208] The pharmaceutical or veterinary compositions according to the present invention are intended for parenteral administration, intravenous administration, intra-arterial administration, intraperitoneal administration, intramuscular administration, intraocular administration, intracranial administration, intracerebral administration, intraosseous administration, intraventricular administration, intrathecal administration, or subcutaneous administration, administration from a drug-eluting stent, administration by infusion technique, or topical administration (in a form suitable for the epidermis, e.g., cream or ointment, inhalation, eye drops, ear drops, or through mucous membranes in the body), and therefore may or may not be formulated in a manner suitable for such administration. Sterile injection solutions can be prepared by incorporating the required amount of the active compound together with various other components listed above in a suitable solvent, followed by sterilization. Pharmaceutical compositions may be best used in the form of a sterile aqueous solution that may contain other substances, e.g., salts or glucose in sufficient quantities to make the solution isotonic with blood. If necessary, the aqueous solution may be buffered (preferably to a pH of 3-9). The preparation of suitable pharmaceutical formulations under sterile conditions can be easily carried out by standard pharmaceutical techniques well known to those skilled in the art.

[0209] Alternatively, the pharmaceutical or veterinary composition according to the present invention may be formulated in powder form, such as a sterile powder which may be a freeze-dried powder.

[0210] The therapeutically effective dose of AnxA5 protein for administration to patients, such as human patients, may be 0.01 to 1000 mg of AnxA5 protein per adult, administered as a single or divided dose based on the daily dose level (e.g., approximately 0.001 to 20 mg per kg of patient body weight, e.g., 0.01 to 10 mg / kg, e.g., greater than 0.1 mg / kg and less than or equal to 20, 10, 5, 4, 3, or 2 mg / kg, e.g., approximately 1 mg / kg).

[0211] In any case, the physician will determine the actual dosage that would be most suitable for any given individual patient, which will vary depending on the patient's age, weight, and response. The dosages mentioned above are shown as examples of average cases. Naturally, there may be individual cases where a higher or lower dosage range is advantageous, and such cases are within the scope of this invention.

[0212] For veterinary use, the compounds of the present invention are administered as suitably acceptable formulations in accordance with normal veterinary practice, and the veterinarian determines the dosage regimen and route of administration that would be most appropriate for the particular animal.

[0213] I. Product characteristics The process of the present invention provides the AnxA5 product as defined above. Therefore, the product produced by the claimed process is also a further fifth aspect of the present invention.

[0214] In further embodiments, any process according to an aspect of the present invention may provide a product containing AnxA5 protein having a purity suitable for use as an injectable pharmaceutical in humans. The processes described herein typically remove process-related impurities to well below acceptable levels, for example, host cell proteins to less than 20 ng per mg of AnxA5 protein, DNA to less than 10 pg per mg of AnxA5 protein, and endotoxins to less than 1 EU per mg of AnxA5 protein.

[0215] In further embodiments, any process according to an aspect of the present invention may provide a product containing non-AnxA5 proteins, particularly host cell proteins (other than recombinantly expressed AnxA5 proteins), at levels of less than or less than 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 ng per mg of AnxA5 protein. The FDA and EMA require that host cell protein levels be less than 100 ng / mg, and the applicant has demonstrated that host cell protein levels are less than 20 ng / mg. Host cell protein content can be measured, for example, by using an anti-host cell protein antibody by the ELISA sandwich technique or other EMA and FDA-approved methods.

[0216] In further embodiments, any process according to an aspect of the present invention may provide a product containing an endotoxin content of less than 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 35, 20, 15 per 1 mg of AnxA5 protein, and / or preferably, the process provides a product in a unit dosage form, which contains less than 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 35, 20, 15 per unit dose, preferably less than 10, 5, or 1 EU. The FDA and EMA require that the endotoxin content be less than 100 EU / dose (the maximum permissible dose is 350 EU / dose), and this required amount corresponds to less than 10 EU / mg in a 10 mg dose. Within these parameters, a unit dosage form may contain approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg of AnxA5 protein or more (e.g., in the range of 0.1 to 1000 mg, or 1 to 100 mg). Endotoxins can be measured, for example, by using LAL-based techniques or other EMA and FDA-approved methods.

[0217] In further embodiments, any process according to an aspect of the present invention involves less than 1,000 pg per 1 mg of AnxA5 protein, preferably less than 500 pg per 1 mg of AnxA5 protein, less than 400 pg per 1 mg of AnxA5 protein, less than 300 pg per 1 mg of AnxA5 protein, less than 200 pg per 1 mg of AnxA5 protein, less than 100 pg per 1 mg of AnxA5 protein, less than 50 pg per 1 mg of AnxA5 protein, less than 40 pg per 1 mg of AnxA5 protein, less than 30 pg per 1 mg of AnxA5 protein, Products may provide nucleic acid (e.g., DNA) levels such as less than 20 pg per mg of AnxA5 protein, less than 15 pg per mg of AnxA5 protein, less than 10 pg per mg of AnxA5 protein, less than 9 pg per mg of AnxA5 protein, less than 8 pg per mg of AnxA5 protein, less than 7 pg per mg of AnxA5 protein, less than 6 pg per mg of AnxA5 protein, less than 5 pg per mg of AnxA5 protein, and, for example, host cell nucleic acid (e.g., DNA) levels of approximately 4 pg per mg of AnxA5 protein. DNA can be measured, for example, by using quantitative polymerase chain reaction (qPCR) techniques or other EMA and FDA approved methods.

[0218] In a particularly preferred embodiment, any process of any aspect of the present invention may provide a product having any one or more properties selected from the following enumerated items: - Typically, the concentration of AnxA5 protein is around 8-12 g / L. Host cell protein levels of -100 ng / mg or less, more preferably 20 ng / mg (determined by ELISA), - Host cell DNA level of 100 pg / mg or less, more preferably 10 pg / mg - 35 EU / mg or less, more preferably 1 EU / mg endotoxin, - Purity of over 95% determined by size exclusion chromatography. Bioburden less than -1 cfu / mL (as determined by Ph.Eur.2.6.12), - A colorless and transparent appearance without visible particles, and - The main band detected by Western plot analysis corresponds to the Annexin A5 reference.

[0219] In a further particularly preferred embodiment, the AnxA5 protein in the product may have a low level of gluconoylation. In this context, “low” can then mean that the level of gluconoylation is less than the level of gluconoylation of the AnxA5 protein expressed in E. coli strain BL21(DE3) (e.g., widely available and described in Marder et al., 2014, BMC Biotechnology, 14:33) (e.g., less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of that level). For example, the level of gluconoylated AnxA5 protein in the product can be in the range of 0.5–30%, or 0.5–20%, or 0.5–15%, or 0.5–10% of the total content of AnxA5 protein in the product. In other words, the level of gluconoylated AnxA5 protein in the product can be below 40%, for example, below 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, preferably substantially 0%. The gluconoylated variant of Anx5 can be measured and quantified, for example, by using ULC or HPLC chromatography equipment with a suitable anion exchange or reversed-phase column.

[0220] Accordingly, a fifth aspect of the present invention provides a composition comprising the AnxA5 protein, which is a direct or indirect product (or can be obtained directly or indirectly) of a process according to any of the first, second, third, or fourth aspects of the present invention. Optionally, the composition is pharmaceutically acceptable and / or veterinarily acceptable.

[0221] J. Medical and veterinary uses A sixth aspect of the present invention also provides a composition of the fifth aspect of the present invention for use in pharmaceuticals. In other words, a sixth aspect of the present invention provides a method comprising administering a therapeutically effective amount of the composition of the fifth aspect of the present invention to a human or animal in need of treatment.

[0222] In a particular embodiment of the sixth aspect of the present invention, the composition of the fifth aspect of the present invention may be used for the following purposes: (a) For the prevention or reduction of the risk of thrombosis (such as atherothrombosis) and / or plaque rupture, or for administration to patients belonging to a risk group, including but not limited to patients with systemic lupus erythematosus (SLE) and / or patients with upper respiratory tract infections or other infections (including pneumococcal infections) that may cause elevated levels of antiphospholipid-related antibodies, or for the treatment (actively or prophylactically) or reduction of the risk of thromboembolism, hemorrhagic or vasculitic stroke, myocardial infarction, angina pectoris or intermittent claudication, unstable angina, other forms of severe angina, or transient ischemic attack (TIA), such as those further described in WO2005 / 099744 (the contents of which are incorporated herein by reference), (b) For the treatment, prevention or reduction of the risk of vascular dysfunction, angina pectoris, ischemic heart disease, peripheral artery disease, systolic hypertension, migraine, type 2 diabetes and erectile dysfunction, for the reduction of ischemic pain and / or for the treatment of vascular rupture, such as those described in WO2009 / 077764 (the contents of which are incorporated herein by reference), (c) For the prevention or treatment of restenosis (especially neointima formation or thickening), or vasculitis, such as those described in WO2009 / 103977 (the contents of which are incorporated herein by reference), (d) For use in inhibiting the activity of cardiolipin oxidized (oxCL), and in the following diseases in mammals, namely, cardiovascular disease (CVD), type 2 diabetes, Alzheimer's disease, dementia in general, rheumatic disease, atherosclerosis, hypertension, acute and / or chronic inflammatory conditions, myocardial infarction, acute coronary syndrome, stroke, transient ischemic attack (TIA), claudication, angina pectoris, type 1 diabetes, rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's syndrome, lupus erythematosus, dermatomyositis, Sjögren's syndrome, erythrorhizosis To treat, prevent and / or reduce the risk of developing cardiovascular diseases, autoimmune diseases or inflammatory conditions, including but not limited to lupus morbidity, multiple sclerosis, myasthenia gravis, asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), arthritis including osteoarthritis, idiopathic inflammatory myopathy (IIM), dermatomyositis (DM), polymyositis (PM), inclusion body myositis, allergic disorders and / or osteoarthritis, as described in WO2010 / 069605 (the contents of which are incorporated herein by reference), and (e) For the prevention and / or reduction of perioperative or postoperative complications following surgical intervention, such as complications following vascular surgery, particularly peripheral vascular surgery, as described in WO2012 / 136819 (the contents of which are incorporated herein by reference).

[0223] In a further embodiment of the sixth aspect of the present invention, the composition of the fifth aspect of the present invention may be used in a prophylactic or therapeutic method to treat, prevent or reduce the risk of hematological disorders, including but not limited to sickle cell anemia.

[0224] In a further embodiment of the sixth aspect of the present invention, the composition of the fifth aspect of the present invention may be used in prophylactic or therapeutic methods to treat, prevent or reduce the risk of, acute and chronic vasculitis, primary or secondary vasculitis, including but not limited to vasculitis with an autoimmune element and / or drug-induced vasculitis. Accordingly, the present invention also provides prophylactic or therapeutic methods to treat, prevent or reduce the risk of, vasculitis, Behçet's disease, cutaneous vasculitis, eosinophilic granulomatosis with polyangiitis (EGPA), giant cell arteritis, granulomatosis with polyangiitis (GPA), immunoglobulin A-associated vasculitis (IgAV), microscopic polyangiitis (MPA), polyarteritis nodosa (PAN), rheumatoid arthritis polymyalgia, and Takayasu's arteritis. Rheumatoid arthritis polymyalgia may be a particular target.

[0225] In a further embodiment of the sixth aspect of the present invention, the composition of the fifth aspect of the present invention may be used in a preventive or therapeutic method for treating, preventing or reducing the risk of retinal vein occlusion.

[0226] In a further embodiment of the sixth aspect of the present invention, the composition of the fifth aspect of the present invention may be used in a preventive or therapeutic method to (i) prevent or reduce the transmission of a viral infection, (ii) prevent or protect against a viral infection, or (iii) treat a viral infection in a subject, wherein the viral infection is caused by a virus selected from the group consisting of: (a) Viruses capable of causing hemorrhagic fever (VHF), and (b) A virus that presents phosphatidylserine (PS) and mediates cell infection and / or intracellular translocation via PS binding.

[0227] Accordingly, in a further embodiment, the present invention provides a composition of a fifth aspect of the present invention for use in a preventive or therapeutic method for preventing or reducing the transmission of a viral infection in a subject, wherein the viral infection is caused by a virus capable of causing hemorrhagic fever (VHF).

[0228] In other words, the present invention provides a preventive or therapeutic method for preventing or reducing the transmission of a viral infection in a subject, the viral infection being caused by a virus capable of causing hemorrhagic fever (VHF), and the method comprises administering a therapeutically effective amount of a composition of a fifth aspect of the present invention to the subject.

[0229] In other words, this embodiment provides a composition of a fifth aspect of the present invention for use in the manufacture of a drug for prophylactic or therapeutic purposes, by preventing or reducing the transmission of a viral infection in a subject, wherein the viral infection is caused by a virus capable of causing hemorrhagic fever (VHF).

[0230] In another embodiment, the present invention provides a composition of a fifth aspect of the present invention for use in a prophylactic or therapeutic method to prevent or reduce the transmission of a viral infection in a subject, the viral infection being caused by a virus that presents phosphatidylserine (PS) and mediates cell infection and / or intracellular translocation via PS binding.

[0231] In other words, the present invention provides a preventive or therapeutic method for preventing or reducing the transmission of a viral infection in a subject, the viral infection being caused by a virus that presents phosphatidylserine (PS) and mediates cell infection and / or intracellular translocation via PS binding, the method comprising administering a therapeutically effective amount of a composition of a fifth aspect of the present invention to the subject.

[0232] In other words, this embodiment provides a composition of a fifth aspect of the present invention for use in the manufacture of a drug for prophylactic or therapeutic purposes, by preventing or reducing the transmission of a viral infection in a subject, wherein the viral infection is caused by a virus that presents phosphatidylserine (PS) and mediates cell infection and / or intracellular translocation via PS binding.

[0233] In another embodiment, the present invention provides a composition of a fifth aspect of the present invention for use in prophylactic or therapeutic methods for preventing or protecting against a viral infection in a subject, wherein the viral infection is caused by a virus capable of causing hemorrhagic fever (VHF).

[0234] In other words, the present invention provides a prophylactic or therapeutic method for preventing or protecting against a viral infection in a subject, which is caused by a virus capable of causing hemorrhagic fever (VHF), and the method comprises administering a therapeutically effective amount of a composition of a fifth aspect of the present invention to the subject.

[0235] To put it another way, this embodiment provides a composition of a fifth aspect of the present invention for use in the manufacture of a drug for prophylactic or therapeutic purposes, by preventing or protecting against a viral infection in a subject, wherein the viral infection is caused by a virus capable of causing hemorrhagic fever (VHF).

[0236] In another embodiment, the present invention provides a composition of a fifth aspect of the present invention for use in prophylactic or therapeutic methods for preventing or protecting against a viral infection in a subject, wherein the viral infection is caused by a virus that presents phosphatidylserine (PS) and mediates cell infection and / or intracellular translocation via PS binding.

[0237] In other words, the present invention provides a prophylactic or therapeutic method for preventing or protecting against a viral infection in a subject, the viral infection being caused by a virus that presents phosphatidylserine (PS) and mediates cell infection and / or intracellular translocation via PS binding, the method comprising administering a therapeutically effective amount of a composition of a fifth aspect of the present invention to the subject.

[0238] In other words, this embodiment provides a composition of a fifth aspect of the present invention for use in the manufacture of a drug for prophylactic or therapeutic purposes, by preventing or protecting against viral infection in a subject, wherein the viral infection is caused by a virus that presents phosphatidylserine (PS) and mediates cell infection and / or intracellular translocation via PS binding.

[0239] In another embodiment, the present invention provides a composition of a fifth aspect of the present invention for use in a preventive or therapeutic method for treating a viral infection in a subject, the viral infection being caused by a virus capable of causing hemorrhagic fever (VHF).

[0240] In other words, the present invention provides a preventive or therapeutic method for treating a viral infection in a subject, which is caused by a virus capable of causing hemorrhagic fever (VHF), and the method comprises administering a therapeutically effective amount of a composition of a fifth aspect of the present invention to the subject.

[0241] To put it another way, this embodiment provides a composition of a fifth aspect of the present invention for use in the manufacture of a drug for a preventive or therapeutic method of treating a viral infection in a subject, wherein the viral infection is caused by a virus (VHF) capable of causing hemorrhagic fever.

[0242] In another embodiment, the present invention provides a composition of a fifth aspect of the present invention for use in a prophylactic or therapeutic method for treating a viral infection in a subject, which is caused by a virus that presents phosphatidylserine (PS) and mediates cell infection and / or intracellular translocation via PS binding.

[0243] In other words, the present invention provides a preventive or therapeutic method for treating a viral infection in a subject, which is caused by a virus that presents phosphatidylserine (PS) and mediates cell infection and / or intracellular translocation via PS binding, and the method comprises administering a therapeutically effective amount of a composition of a fifth aspect of the present invention to the subject.

[0244] In other words, this embodiment provides a composition of a fifth aspect of the present invention for use in the manufacture of a drug for prophylactic or therapeutic purposes by treating a viral infection in a subject, wherein the viral infection is caused by a virus that presents phosphatidylserine (PS) and mediates cell infection and / or intracellular translocation via PS binding.

[0245] Further embodiments of the present invention provide a composition according to a fifth aspect of the present invention for use in a method of treating a subject infected with or suspected of being infected with a hemorrhagic fever-causing pathogen, such as a hemorrhagic fever-causing virus (VHF) or hemorrhagic fever-causing bacteria (BHF).

[0246] In other words, this embodiment provides a method for treating a subject who is infected with or suspected of being infected with a hemorrhagic fever-causing pathogen, such as a hemorrhagic fever-causing virus (VHF) or hemorrhagic fever-causing bacteria (BHF), the method comprising administering a therapeutically effective amount of a composition of the fifth embodiment of the present invention to the subject.

[0247] In other words, this embodiment provides a composition of a fifth aspect of the present invention for use in the manufacture of a drug for the treatment of a subject infected with or suspected of being infected with a hemorrhagic fever-causing pathogen, such as a hemorrhagic fever-causing virus (VHF) or hemorrhagic fever-causing bacteria (BHF).

[0248] Another embodiment of the present invention provides a composition of a fifth aspect of the present invention for use in a method of treating a subject that has been in contact with another subject that is infected with or suspected to be infected with a hemorrhagic fever-causing pathogen, such as a hemorrhagic fever-causing virus (VHF) or hemorrhagic fever-causing bacteria (BHF).

[0249] In other words, this embodiment provides a method for treating a subject who has been in contact with another subject who is infected with or suspected to be infected with a hemorrhagic fever-causing pathogen, such as a virus (VHF) or bacteria (BHF), the method comprising administering a therapeutically effective amount of a composition of the fifth embodiment of the present invention to the subject.

[0250] In other words, this embodiment provides a composition of a fifth aspect of the present invention for use in the manufacture of a drug for the treatment of a subject that has been in contact with another subject that is infected with or suspected to be infected with a hemorrhagic fever-causing pathogen, such as a virus (VHF) or bacteria (BHF).

[0251] Further embodiments of the present invention provide a composition of a fifth aspect of the present invention for use in a method of treating a subject that has come into contact with biomaterial present in or produced by another subject that is infected with or suspected to be infected with a hemorrhagic fever-causing pathogen, such as a hemorrhagic fever-causing virus (VHF) or hemorrhagic fever-causing bacteria (BHF).

[0252] In other words, this embodiment provides a method for treating a subject that has been in contact with biomaterial present in or produced by another subject that is infected with or suspected to be infected with a hemorrhagic fever-causing pathogen, such as a hemorrhagic fever-causing virus (VHF) or hemorrhagic fever-causing bacteria (BHF), the method comprising administering a therapeutically effective amount of a composition of the fifth embodiment of the present invention to the subject.

[0253] In other words, this embodiment provides a composition of a fifth aspect of the present invention for use in the manufacture of a drug for the treatment of a subject that has come into contact with biomaterial present in or produced by another subject that is infected with or suspected to be infected with a hemorrhagic fever-causing pathogen, such as a hemorrhagic fever-causing virus (VHF) or hemorrhagic fever-causing bacteria (BHF).

[0254] In these embodiments of the present invention described above, the pathogen capable of causing hemorrhagic fever may be VHF.

[0255] Viral hemorrhagic disorders (VHFs) are a diverse group of animal and human diseases that can be caused by at least five distinctly different families of RNA viruses, namely the Arenaviridae, Filoviridae, Bunyaviridae, Flaviviridae, and Rhabdoviridae families. All types of VHFs may be characterized by fever and hemorrhagic disorders, and all can progress to high fever, shock, and death in many cases.

[0256] Subjects suspected of being infected with a pathogen capable of causing hemorrhagic fever, such as a virus capable of causing viral hemorrhagic fever (VHF) or a bacterium capable of causing bacterial hemorrhagic fever (BHF), can be subjects who have been in contact with this disease in the past (e.g., based on employment as a healthcare worker or due to infection of a family member), and / or can be subjects presenting one or more signs or symptoms of infection before a definitive diagnosis.

[0257] The signs or symptoms of susceptible VHF typically include fever and increased easy bleeding (bleeding tendency). The onset of VHF symptoms is also often caused by flushing of the face and chest, small red or purple spots (petechiae), obvious bleeding, swelling caused by edema, low blood pressure (hypotension), and shock. Fatigue, muscle pain (myalgia), headache, vomiting, and diarrhea occur frequently. The severity of symptoms varies depending on the type of virus, among which the "VHF syndrome" (leading to shock due to capillary leakage, bleeding tendency, and circulatory failure) appears in the majority of patients with filovirus hemorrhagic fever (e.g., Ebola and Marburg), CCHF, and South American hemorrhagic fever, but also appears in a minority of patients with dengue fever, RVF, and Lassa fever.

[0258] In a sixth aspect of the present invention, the VHF can be Ebola, and the subject can present one or more symptoms of Ebola, such as initial clinical symptoms such as excessive or profuse sweating, fever, muscle pain, general fatigue, and / or chills; and / or influenza-like symptoms optionally accompanied by gastrointestinal symptoms; spotted papules, petechiae, conjunctival hemorrhage, epistaxis, melena, hematemesis, shock and / or encephalopathy; leukopenia (e.g., related to increased lymphocytic apoptosis), thrombocytopenia, increased levels of aminotransferase, thrombin and / or partial thromboplastin time, fibrin degradation products detectable in the blood, and / or disseminated intravascular coagulation (DIC).

[0259] Definitive diagnosis is usually made in a standard laboratory with advanced biological containment capabilities. Laboratory findings vary somewhat between viruses, but generally include decreased total white blood cell count (especially lymphocytes), decreased platelet count, increased serum liver enzymes, and decreased blood clotting ability, measured as increased prothrombin (PT) and activated partial thromboplastin time (PTT). Hematocrit may be elevated. Serum urea and creatinine may be elevated, depending on the patient's hydration status. Bleeding time tends to be prolonged.

[0260] For example, because Ebola is a BSL-4 pathogen, confirmed clinical laboratory diagnosis of viremia during the acute phase of Ebola virus infection is possible in suitable laboratory facilities. The assays that can be used are based on the stage of the disease.

[0261] During acute illness, assays include a) virus isolation using Vero or Vero E6 cell lines, b) RT-PCR and real-time quantitative PCR assays with appropriate false-negative and false-positive controls, c) antigen capture ELISA, and d) IgM ELISA.

[0262] Later in the course of the disease, available tests include a) IgM and IgG ELISA using true viral antigens, and in fatal cases, autopsy tissue may be used for a) antigen detection using immunohistochemical techniques, b) detection of Ebola antigen with immunohistochemistry assistance (Zaki et al, J Infect Dis, 1999;179(Suppl.1):S36e47. (Its contents are incorporated herein by reference in their entirety)), and c) in situ hybridization techniques for detection of viral RNA.

[0263] The details of each of these techniques are summarized in Saijo et al, Clin Vaccine Immunol 2006;13:444e51, and its contents are incorporated herein by reference in their entirety.

[0264] ELISA-based assays have been standardized by the CDC for the detection of Ebola virus-specific antibodies. These assays have been shown to be highly sensitive and capable of detecting antibodies in the serum of individuals exposed to Ebola 10 years prior to the outbreak. Cellular plaque assays and endpoint titration assays (TCID50) have also been developed for the detection and quantification of filoviruses for use in preclinical studies (Shurtleff et al, Viruses 2012;4:3511e30; Smither et al, J Virol Methods 2013;193:565e71 (their contents are incorporated herein by reference in their entirety)).

[0265] For example, VHF could be a virus belonging to a family selected from Filoviridae, Arenaviridae, Bunyaviridae, Flaviviridae, or Rhabdoviridae.

[0266] The Arenaviridae family includes viruses that cause Lassa fever, Lujo virus, and hemorrhagic fever in Argentina, Bolivia, Brazil, and Venezuela.

[0267] The Bunyaviridae family includes members of the Hanta virus genus, which causes hemorrhagic fever with renal syndrome (HFRS); the Crimean-Congo hemorrhagic fever (CCHF) virus derived from the Nairo virus genus; the Garissa and Ilesha viruses derived from the Orthobunya virus genus; and the Rift Valley fever (RVF) virus derived from the Phlebo virus genus.

[0268] The Filoviridae family includes the Ebola virus and the Marburg virus.

[0269] The Flaviviridae family includes two viruses in the tick-borne encephalitis group that cause dengue, yellow fever, and VHF: Omsk hemorrhagic fever virus and Kyasanur forest disease virus.

[0270] The isolation of a member of the Rhabdoviridae family responsible for two fatal and two non-fatal cases of hemorrhagic fever in the Bas-Congo district of the Republic of Congo was also reported. The non-fatal cases occurred in healthcare workers who were treating the other two patients, suggesting the possibility of human-to-human transmission.

[0271] Therefore, for example, in one particularly targeted embodiment, the present invention may be applied to viruses of the Filoviridae family, such as the Ebola virus and the Marburg virus. In another particularly targeted embodiment, the present invention may be applied to viruses of the Flaviviridae family, such as the dengue virus.

[0272] Accordingly, the present invention realizes a composition of a fifth aspect of the present invention for use in the above-described preventive or therapeutic methods for (i) preventing or reducing the transmission of Ebola infection, (ii) preventing or protecting against Ebola infection, or (iii) treating Ebola infection in a subject that is infected with or suspected of being infected with the Ebola virus, or has had or is expected to have had contact with another subject that is infected with or suspected of being infected with the Ebola virus, or has had or is expected to have had contact with biomaterial present in or produced by another subject that is infected with or suspected of being infected with the Ebola virus.

[0273] Accordingly, the present invention realizes a composition of a fifth aspect of the present invention for use in the above-described preventive or therapeutic methods for (i) preventing or reducing the transmission of Marburg infection, (ii) preventing or protecting against Marburg infection, or (iii) treating Marburg infection in which the subject has been or is suspected of being infected with the Marburg virus, or has been or is expected to be in contact with another subject that has been or is suspected of being infected with the Marburg virus, or has been or is expected to be in contact with biomaterial present in or produced by another subject that has been or is suspected of being infected with the Marburg virus.

[0274] Accordingly, the present invention realizes a composition of a fifth aspect of the present invention for use in the above-described preventive or therapeutic methods for (i) preventing or reducing the transmission of dengue virus infection, (ii) preventing or protecting against dengue virus infection, or (iii) treating dengue virus infection in which the subject has been or is suspected of being infected with dengue virus, or has been or is expected to be in contact with another subject that has been or is suspected of being infected with dengue virus, or has been in contact with biomaterial present in or produced by another subject that has been or is suspected of being infected with dengue virus.

[0275] The present invention also provides a composition according to a fifth aspect of the present invention for use in the above-described method for treating, delaying the onset and / or progression of an infection caused by a target VHF or BHF.

[0276] The present invention also provides a composition according to a fifth aspect of the present invention for use in the above-described method for preventing, reducing, delaying the onset of, or delaying the progression of direct and / or indirect bacterial or viral damage to the immune and / or vascular system in a subject caused by BHF or VHF.

[0277] For example, the present invention may be used, for example, in the context of Ebola infection, to prevent, reduce, delay the onset of, or delay the progression of direct and / or indirect bacterial or viral damage to the immune system in a subject. For example, bacterial or viral damage may be selected from damage to the innate immune response, damage to the acquired humoral response, damage to dendritic cells, damage to the regulatory function of the production of inflammatory factors such as interferon production (including IL1 production), damage to macrophages, and / or damage to monocytes.

[0278] The present invention may be used to prevent, reduce, delay the onset of, or delay the progression of blood leakage (bleeding), hypotension, decrease in blood pressure, shock, or death in a subject.

[0279] The present invention may be used to prevent, reduce, delay the onset of, or delay the progression of nitric oxide damage to the vascular endothelium of a subject induced by a virus.

[0280] The present invention provides a composition according to a fifth aspect of the present invention for use in a method for preventing, reducing, delaying the onset of, or delaying the progression of damage, activation, death, and / or disruption of the integrity of vascular endothelium or its endothelial cells in a subject infected or suspected of being infected with a pathogen capable of causing a hemorrhagic fever such as VHF or BHF. The integrity of the vascular endothelium or its endothelial cells may be determined, for example, by the degree of cellular or vascular epithelial leakage and / or by the detection of one or more bleeding events, or the formation of edema and / or dehydration symptoms in the subject.

[0281] The present invention provides a composition according to a fifth aspect of the present invention for use in a method of preventing, reducing, delaying the onset or progression of damage, activation, death, and / or destruction of the integrity of vascular endothelium or its endothelial cells in an object that has been in contact with or is expected to be in contact with another object that has been infected with or is suspected to be infected with a pathogen capable of causing hemorrhagic fever such as VHF or BHF.

[0282] The present invention provides a composition according to a fifth aspect of the present invention for use in a method of preventing, reducing, delaying the onset or progression of damage, activation, death, and / or destruction of the integrity of vascular endothelium or its endothelial cells in another subject that has been in contact with or is expected to be in contact with biomaterial present in or produced by another subject that is infected with or suspected to be infected with a pathogen capable of causing hemorrhagic fever such as VHF or BHF.

[0283] Further embodiments of the present invention realize a composition of a fifth aspect of the present invention for use in prophylactic or therapeutic methods described above with reference to various embodiments of the present invention, wherein the viral infection is caused by a virus that presents phosphatidylserine (PS) and mediates cell infection and / or intracellular translocation via PS binding. Alternatively, the viral infection may be caused by a virus that presents one or more other types of phospholipids to which annexin A5 binds and / or other moieties to which annexin A5 binds.

[0284] Viruses that present phosphatidylserine (PS) and mediate cell infection and / or intracellular translocation via PS binding may include, in particular, inclusion viruses containing phosphatidylserine (PS) within the viral envelope, especially within the outer layer. The presentation of PS by a virus can be determined by methods known in the art, for example, by ELISA studies measuring the binding of annexin A5 to the virus. Preferred methods may include, for example, ELISA measurements of hemagglutinin (HA)-tagged annexin A5 binding to anti-HA antiserum, such as those described in Moller-Tank, et al, 2013, J. Virol., 87(15), 8327-8341 (the contents of which are incorporated herein by reference).

[0285] The group of viruses that are particularly targeted by the present invention includes those that mediate cell infection and / or internal migration through binding to phosphatidylserine-mediated virus entry enhancing receptors (PVEERs). PVEERs were discussed in Moller Tank, et al, 2013, J. Virol., 87(15), 8327-8341, and examples include T cell immunoglobulin and mucin 1 (TIM-1) receptors. Further examples may include TIM-4, Gas6 or protein S / Axl, Mer, and Tyro3, ​​as well as MFG-E8 / integrin αvβ3 or αvβ5.

[0286] Ebola is an example of a particularly targeted virus that presents phosphatidylserine (PS) and mediates cell infection and / or intracellular translocation via PS binding to TIM-1. Moller Tank, et al, 2013, J. Virol., 87(15), 8327-8341.

[0287] The present invention recognizes that Annexin A5 and compositions of a fifth aspect of the present invention may be used to inhibit or block PS-mediated cell infection and / or intracellular translocation of viruses such as Ebola virus via PVEERs such as TIM-1, thereby being useful in prophylactic or therapeutic methods to (i) prevent or reduce the transmission of viral infection, (ii) prevent or protect against transmission of viral infection, or (iii) treat viral infection in a subject, which is caused by a virus that presents phosphatidylserine (PS) and mediates cell infection and / or intracellular translocation via PS binding.

[0288] Viruses that present phosphatidylserine (PS) may be selected from a group consisting of, for example, viruses of the Filoviridae family (such as Ebola and Marburg), the Flaviviridae family, hepatitis A, alphaviruses, baculoviruses, and arenaviruses. Viruses may be infectious in humans, or only in humans. Viruses may be infectious in non-human animals, or only in non-human animals, such as one or more animals selected from dogs, cats, cattle, sheep, pigs, goats, rodents, camels, domestic animals, and wild animals.

[0289] PVEERs such as TIM-1 may be involved in the internal translocation of viruses to various cell types. In one embodiment, the cell types of particular interest in protection and / or therapy in the present invention may include one or more cell types selected from the group consisting of epithelial cells (including vascular epithelial cells), mast cells, B cells, and T cells such as CD4+ cells or CD8+ cells and especially activated CD4+ cells.

[0290] TIM-1, also known as HAVCR1 and KIM-1, has been identified as a susceptibility gene for human asthma (Mclntire et al, 2003, Nature 425:576). The amino acid sequence of one published human TIM-1 protein is shown below. MHPQVVILSLILHLADSVAGSVKVGGEAGPSVTLPCHYSGAVTSMCWRGSC SLFTCQNGIVWTNGTHVTYRKDTRYKLLGDLSRRDVSLTIENTAVSDSGVYC CRVEHRGWFNDMKITVSLEIVPPKVTTTPIVTTVPTVTTVRTSTTVPTTTTVPMTTVPTTTVPTTMSIPTTTTVLTTMTVSTTTSVPTTTSIPTTTSVPVTTTVSTFVPP MPLPRQNHEPVATSPSSPQPAETHPTTLQGAIRREPTSSPLYSYTTDGNDTVTE SSDGLWNNQTQLFLEHSLLTANTTKGIYAGVCISVLVLLALLGVIIAKKYFF KKEVQQLSVSFSSLQIKALQNAVEKEVQAEDNIYIENSLYATD(Sequence ID 2).

[0291] TIM-1 is a type I membrane protein with an extracellular region containing an IgV domain, a mucin-rich domain, and a short membrane-proximal stalk containing an N-linked glycosylation site (Ichimura et al, 1998, J, Biol, Chem. 273(7):4135-42). The TIM-1 IgV domain has a disulfide-dependent conformation, where the CC' loop folds over the GFC β chain, creating a unique gap formed by the CC' and FG loops (Santiago et al, 2007, Immunity 26(3):299-310). The gap constructed by the CC' and FG loops is a binding site for phosphatidylserine (Kobayashi et al, 2007, Immunity 27(6):927-40). Antibodies directed to the CC' / FG gap of the TIM-1 IgV domain inhibit TIM-1 binding to phosphatidylserine and dendritic cells, exhibiting therapeutic activity in vivo in a humanized mouse model of allergic asthma (Sonar et al, 2010, J.Clin.Invest.120:2767-81).

[0292] Further embodiments of the present invention are based on the use of compositions of a fifth aspect of the present invention to prevent, inhibit, or reduce the ability of the IgV domain of TIM-1 and other PVEERs to bind to PS presented thereto. The AnxA5 protein in the compositions of the fifth aspect of the present invention preferably also has the ability to bind to PS and, in this embodiment of the present invention, is competitive with PVEERs for binding to PS.

[0293] Therefore, in a further embodiment, a composition of the fifth aspect of the present invention may be used in a method for inhibiting phosphatidylserine binding to TIM-1 (or other PVEER).

[0294] For example, this may be useful prophylactically or therapeutically in relation to inhibiting, reducing, or preventing cell infection by viruses that mediate cell infection and / or intracellular translocation via phosphatidylserine (PS).

[0295] Alternatively, this may be useful prophylactically or therapeutically in relation to addressing other conditions involving the binding of PS to TIM-1 (or other PVEERs). TIM-1-related disorders are discussed further below.

[0296] Accordingly, in another embodiment, the present invention provides a method for inhibiting or reducing the binding of TIM-1 or other PVEERs to phosphatidylserine, the method comprising contacting a first cell expressing TIM-1 or other PVEER with an amount of a composition of a fifth aspect of the present invention effective in inhibiting or reducing the binding of the first cell to a second cell containing phosphatidylserine on its cell surface, or to a virus presenting phosphatidylserine (PS) on its surface. The method may be an in vivo or in vitro method. In the case of an in vivo method, it may be for treating or preventing a pathological condition in which the binding of PS to TIM-1 or other PVEERs is involved.

[0297] In other words, this embodiment of the present invention also provides a composition of a fifth aspect of the present invention for use in prophylactic or therapeutic methods to inhibit or reduce the binding of TIM-1 or other PVEERs to phosphatidylserine in patients requiring treatment.

[0298] In another embodiment, the present invention provides a method for inhibiting or reducing the binding of PS to TIM-1 or other PVEERs on dendritic cells, the method comprising contacting dendritic cells expressing TIM-1 or other PVEERs with an amount of a composition of the fifth embodiment of the present invention effective in inhibiting or reducing the binding of PS to dendritic cells. The method may be in vivo or in vitro. If it is an in vivo method, it may treat or prevent a pathological condition in which the binding of PS to TIM-1 or other PVEERs on dendritic cells is involved.

[0299] In other words, this embodiment of the present invention also provides a composition of a fifth aspect of the present invention for use in prophylactic or therapeutic methods to inhibit or reduce the binding of PS to TIM-1 or other PVEERs on dendritic cells in patients requiring treatment.

[0300] Furthermore, a method for treating or preventing an inflammatory or autoimmune condition is disclosed, which comprises administering a pharmaceutical composition comprising a therapeutically effective amount of a composition according to a fifth aspect of the present invention to a mammal having an inflammatory or autoimmune condition.

[0301] In other words, this embodiment of the present invention also provides a composition of a fifth aspect of the present invention for use in prophylactic or therapeutic methods for preventing, treating or reducing inflammatory or autoimmune conditions.

[0302] Furthermore, a method for treating or preventing asthma is disclosed, which includes administering a pharmaceutical composition comprising a composition according to a fifth aspect of the present invention to a mammal having asthma.

[0303] In other words, this embodiment of the present invention also provides a composition of a fifth aspect of the present invention for use in preventive or therapeutic methods for preventing, treating or reducing asthma.

[0304] Also disclosed are methods for treating or preventing atopic disorders, which include administering to a mammal having an atopic disorder a pharmaceutical composition comprising a therapeutically effective amount of a composition according to a fifth aspect of the present invention. Atopic disorders may include, for example, atopic dermatitis, contact dermatitis, urticaria, allergic rhinitis, angioedema, latex allergy, or allergic lung disorders (e.g., asthma, allergic bronchospergillosis, or hypersensitivity pneumonitis).

[0305] In other words, this embodiment of the present invention also provides a composition of a fifth aspect of the present invention for use in prophylactic or therapeutic methods for preventing, treating or reducing atopic disorders.

[0306] A composition according to a fifth aspect of the present invention, used to treat or prevent a variety of TIM-1-related disorders, including immunological disorders such as inflammatory disorders and autoimmune disorders, and other PVEER-related disorders, as described herein.

[0307] The term "treat" includes administering any of the substances or compositions described herein in an amount, manner, and / or form effective in improving a condition, symptom, or parameter associated with the disorder, or in preventing the progression or worsening of the disorder (including secondary injuries caused by the disorder), either to a statistically significant extent or to an extent detectable by a person skilled in the art.

[0308] To subjects who are at risk of, diagnosed with, or have one of these disorders, a composition of the fifth aspect of the present invention may be administered in an amount and over such a duration to provide an overall therapeutic effect. The composition of the fifth aspect of the present invention may be administered alone (monotherapy) or in combination with other agents (combination therapy), either mixed or separately, simultaneously or sequentially. In the case of combination therapy, the amount and timing of administration may be such as to provide, for example, an additive or synergistic therapeutic effect. Furthermore, administration of the composition of the fifth aspect of the present invention (with or without the second agent) may be used as primary treatment, e.g., first-line treatment, or as secondary treatment, e.g., for subjects who have had an insufficient response to previously administered therapies (i.e., therapies other than therapy with the AnxA5 protein).

[0309] Diseases or conditions treatable with compositions of the fifth aspect of the present invention as described herein include, for example, ischemia-reperfusion injury (e.g., organ ischemia-reperfusion injury such as hepatic or renal ischemia-reperfusion injury), allergies, asthma, inflammatory bowel disease (IBD), Crohn's disease, transplant rejection, pancreatitis, and delayed-type hypersensitivity (DTH).

[0310] Additional diseases or conditions treatable with the compositions of the fifth aspect of the present invention described herein include, for example, autoimmune disorders.

[0311] Systemic lupus erythematosus (SLE) is a TH-2-mediated autoimmune disorder characterized by high levels of autoantibodies directed against intracellular antigens such as double-stranded DNA, single-stranded DNA, and histones.

[0312] Other organ-specific or systemic autoimmune diseases suitable for treatment with the compositions of the fifth aspect of the present invention described herein include myasthenia gravis, autoimmune hemolytic anemia, Chagas disease, Graves' disease, idiopathic thrombocytopenic purpura (ITP), Wegner granulomatosis, polyarteritis nodosa, and rapidly progressive crescentic glomerulonephritis. See, for example, Benjamini et al., 1996, Immunology, A Short Course, Third Ed. (Wiley-Liss, New York). In addition, rheumatoid arthritis (RA) is suitable for treatment with Annexin A5 as described herein.

[0313] Additional TIM-1-related diseases or conditions treatable with the compositions of the fifth aspect of the present invention described herein include, for example, graft-versus-host disease (GVHD). GVHD is an example of a T cell-mediated condition that can be treated with Annexin A5 as described herein. GVHD is triggered when donor T cells recognize a host antigen as heterologous. Often a fatal consequence of bone marrow transplantation (BMT) in human patients, GVHD can be acute or chronic. Acute and chronic forms of GVHD are examples of antigen-specific Th1 and Th2 response developments, respectively. Acute GVHD occurs within the first two months after BMT and is characterized by donor cytotoxic T cell-mediated injury to the skin, intestines, liver, and other organs. Chronic GVHD appears later (more than 100 days after BMT) and is characterized by damage to the skin, kidneys, and other organs caused by overproduction of immunoglobulins (Ig), including autoantibodies, and Ig deposition. Nearly 90% of patients with acute GVHD progress to chronic GVHD. Chronic GVHD appears to be a Th2 T cell-mediated disease (De Wit et al, 1993, J.Immunol. 150:361-366). Acute GVHD is a Th1-mediated disease (Krenger et al, 1996, Immunol. Res. 15:50-73, Williamson et al, 1996, J.Immunol. 157:689-699). T cell-mediated cytotoxic activity is characteristic of acute GVHD. The results of donor anti-host cytotoxic activity can be seen in various ways. Firstly, host lymphocytes are rapidly destroyed, such as when mice experiencing acute GVHD are severely immunosuppressed. Secondly, once donor lymphocytes engraft and proliferate within the host spleen, their cytotoxic activity can be directly measured in vitro by utilizing cell lines that express host antigens that can be recognized (as heterologous) by the donor cells. Thirdly, the disease becomes lethal as further tissues and cell populations are destroyed.

[0314] Additional TIM-1-related disorders or conditions treatable with the compositions of the fifth aspect of the present invention described herein include, for example, atopic disorders. Atopic disorders are characterized by the expression of cytokines, chemokines, and other molecules characteristic of the Th2 response, particularly IL-4, IL-5, and IL-13 cytokines, by immune system cells, including activated T cells and APCs. Such atopic disorders are therefore readily treatable with the compositions of the fifth aspect of the present invention described herein. Atopic disorders include airway hypersensitivity and distress syndrome, atopic dermatitis, contact dermatitis, urticaria, allergic rhinitis, angioedema, latex allergy, and allergic lung disorders (e.g., asthma, allergic bronchopulmonary aspergillosis, and hypersensitivity pneumonitis).

[0315] Additional TIM-1-related diseases or conditions treatable with the compositions of the fifth aspect of the present invention described herein include, for example, numerous immune or inflammatory disorders. Immune or inflammatory disorders include allergic rhinitis; autoimmune hemolytic anemia; acanthosis nigricans, Addison's disease; alopecia areata; alopecia generalis; amyloidosis; anaphylactic purpura; anaphylactic reactions; aplastic anemia; ankylosing spondylitis; cranial arteritis; giant cell arteritis; Takayasu's arteritis; temporal arteritis; telangiectatic ataxia; autoimmune oophoritis; autoimmune orchitis; autoimmune polyglandular endocrine insufficiency; Behçet's disease; Berger's disease; Buerger's disease; bronchitis; bullous pemphigoid; chronic mucocutaneous candidiasis; Kaplan's syndrome; and postmyocardial infarction. Syndrome; Post-pericardiotomy syndrome; Carditis; Celiac Sprue; Chagas disease; Chediak-Higashi syndrome; Churg-Strauss disease; Cogan syndrome; Cold agglutinin disease; CREST syndrome; Crohn's disease; Cryoglobulinemia; Idiopathic fibrous alveolitis; Herpetiform dermatitis; Dermatomyositis; Diabetes mellitus; Diamond-Blackfan syndrome; DiGeorge syndrome; Lupus discoid; Eosinophilic fasciitis; Episcleritis; Erythema elevata; Erythema annulare; Erythema multiforme; Erythema nodosum; Familial Mediterranean fever; Felty's syndrome; Pulmonary fibrosis; Anaphylactic-like reactions Glomerulonephritis; autoimmune glomerulonephritis; post-streptococcal glomerulonephritis; post-transplant glomerulonephritis; membranous glomerulopathy; Goodpasture syndrome; immune-mediated granulocytopenia; granuloma annulare; allergic granulomatosis; granulomatous myositis; Graves' disease; Hashimoto's thyroiditis; neonatal hemolytic disease; idiopathic hemochromatosis; Henoch-Schönlein purpura; chronic active hepatitis and chronic progressive hepatitis; histiocytosis X; eosinophilic syndrome; idiopathic thrombocytopenic purpura; Job's syndrome; juvenile dermatomyositis; juvenile rheumatoid arthritis (juvenile chronic arthritis); Kawasaki disease; Keratitis; keratoconjunctivitis sicca; Landry-Guillain-Barré syndrome; lepromatous leprosy; Loeffler syndrome; lupus; Lyell's syndrome; Lyme disease; lymphomatous granulomatosis; systemic mastocytosis; mixed connective tissue disease; mononeuropathy; Muckle-Wells syndrome; mucocutaneous lymphadenopathy; multicentric retiohistiocytosis; multiple sclerosis; myasthenia gravis; mycosis fungoides; systemic necrotizing vasculitis; nephrotic syndrome; overlap syndrome; panniculitis; paroxysmal cold hemoglobinuria; paroxysmal nocturnal hemoglobinuria; bullous pemphigoid; pemphigus;Pemphigus erythematous; Pemphigus foliaceus; Pemphigus vulgaris; Pigeon breeder's disease; Polyarteritis nodosa; Polymyalgia rheumatica; Polymyositis; Idiopathic polyneuritis; Familial polyneuritis of Portugal; Preeclampsia / Eclampsia; Primary biliary cirrhosis; Systemic progressive sclerosis (scleroderma); Psoriasis; Psoriatic arthritis; Alveolar proteinosis; Pulmonary fibrosis, Raynaud's phenomenon / syndrome; Riedel's thyroiditis; Reiter's syndrome, Relapsing polychondritis; Rheumatic fever; Rheumatoid arthritis; Sarcoidosis; Scleritis; Sclerosing cholangitis; Serum sickness; Sézary syndrome; Sjögren's syndrome; Stevens-Johnson syndrome; Still's disease; Subacute sclerosing panencephalitis; Sympathetic ophthalmitis; Systemic lupus erythematous; Transplant rejection (yransplant) This includes, but is not limited to, rejection; ulcerative colitis; undifferentiated connective tissue disease; chronic urticaria; cold urticaria; uveitis; vitiligo; Weber-Christian disease; Wegener's granulomatosis; or Wiscott-Aldrich syndrome.

[0316] The present invention will now be described with reference to one or more non-limiting embodiments. [Examples]

[0317] The following embodiments are included to demonstrate specific embodiments of the present invention. Those skilled in the art will understand that the techniques disclosed in the following embodiments are representative of techniques found by the inventors to work well in carrying out the present invention and may therefore be considered to constitute a preferred mode for its implementation. However, those skilled in the art will understand that many modifications can be made in the specific embodiments disclosed without departing from the spirit and scope of the present invention, and that similar or comparable results can still be obtained.

[0318] Comparative Example 1 The process described by Marder et al., 2014, BMC Biotechnology, 14:33, describes the processing of a 1L culture, which consists of two 30-minute centrifugations of 38,900g. In the first centrifugation step, annexin A5 bound to cell debris is precipitated, and in the second centrifugation step, cell debris is precipitated while maintaining annexin A5 in solution.

[0319] To calculate the impact of scaling Marder et al.'s process from 1 L to a commercially relevant 1000 L culture volume, the following analysis is provided.

[0320] Based on the selection of the best centrifuges currently available with the best rotor for maximum throughput, they can hold 6 x 250 ml = 1.5 liters and achieve 30,200 to 38,400 g. Examples include the expensive carbon fiber lightweight rotor (Fiberlite F14-6 x 250y fixed-angle rotor) for use in Thermo Scientific™ Sorvall™ LYNX ultrafast centrifuges, or the JLA-16.250 rotor, fixed-angle, aluminum, biosafety lid, 6 x 250 mL, 38,400 g for use in Beckmancoulters Avanti JXN-26. When using such high-end, high-performance centrifuges, it would take approximately 45 minutes to load the centrifuge, start it, accelerate to the required speed, spend 30 minutes at maximum G force, then carefully break the pellets until they come to a standstill without disturbing them, and then empty the rotor.

[0321] In other words, the best centrifuges currently available can process 1.5 liters per 45 minutes.

[0322] In a section titled "Purification," Marder et al. reported that they suspended 3 g of wet weight cells in 30 mL of buffer before sonication and centrifugation. Therefore, the wet weight (WCW) concentration of cells used by Marder et al. during centrifugation was (3 g in 30 ml of buffer) = 9.1% WCW.

[0323] Marder et al. also (under the heading "Bioreactor cultivation" on page 2) reported a biomass concentration of 27.48 g(DCW)L -1 The average value (SD=1.96) is reported. Therefore, the cell dry weight (DCW) concentration in Marder's fermenter was 27.48 grams / L = 2.748%. It is known that 1 gram DCW = approximately 4 grams of cell wet weight (WCW). Therefore, in the cell-enriched fermenter, there was a WCW concentration of 2.748 × 4 = 11.0%. If we scale this up to a maximum culture volume of 1000L and conservatively assume a 5% cell loss during sampling from 1000L, the WCW in a 1000L tank using Marder's method would be 1000 × 11% × 0.95 = 104.5 kg WCW.

[0324] Marder's centrifugation method used a 9.1% WCW concentration during the centrifugation process. Therefore, 104.5 kg of WCW from 1000 L of culture needed to be diluted to a 9.1% WCW concentration, which required a total centrifugation volume of 1148 L.

[0325] Assuming generously that the bio-manufacturing facility has two high-end, high-performance centrifuges, one of which can be used to pelletize annexin together with cell debris (first centrifugation), while the other can operate in parallel with a second centrifugation while the annexin is in solution and the cell debris is being pelletized, then the following applies:

[0326] The total time required to centrifuge 1148 L of solution (the centrifuge can process 1.5 liters per 45 minutes) is 1148 / 1.5 = 766 centrifuges at 45 minutes each = 34,470 minutes = 574.5 hours.

[0327] Assuming the bio-manufacturing facility operates 12 hours per day, processing WCW from a 1000L tank using Marder et al.'s method would take 48 days of work, or 10 weeks of centrifugation alone (assuming 5 working days per week).

[0328] In total, fermentation, downstream processing, and other operations will require approximately two additional weeks. Assuming that the bio-manufacturing facility is fully occupied with that one process, and therefore other production cannot be carried out within the same facility during that time, this would mean 12 weeks at the manufacturing plant for cell preparation and processing from 1000L cultures. This is despite the generous assumption that two centrifuges are available. If only one centrifuge is used, production would take 22 weeks per 1000L batch.

[0329] In contrast, as discussed below, the method of the present invention can process a 1000L culture in just two weeks, that is, about six times faster (and also provides a much higher quality product with a much higher yield than the product of the Marder et al. process).

[0330] Because the manufacturing plant is occupied and other production cannot be carried out within the same plant during that time, manufacturing costs are directly proportional to the manufacturing time.

[0331] The yield of annexin A5 in the process of the present invention is calculated to be 2 to 3 times higher per batch than the product produced by Marder et al. This means that the production cost per gram of annexin A5 protein is (6 × 2 - 3 =) 12 to 18 times higher in Marder's process.

[0332] Furthermore, the purity of the protein from Marder's process would likely be unsuitable for human use. Despite careful centrifugation, only one anion exchange chromatography step was used, which falls far short of the requirements for achieving sufficient purity with respect to both in-process impurities (particularly endotoxins) and product-related variants. Marder provides no data regarding endotoxin levels or other impurities, further suggesting a lack of suitability for pharmaceutical use.

[0333] In addition, the extremely slow centrifugation operation in the Marder process requires exposing the annexin A5 protein to an unstable environment for an extended period. This is a further drawback of the long manufacturing operation, which is likely to result in product degradation or denaturation and has a negative impact on product quality.

[0334] Example 1 TIFF2023088961000002.tif240170TIFF2023088961000003.tif54170

[0335] introduction: A 320-amino acid sequence containing recombinant approximately 36 kDa protein annexin A5 is expressed in the cytoplasm of E. coli BL21 / pHIP.ANXA5. Recombinant annexin A5 is produced primarily in its soluble form. A thermoinducible expression plasmid, pHIP, containing the coding sequence for annexin A5, is used. The selective marker is the kanamycin resistance gene. The MCB for each clone has been established and extensively characterized.

[0336] The manufacturing process can be scaled up from a laboratory-scale equivalent to a 3L fermentation volume to a large-scale equivalent to a 100L fermentation volume.

[0337] The developed process involves efficient anion exchange capture from crude lysate, followed by an affinity step with immobilized heparin in the presence of calcium. This intermediate affinity step is highly specific to annexin A5. High-solubility anion exchange chromatography is used as the final refining step. The refining step allows for the separation of product-related impurities. Formulation is carried out by ultradialysis filtration using a 10 kD NMWCO cassette.

[0338] This embodiment describes and evaluates planned adaptations / modifications in the manufacturing process, determines operational parameters and necessary measures to ensure successful import, and defines sufficient criteria for determining that success. Successful import is indicated by the performance of downstream process operations that implement adaptations to laboratory-scale procedures with respect to scaling, resulting in comparable yield and quality DS.

[0339] The overall project objective is the development of a cGMP manufacturing process for annexin A5.

[0340] Procedure-Process Comparison and Evaluation This section evaluates the process parameters, raw materials, consumables, buffers, and equipment used.

[0341] Figure 2 shows a schematic overview of the complete process for the production of Annexin A5.

[0342] Table 1 compares and evaluates the raw materials used in the 3L and 100L processes. Table 1: Comparison of raw materials used in 3L and 100L processes TIFF2023088961000004.tif239170TIFF2023088961000005.tif31170

[0343] Table 2 compares and evaluates the consumables used in the 3L and 100L processes. Consumables (sampling devices and tubing) should not affect the product quality and yield of the DSP process. All materials used meet the required specifications. Table 2: List of consumables TIFF2023088961000006.tif152170

[0344] Review: ●All consumables used are single-use or made from materials specifically designed for the product. ● The bags used for buffer storage and as intermediate product containers were from Sartorius, featuring a PE / EVOH layer (CX5-14 film) throughout the entire process. These bags were validated by the manufacturer for their sterility, low endotoxin content, and leachability and extractability. ● All other consumables used, including materials that come into contact with the product such as tubing, connectors, sample collection systems, or sample containers, are suitable for their intended purpose in each step of this process. This typically includes USP Class VI certification, sterility, and / or low endotoxin levels, where applicable. Platinum-hardened silicone tubing is used throughout the DSP, with the exception of C-Flex tubing integrated with bags. All consumables used are free of animal-derived components or are available with TSE certification.

[0345] Table 3 compares the equipment used in the 3L and 100L processes for the production of Annexin A5. Table 3: List of equipment used TIFF2023088961000007.tif233170

[0346] The media, buffer, and solution are as shown in Table 4. The buffer specifications apply only to conductivity and are based on the preparation of the test buffer. The buffers are prepared before this process, tested according to their specifications, microfiltered (0.2 μm filter), and stored (retention time of 3 months or less at room temperature). A sample of the buffer is taken at the time of use (for reference; decomposition: endotoxins, bioburden). Table 4: List of media and solutions TIFF2023088961000008.tif235170TIFF2023088961000009.tif167170

[0347] A scalable supply batch fermentation process was developed and scaled up to production units. The downstream purification process includes three chromatography steps. After benzonase treatment, the filtered and diluted feed stream is applied to AX chromatography (Q Sepharose XL, GE Healthcare) as the first capture step. The eluate is conditioned by dilution to enable intermediate purification using affinity chromatography (Heparin Hyper DM, Pall). The AF pool is diluted and applied to the final AX chromatography step (Source15 Q, GE Healthcare). Finally, concentration and buffer exchange are performed by UF / DF.

[0348] We successfully conducted trial runs with a DSP equivalent to a 3L fermentation volume, demonstrating sufficient process performance for all process stages.

[0349] The target scope is defined by a small-scale process, and this is used to evaluate the results of scaling up.

[0350] Process comparison The following section details the laboratory-scale downstream process (DSP) based on trial runs conducted at a process scale equivalent to a 3L fermentation volume. Generally, apart from the load on the first capture step, the chromatographic process is performed at a laboratory scale using the AektaExplorer system. At large scale, all chromatographic processes are carried out using the Bioprocess system. The scaling factor for the DSP is 33 (3L USP From 100L USP to).

[0351] 1.1.1 Resuspension of biomass, treatment with benzonase, and cell disruption After fermentation, the biomass is collected by centrifugation and stored at -20°C. Downstream processing is initiated by thawing the biomass and resuspending it in homogenization buffer 1. Before homogenization, homogenization buffer (3,300 U / L) is added. USP or 1.850 U / L再懸濁させたバイオマス Pre-diluted benzonase is added to the resuspended cells in a solution. The resuspension ratio is set to 1 g biomass / 10 mL. Homogenization is performed for 3 cycles at 600 bar to achieve a high degree of homogenization beneficial for the subsequent capture step. Since a temperature increase of up to 40°C is desired to enable optimal digestion of nucleic acids by benzonase, no active cooling is required during homogenization. On a small scale, a temperature range of 36-40°C was obtained.

[0352] The process flowchart for biomass resuspension, benzonase treatment, and cell disruption is shown below. TIFF2023088961000010.tif153170

[0353] 1.1.2 Clarification, conditioning, and capture chromatography Following homogenization, the lysate is clarified by filtration using a Cuno 60 SP (0.6-0.2 μm) depth filter. This step is performed to reduce the nucleic acid content and to obtain a particle-reduced solution suitable for capture chromatography. The depth filter is pre-washed with water according to the manufacturer's instructions.

[0354] After filtration, dilute the lysate 2-fold with 1% Tween 80. Add EDTA to a final concentration of 2 mM.

[0355] This conditioned pool is applied offline to AX capture chromatography using a peristaltic pump. The AX capture column is equilibrated with two column volumes (CV) (20 mM Tris pH 7.4, 0.1% Tween 80, 25 mM NaCl) at a linear pump speed of 200 cm / h.

[0356] After loading, the column is washed offline with equilibration buffer for 5 CV, then transferred to the chromatography system and washed again for 5 CV. Annexin A5 elution is performed by stepwise elution for 9 CV using high-concentration salts (20 mM Tris pH 7.4; 0.1% Tween 80; 300 mM NaCl). Fractionation is performed in 0.1 absorption unit (AU) increments using UV elution. 280nm The signal is defined from the rise to the 0.2 AU peak at the descending peak. The total elution peak is further processed.

[0357] The column is regenerated and cleaned using a two-step CIP procedure (Step 1: 3 CV in 2M NaCl, 100 cm / h; upward flow; Step 2: 3 CV in 1M NaOH; incubation for over 15 hours, 40 cm / h upward flow). The column is then finally stored in 20 mM NaOH.

[0358] Figure 3 provides a process flowchart for AX capture chromatography.

[0359] In general, the capture step can be considered a conditioning step that enhances the performance of the intermediate step. It concentrates the product and significantly alters the matrix of the load. Furthermore, a strong reduction of endotoxins (approximately 97%) and a moderate reduction of DNA and HCP were observed.

[0360] 1.1.3 Intermediate affinity chromatography The resulting AX elution pool (250 mL / L) USP The solution was filtered before intermediate chromatography (Sartopore2 0.45~0.2 μm).

[0361] The filtered AX pool was then diluted eightfold (dilution buffer: 20 mM Tris pH 7.4; 0.1% Tween 80; 2 mM CaCl2). Dilution with calcium allows annexin A5 to bind to immobilized heparin chromatography. This interaction is slower compared to ionic interactions. Contact time is critical, and therefore chromatography should be performed at a rate of 100 cm / h or less.

[0362] Two washing steps are performed. Washing step 1 is carried out with 15 CV (20 mM Tris pH 7.4; 0.1% Tween 80; 2 mM CaCl2), followed by a second washing step with 2 CV using a calcium-free buffer (20 mM Tris pH 7.4; 0.1% Tween 80).

[0363] Elution is performed by stepwise elution using a buffer containing EDTA (20 mM Tris pH 7.4; 0.1% Tween 80; 10 mM EDTA; 25 mM NaCl) to chelate calcium ions. The chelation reaction specifically elutes annexin A5, which can only bind to heparin in the presence of calcium. To enable concentrated elution, the flow rate was reduced to 60 cm / h or less during elution. The complete elution peak was collected, for example, starting with an increase in the UV signal at 0.05 AU and ending at a descending peak at 0.05 AU, which corresponds to approximately 7 CV. The elution profile shows a single sharp peak.

[0364] The column is regenerated and cleaned using a two-step CIP procedure (Step 1: 3 CV in 2M NaCl, 100 cm / h; upward flow; Step 2: 3 CV in 0.1M NaOH; incubation for over 15 hours, 40 cm / h upward flow). The column is finally stored in 1M NaCl in 25% EtOH.

[0365] Figure 1 shows a process flowchart for intermediate affinity chromatography.

[0366] The intermediate step is the most powerful purification step in this process scheme. Annexin A5 binds to calcium ions. In this calcium-bound state, the product can form a highly specific bond with heparin. Only the correctly folded annexin A5 form, which has the ability to complex with calcium, can bind to heparin. Thus, the affinity chromatography step can distinguish between the correctly folded and misfolded products. In addition, the intermediate step achieves a high removal coefficient when the highly specific interaction is combined with a specific elution mode by the chelation reaction of calcium with EDTA. Therefore, a strong reduction of endotoxins (a further reduction of approximately 99%) and HCP is observed, along with a moderate reduction in DNA content.

[0367] The combination of the endotoxin reduction effects of the first AX capture step (approximately 97%) and the intermediate affinity chromatography step (approximately 99%) provides an annexin A5 product in which the endotoxin level is reduced to approximately 0.03% of the level in the clarified product prior to the first AX step.

[0368] 1.1.4 Refining AX chromatography The resulting AF elution pool (300 mL / L) USP The solution is diluted 2-fold (35 mM Tris pH 8; 0.1% Tween 80; 12.5 mM MgCl2) and filtered before refining chromatography (Sartopore 2 0.45-0.2 μm). Dilution reduces the conductivity of the AX load and also causes free EDTA molecules to complex with Mg ions. Otherwise, free EDTA will bind to the column, thereby reducing the volume in this step, and also reducing separation.

[0369] The refined resin is Source15 Q, which has an average resin diameter of 15 μm. It is a highly soluble refined resin with the disadvantage of high back pressure. Therefore, chromatography is performed at 100 cm / h. To allow for proper solubility, the loading should be less than 16 g / L of resin.

[0370] Washing after loading is performed with buffer A (20 mM Bis-Tris pH 7; 25 mM NaCl) over 3 CV. Elution is performed using a linear gradient from 33 CV to 100% B (20 mM Bis-Tris pH 7; 180 mM NaCl). This chromatographic step is primarily designed for the removal of product-related impurities. Various forms of annexin A5 elute from 40–100% B, starting with a main peak, followed by a second reduced peak and several smaller peaks.

[0371] Starting at 0.05 AU, the first main peak is collected up to the valley between Peak 1 and Peak 2 (corresponding to approximately 7 CV).

[0372] Perform a two-step CIP procedure to regenerate and clean the column (Step 1: 3 CV in 2M NaCl, 100 cm / h; upward flow; Step 2: 3 CV in 1M NaOH; incubation for over 15 hours, 40 cm / h upward flow). Finally, store the column in 25 mM NaCl.

[0373] Recent results from small-scale experiments suggest a positive effect of Tween80. Processes using 0.1% Tween80 increased the product yield after the intermediate process by approximately 30%. To ensure adequate solubility, the load on the refining process is limited to 16 g / L resin. The improved yield also has implications for scaling up scenarios. The calculated column dimensions for the refining process were designed for two cycles. 100L USP When processing a total volume from a large scale, a scenario of 4-5 cycles is required to achieve increased yield.

[0374] Figure 2 shows a process flowchart for the AX refining chromatography step.

[0375] The refining process is primarily carried out to reduce impurities associated with the product, such as the separation of various annexin A5 isoforms. In addition, the refining process achieves the highest removal coefficient in the process for residual DNA and strongly reduces HCP. Endotoxins, which are already at low levels after the intermediate process, are further reduced by approximately 99%, bringing the endotoxin level down to about 0.0003% of the level in the clarified product before the first AX process.

[0376] 1.1.5 Ultrafiltration / diafiltration and formulation of Annexin A5 To increase the product concentration and change the buffer, transfer the AX pool directly to UF / DF. Following the buffer change, add Tween80 to a final concentration of 0.05%, and then filter the active pharmaceutical ingredient sterile.

[0377] In the first process step, the AX pool is concentrated 6 to 8 times. Subsequently, the buffer is changed in a formulation buffer (20 mM Bis-Tris, 150 mM NaCl, 1 mM CaCl2 pH 7 or pH 7.4) without Tween, at 8 to 10 diafiltration volumes. After the buffer change, a second concentration is performed to obtain a final concentration of 12 g / L. This allows for reaching a final concentration of 10 g / L after sterile filtration by adding the first washing solution to the cassette to a final concentration of 0.05% and then adding Tween 80. To minimize coating layer formation, the UF / DF step is performed at a low TMP of 0.9 to 1.1 bar.

[0378] Figure 3 shows a process flowchart for ultrafiltration / dialysis and formulation of Annexin A5.

[0379] Two target values ​​and tolerance criteria To evaluate the performance and scalability of this process compared to small-scale DSP execution, the following target values ​​and tolerances are defined. The target values ​​are defined based on commissioning IPC / bulk analysis. Key process steps are identified, and critical process parameters are characterized to enhance the reliability of process performance.

[0380] 2.1 Key Process Parameters Table 5 shows the key process parameters. Achieving the target range during the implementation of this process indicates a successful process scaling up. Table 5: Process parameters and their respective target values ​​for each process step. TIFF2023088961000011.tif159170

[0381] 2.2 Important Process Parameters High homogenization of the suspension before capture chromatography is crucial. To achieve this, the use of three homogenization cycles is preferable. Furthermore, increasing the temperature during homogenization is preferable to obtain a lysate temperature in the range of 37°C. This is important for the benzonase activity, which has a direct impact on the filtration process and capture performance.

[0382] Pooling from the refining process is also important, as this process is used to separate product-related impurities. To minimize the formation of a coating layer, the UF / DF process is carried out under moderate conditions with respect to TMP. Table 6: Important process parameters TIFF2023088961000012.tif58170

[0383] 2.3 In-Process Control Table 7 shows the in-process control. Achieving the target range during this process execution indicates a successful process scaling. The target range is set based on observations from the previous small-scale execution, which was done solely through implemented changes. TIFF2023088961000013.tif234170TIFF2023088961000014.tif246170TIFF2023088961000015.tif244170TIFF2023088961000016.tif238170 TIFF2023088961000017.tif232170TIFF2023088961000018.tif224170TIFF2023088961000019.tif238170TIFF2023088961000020.tif111170

[0384] 3 Conclusion The manufacturing process described above is well-suited for large-scale production without bottlenecks, even for volumes of 10,000L or more, if required.

[0385] Following commissioning at a scaled-up 200L GMP experimental plant, the process delivered a product containing 1.8 pg of host cell DNA per 1 mg of AnxA5 protein, 16.6 ng of host cell protein per 1 mg of AnxA5 protein, and 0.1 EU per 1 mg of AnxA5 protein.

[0386] Throughout this manufacturing process, the annexin A5 protein is maintained in its active form in solution unless it is transiently bound to the chromatography resin.

[0387] When applied to a 1,000L culture, the overall process time in the manufacturing plant would be one week for fermentation, harvesting, cell disruption, and processing before chromatography, and one consecutive week for downstream processing. The entire process under GMP would take two weeks. This is scale-independent, perfectly conforms to industrial standards, and will be adaptable to any CMO or pharmaceutical manufacturer.

[0388] As mentioned above, in comparison, Marder et al.'s process would take approximately 12 weeks to process a 1,000L culture.

[0389] Furthermore, the yield in the present invention's process is 2 to 3 times higher per batch than that of Marder et al. This means that the manufacturing cost per gram of Annexin A5 active pharmaceutical ingredient is (6 - 8 × 2 - 3 =) 12 to 24 times higher in Marder's process.

[0390] Furthermore, in addition to providing a faster and higher yield purification process than Marder et al.'s process, the process of the present invention also provides a higher purity product. As discussed above in Comparative Example 1, the purity of the protein from Marder's process would not be suitable for human use. Despite careful centrifugation, Marder's process uses only one anion exchange chromatography step, which falls far short of the requirements for achieving sufficient purity with respect to both in-process impurities (particularly endotoxins) and product-related variants. Marder does not provide any data regarding endotoxin levels or other impurities, further suggesting a lack of suitability for pharmaceutical use.

[0391] In contrast, the process of this application provides a highly pure annexin A5 protein product having the following enumerated characteristics: - Typically, the concentration is around 8-12 g / L. - Host cell protein levels of less than 100 ng / mg, more typically less than 20 ng / mg (determined by ELISA), - Host cell DNA levels of 100 pg / mg or less, more typically less than 10 pg / mg. - Endotoxins less than 35 EU / mg, more typically less than 1 EU / mg. - Purity of over 95% determined by size exclusion chromatography. Bioburden less than -1 cfu / mL (as determined by Ph.Eur.2.6.12), - A colorless and transparent appearance without visible particles, and - The main band detected by Western plot analysis corresponds to the Annexin A5 reference.

[0392] This process was repeated for the second refining step using Capto Q ImpRes instead of Source 15Q. This provided a more efficient process because the Capto Q ImpRes anion exchange resin has a high binding capacity, can withstand high flow rates without back pressure, can be filled at higher bed heights, and is less expensive. The quality and purity of the final product were maintained.

[0393] Compared to Source 15Q, Capto Q ImpRes resin has the following characteristics: ●Grams / liter resin unit, more than double the capacity ● Withstands more than twice the flow velocity at the same back pressure. ●Typically, they can be filled with a higher bed height, approximately 35-60% higher, which provides a higher capacity for any given column bed area, and ● The cost is less than half the purchase price per liter of resin.

[0394] Example 2 This example illustrates a comparison of anion exchange (AX) capture and affinity capture by heparin chromatography.

[0395] AX capture and heparin affinity capture chromatography were compared in terms of annexin A5 step yield and purity in the capture eluate. Both strategies were compared in batch experiments under optimized conditions.

[0396] Test parameters: AX chromatography: Batch mode 500 μl resin (75% slurry) Buffer solution AX A: 20 mM sodium phosphate, pH 7, 5 mM EDTA, 250 mM NaCl Buffer AX B: 20 ​​mM sodium phosphate, pH 6.5, 5 mM EDTA AX chromatography: Load: 10 mL of pre-filtered lysate CIP: 1M ​​NaOH Heparin affinity chromatography: Batch mode 500 μl resin (75% slurry) Load: 10 mL of pre-filtered lysate; + 10 mM CaCl2 Buffer solution AF A: 50mM Tris pH 7.4, 5mM CaCl2 Buffer solution AF B: 50mM Tris pH 7.4, 40mM EGTA, 50mM NaCl CIP: 3M NaCl Table 7: Comparison of AX and heparin affinity chromatography capture TIFF2023088961000021.tif64170

[0397] These results demonstrate that AX capture prior to affinity chromatography does not significantly improve purity, but has a considerable impact on the yield of the heparin process. Furthermore, expensive affinity resins have a long lifespan when used as an intermediate step. Yield is therefore extremely important.

[0398] Capture by AX can be considered a conditioning step that enables the effective use of the highly specific affinity step by heparin chromatography.

[0399] Example 3 The partially purified annexin A5 product was obtained using the same method as in Example 1 up to the first anion exchange chromatography capture step.

[0400] In short, recombinant E. coli expressing annexin A5 was resuspended with 3200 U of bezonase in homogenization buffer (50 mM Tris, 1 mM MgCl, 1% Tween 20 pH 7.5) and homogenized for 3 cycles at 600 bar pressure. The temperature after homogenization was measured at 36°C. A clarification step was performed using Cuno 60 SP 0.6-0.2 μm, and the clarified solution was diluted 1:2 in 1% Tween 20 by adding EDTA. After conditioning for capture, the solution had a pH of 6.9 and a conductivity of 2.7 mS / cm. Anion exchange was performed using Q Sepharose XL (GE), followed by washing with buffer A (20 mM Tris, 25 mM NaCl, 0.1% Tween 20, pH 7.4), and then elution with buffer B (20 mM Tris, 300 mM NaCl, 0.1% Tween 20, pH 7.4). The resulting product was then sterile filtered through a 0.2 μm filter.

[0401] The resulting sterile filtered anion exchange product was purified using heparin affinity chromatography, and various conditions were tested.

[0402] The heparin affinity chromatography conditions used were as follows: TIFF2023088961000022.tif65170

[0403] The captured elution pool was sterile filtered using a Sartopore 2 filter with a diameter of 0.45–0.2 μm. This pool was diluted eightfold with (1050 mL) buffer A from heparin chromatography. The resulting pool had a pH of 7.4 and a conductivity of 6.8 mS. Elution was performed using a reduced flow rate of 60 cm / h.

[0404] To determine the effect of Tween80, tests 1 and 2 were performed using buffer A for washing and buffer B for elution. Test 1: Buffer A: 20 mM Tris, 25 mM NaCl, 2 mM CaCl2, 0.1% Tween20, pH 7.4 Buffer B 20mM Tris, 10mM EDTA, 25mM NaCl, 0.1% Tween20, pH7.4 Test 2: Buffer A: 20mM Tris, 25mM NaCl, 2mM CaCl2, 0.1% Tween20, 0.1% Tween80, pH 7.4 Buffer B: 20 ​​mM Tris, 10 mM EDTA, 100 mM NaCl, 0.1% Tween 20, 0.1% Tween 80, pH 7.4 The results for Test 1 are shown in Figure 7A, and the results for Test 2 are shown in Figure 7B.

[0405] The results show that, in contrast to the eluate separated under standard conditions (Test 1), the addition of Tween80 shifted the eluate to a single peak. Tween80 appears to stabilize annexin A5. A possible explanation for this change in elution behavior is that theoretical precipitation on the column is prevented. Two main positive effects of using Tween80 in the heparin affinity chromatography process can be noted. - Reduced pressure: The pressure on the column, which increased from 0.5 to 2-3 bar under load, was clearly reduced to 0.5 bar. This is particularly beneficial for large-scale applications. Some sedimentation observed after long-term incubation may be the reason for this pressure increase. - Prevention of precipitation: A second positive effect is observed in elution. Of the eluate fractions, the highly concentrated main peak elution fraction tends to precipitate. Assuming this effect is related to the very high concentration of annexin A5 in the main peak, no further precipitation was observed after pooling of the elution fraction, and moreover, the precipitation appeared to be reversible. Precipitation in the main peak could not be prevented even by increasing the salt concentration in the eluate. In contrast, the addition of Tween80 also prevented the formation of precipitate in the main peak elution fraction. Based on these results, the additional addition of Tween80 to all intermediate chromatography buffers appears advantageous because it has a stabilizing effect on annexin A5.

[0406] Example 4 The partially purified annexin A5 product was obtained using the same method as in Example 1 up to the first anion exchange chromatography capture step.

[0407] The 1.25 mL product from the anion exchange step was then mixed with 8.75 mL of various forms of test dilution buffer. The mixture was then incubated at ambient temperature and visually evaluated after 30 minutes, 18 hours, and 4 days.

[0408] The results are shown in the table below. TIFF2023088961000023.tif192170

[0409] These results demonstrate the benefit of adding Tween 80 (i.e., polysorbate 80) in avoiding product precipitation in the sample. This is important in relation to conditioning AnxA5 products before application to chromatographic columns such as affinity chromatography columns, in order to reduce precipitation and prevent an increase in back pressure when delivering the solution to the column.

Claims

1. 1. A process for the recovery and / or purification of a recombinantly expressed intracellular protein comprising the sequence of annexin A5 (AnxA5) from endotoxin-producing host cells having a cell wall, said process comprising releasing said intracellular protein from said host cells; The step of releasing the intracellular AnxA5 protein is carried out in the presence of a homogenization buffer containing a non-ionic detergent; Preferably, the process does not include any centrifugation step for the recovery and / or purification of the AnxA5 protein after its release from the host cells, and / or the AnxA5 protein remains in solution throughout the process except when temporarily bound to any chromatographic resin.

2. 2. The process of claim 1, wherein the non-ionic detergent is a polysorbate, preferably a polysorbate selected from Tween 20 and Tween 80, most preferably Tween 80.

3. The process of claim 1 or 2, wherein the step of releasing the intracellular AnxA5 protein is carried out in the presence of a homogenization buffer containing an amount of non-ionic detergent effective to reduce or prevent the binding between annexin A5 and endotoxin.

4. 4. The process according to any one of claims 1 to 3, wherein the step of releasing the intracellular AnxA5 protein is carried out in the presence of a homogenization buffer comprising 0.01 to 10% (w / w) non-ionic detergent, for example 0.02 to 5% (w / w), 0.05 to 2% (w / w), or about 1% (w / w) non-ionic detergent.

5. the process includes releasing the intracellular AnxA5 protein from the host cell; the concentration of free calcium ions in the homogenization buffer at the time of releasing the intracellular AnxA5 protein from the host cells or after releasing the intracellular AnxA5 protein from the host cells but before any further chromatographic purification is less than 10 mM, preferably less than 5 mM, less than 1 mM, more preferably less than 500 μM, or substantially zero; and / or 5. A process according to any one of claims 1 to 4 for the recovery and / or purification of recombinantly expressed intracellular proteins comprising the sequence of annexin A5 (AnxA5) from host cells having a cell wall, wherein the homogenization buffer contains or is modified to contain a calcium metal ion chelator after the intracellular AnxA5 protein has been released.

6. 6. The process of claim 5, wherein the calcium metal ion chelator is selected from EDTA or a salt thereof, EGTA or a salt thereof, most preferably EDTA.

7. The process of claim 5 or 6, wherein the level of free calcium ions and / or the amount of calcium metal ion chelator is effective to reduce or prevent the binding between annexin A5 and components of the cell wall of the host cell.

8. 8. The process according to any of claims 5 to 7, wherein the homogenization buffer comprises or is adjusted to comprise (before or after the release of the AnxA5 protein) 0.01 to 500 mM, e.g., 0.05 to 100 mM, 0.5 to 20 mM, 1 to 15 mM, 2 to 10 mM, or about 4 mM calcium metal ion chelator, preferably wherein the calcium metal ion chelator is EDTA.

9. 9. The process of claim 5, 6, 7 or 8, wherein the homogenization buffer comprises a non-ionic detergent according to any of the processes of claims 2, 3 or 4.

10. 10. The process of claim 9, wherein the calcium metal ion chelator is EDTA.

11. 11. The process of claim 9 or 10, wherein the non-ionic detergent is Tween 80.

12. 12. The process of any one of claims 1 to 11, wherein the process comprises the recovery and / or purification of recombinantly expressed intracellular AnxA5 protein from a culture of the host cells, the culture having a volume of at least 100 L, 500 L, 1,000 L, 5,000 L, or 10,000 L.

13. 13. The process of any of claims 1 to 12, comprising mixing biomass from said culture of host cells in said homogenization buffer at a concentration of about 10 g biomass per mL of homogenization buffer.

14. 14. The process of any of claims 1 to 13, wherein the step of releasing the intracellular AnxA5 protein from the host cells in the homogenization buffer comprises lysing, disintegrating, homogenizing, sonicating or pressure-treating the host cells so that the cell wall and cell membrane barriers of the host cells are disrupted, thereby releasing the intracellular AnxA5 protein, and optionally this step does not include the use of osmotic shock and / or a freeze-thaw step.

15. The process of claim 14, wherein the step of releasing the intracellular AnxA5 protein from the host cells comprises high-pressure homogenization, for example, one or more cycles of high-pressure homogenization at about 400 bar to about 2,500 bar, preferably three cycles of homogenization at about 600 bar, or two cycles of homogenization at about 800 bar.

16. 16. The process of any of claims 1 to 15, wherein the step of releasing the intracellular AnxA5 protein produces a biomass homogenate comprising the released AnxA5 protein.

17. 17. The process of claim 16, wherein the biomass homogenate further comprises one or more (typically all) of the impurities selected from the group consisting of host cell proteins, host cell wall components, host cell membranes, host cell nucleic acids, and endotoxins.

18. 18. The process of claim 16 or 17, further comprising clarifying the biomass homogenate, thereby producing a clarified product comprising the released AnxA5 protein.

19. 19. The process of claim 18, wherein the step of clarifying the biomass homogenate comprises treating the homogenate with a nuclease, such as nuclease A, preferably nuclease A from Serratia marescens, and optionally the nuclease is included in the homogenization buffer prior to release of the intracellular AnxA5 protein.

20. The process of claim 18 or 19, wherein the step of clarifying the biomass homogenate (preferably following the nuclease treatment of claim 18 or 19) comprises passing the biomass homogenate containing the released AnxA5 protein through a filter (such as a cellulose or polypropylene filter, preferably the filter is a depth filter and / or preferably the filter has a cut-off of less than 4 μm), and the filter effluent is the clarified product containing the released AnxA5 protein.

21. 21. The process of any preceding claim, further comprising subjecting the released AnxA5 protein to an anion exchange resin to perform a first anion exchange step, thereby producing a first anion exchange product comprising the released AnxA5 protein.

22. The process of claim 21, wherein the clarified product containing the released AnxA5 protein produced by the method of claim 20 is subjected to the first anion exchange step, thereby producing a first anion exchange product containing the released AnxA5 protein.

23. The process of claim 21 or 22, wherein prior to the first anion exchange step, one or more parameters of the environment of the released AnxA5 protein are adjusted, selected from the group consisting of pH, conductivity, level of calcium ion chelator and level of non-ionic detergent.

24. 24. The process according to any of claims 21 to 23, wherein the released AnxA5 protein subjected to the anion exchange step is formulated at a pH of about 6.9, a conductivity of about 2.8 mS / cm, a calcium ion chelator concentration of about 1 mM and diluted using a non-ionic detergent, e.g. to obtain a final non-ionic detergent concentration of 0.01 to 1% (w / v), more preferably about 0.1% (w / v).

25. 25. The process of any one of claims 21 to 24, wherein the AnxA5 protein is bound during the anion exchange step, and the first anion exchange product comprising the released AnxA5 protein is produced by applying a wash solution and / or elution buffer to the anion exchange resin to release the bound AnxA5 protein, and optionally the elution buffer comprises NaCl, for example about 300 mM NaCl.

26. 1. A process for the recovery and / or purification of a protein comprising the sequence of annexin A5 (AnxA5) from a solution containing said AnxA5 protein and one or more impurities, the process comprising: subjecting the solution containing the AnxA5 protein and one or more impurities to an anion exchange resin to perform a first anion exchange step, thereby producing a first anion exchange product containing the released AnxA5 protein; subjecting the first anion exchange product directly or indirectly to an affinity chromatography step, thereby producing a first affinity chromatography product comprising the released AnxA5 protein; Preferably, said AnxA5 protein remains in solution throughout said process, including any preceding or subsequent steps, except when transiently bound to any chromatographic resin.

27. 27. The process of claim 26, wherein the affinity chromatography step comprises binding of the AnxA5 protein to immobilized heparin, the binding being facilitated by the presence of calcium ions.

28. 28. The process of claim 27, wherein the AnxA5 protein is eluted from the immobilized heparin using an elution buffer containing a calcium ion chelator such as EDTA.

29. 1. A process for the recovery and / or purification of a protein comprising the sequence of annexin A5 (AnxA5) from a solution containing said AnxA5 protein and one or more impurities, the process comprising: subjecting the solution containing the AnxA5 protein and one or more impurities to a heparin affinity chromatography step in the presence of Tween 80 (preferably in the presence of 0.1% Tween 80), thereby producing a first affinity chromatography product containing the released AnxA5 protein; Preferably, said AnxA5 protein remains in solution throughout said process, including any preceding or subsequent steps, except when transiently bound to any chromatographic resin.

30. 26. The process of any preceding claim, further comprising subjecting the released AnxA5 protein to an affinity chromatography step, thereby producing a first affinity chromatography product comprising the released AnxA5 protein.

31. 31. The process of claim 30, wherein the AnxA5 protein in the first anion exchange product produced by the method of any one of claims 21 to 25 is subjected to the affinity chromatography step.

32. (a) a biomass homogenate comprising the released AnxA5 protein of claim 16 or 17 is clarified by the process of any of claims 18, 19 or 20, thereby producing a clarified product comprising the released AnxA5 protein; (b) performing a first anion exchange step according to any one of claims 21 to 25, whereby the AnxA5 protein in the clarified product is subjected to an anion exchange resin to produce a first anion exchange product comprising the AnxA5 protein; (c) subjecting the AnxA5 protein in the first anion exchange product to an affinity chromatography step according to claim 30 or 31.

33. 33. The process of any of claims 30 to 32, wherein the affinity chromatography step comprises binding of the AnxA5 protein to immobilized heparin, optionally wherein the binding is facilitated by the presence of calcium ions.

34. 34. The process of claim 33, wherein the AnxA5 protein is eluted from the immobilized heparin using an elution buffer containing a calcium ion chelator such as EDTA.

35. 35. The process of any of claims 30 to 34, wherein the first affinity chromatography product comprises the released AnxA5 protein and, optionally, a calcium ion chelator such as EDTA or EGTA in the range of 0.1 to 500 mM, more preferably about 10 mM.

36. 1. A process for the recovery and / or purification of a protein comprising the sequence of annexin A5 (AnxA5) from a composition comprising said AnxA5 protein and a calcium metal ion chelator, comprising: the process comprises subjecting the composition to an anion exchange resin to perform an anion exchange step, thereby recovering and / or purifying the AnxA5 protein from the composition; further characterized in that the anion exchange step is carried out in the presence of an additional selected metal ion; the additional selected metal ion is selected such that the calcium metal ion chelator has a binding affinity for the selected metal ion that is greater than its binding affinity for the anion exchange resin but less than its binding affinity for calcium ions; Preferably, said AnxA5 protein remains in solution throughout said process, including any preceding or subsequent steps, except when transiently bound to any chromatographic resin.

37. 37. The process of claim 36, wherein the calcium metal ion chelator is selected from EDTA or a salt thereof, EGTA or a salt thereof, most preferably EDTA.

38. 38. The process of claim 36 or 37, wherein the calcium metal ion chelator is present in the composition at a concentration of about or greater than at least 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM or more.

39. The selected metal ion is Mg ２＋ 39. The process according to any one of claims 36 to 38, wherein the cation is a divalent cation such as an ion.

40. 40. The process of any of claims 36-39, wherein the selected metal ion is present in an amount effective to reduce or prevent interaction between the calcium ion chelator and the anion exchange resin during the process of subjecting the composition to the anion exchange resin during the anion exchange step.

41. 41. The process of any one of claims 36 to 40, wherein the selected metal ion is present during the anion exchange step in an amount effective to increase binding of the AnxA5 protein to the anion exchange resin in the presence of the calcium ion chelator, thereby reducing loss of AnxA5 protein in the flow-through fraction of the anion exchange step compared to the level of loss observed when the selected metal ion is not present during the anion exchange step.

42. 42. The process of any of claims 36 to 41, wherein the selected metal ion is present during the anion exchange step at a concentration of about 1 to about 100 mM, e.g., about 2 to about 50 mM, about 5 to about 25 mM, about 10 to about 15 mM, or about 12.5 mM (e.g., by adding the selected metal ion to the composition comprising the AnxA5 protein and a calcium metal ion chelator prior to subjecting the composition to the anion exchange resin).

43. The calcium metal ion chelator is EDTA and the selected metal ion is Mg ２＋ ions, preferably Mg ２＋ 43. The process of any of claims 36 to 42, wherein the molar ratio of ions to EDTA is in the range of 0.5:1 to 2:1, most preferably at least 1:1 or higher.

44. The composition, which comprises the AnxA5 protein and a calcium metal ion chelator and is subjected to the anion exchange resin, 44. The process of any one of claims 36 to 43, which is a direct or indirect product of a preceding process, comprising the step of subjecting the AnxA5 protein to an affinity chromatography step and eluting the AnxA5 protein with a calcium ion chelator, thereby producing an affinity chromatography product, which is a composition comprising the AnxA5 protein and a calcium metal ion chelator.

45. 45. The process of claim 44, wherein the preceding affinity chromatography step comprises binding of the AnxA5 protein to immobilized heparin, optionally wherein the binding is facilitated by the presence of calcium ions.

46. 46. ​​The process of claim 45, wherein the AnxA5 protein is eluted from the immobilized heparin using an elution buffer containing a calcium ion chelator such as EDTA or EGTA.

47. 47. The process of any one of claims 44 to 46, wherein there is no dialysis step between the preceding affinity chromatography step and the anion exchange step and / or no removal of calcium ion chelator from the product of the preceding affinity chromatography step prior to the application of the direct or indirect product to the anion exchange step.

48. 48. The process of any one of claims 36 to 47, wherein the selected metal ions are added to the composition prior to or during the anion exchange step.

49. A process according to any one of claims 1 to 35, wherein the process comprises an anion exchange step according to any one of claims 36 to 48.

50. 13. A process for the recovery and / or purification of a recombinantly expressed intracellular protein comprising the sequence of annexin A5 (AnxA5) from a host cell having a cell wall or a culture thereof according to claim 12, comprising: (a) the process according to any one of claims 1 to 4, comprising releasing the intracellular proteins from the host cells in the presence of a homogenization buffer comprising a non-ionic detergent, (b) optionally, the releasing step comprises, in accordance with any one of claims 13 to 17, (c) further optionally, the process comprises clarifying the biomass homogenate according to any of claims 18 to 20; (d) performing a first anion exchange step according to any of claims 21 to 25, thereby subjecting the released AnxA5 protein directly or indirectly to an anion exchange resin, optionally in the presence of a calcium ion chelator, to produce a first anion exchange product comprising the released AnxA5 protein; (e) the process further comprises the step of subjecting the released AnxA5 protein to an affinity chromatography step directly or indirectly according to any of claims 26 to 35, (f) the product of the affinity chromatography step is a composition comprising the AnxA5 protein and a calcium metal ion chelator; (g) the direct or indirect product of the affinity chromatography step, comprising the AnxA5 protein and the calcium metal ion chelator, is subjected to an anion exchange step according to any one of claims 36 to 49; Preferably, none of steps (a) to (g) includes or is interrupted by one or more steps selected from centrifugation and / or dialysis, and more preferably, the AnxA5 protein remains soluble throughout the process except when transiently bound to anion exchange and affinity chromatography solid phases.

51. A process according to any one of claims 1 to 50, preferably comprising at the end of the process according to any one of claims 1 to 50 one or more further steps selected from the group consisting of concentration, buffer change, conditioning and filtration (such as sterile filtration), and optionally a final step of storing the AnxA5 protein-containing product in a sterile container.

52. 52. The process of claim 51, wherein one of the further steps is diafiltration, and optionally the product of the diafiltration step contains the AnxA5 protein at a concentration of at least about 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 50 100 mg / mL or more.

53. 53. The process of claim 51 or 52, wherein the filtration uses a 0.45-0.2 μm filter or a 0.22 μm filter, and is preferably a sterile filtration step.

54. 54. The process of any of claims 51 to 53, wherein sterile filtration is the final purification step before storing the AnxA5 protein-containing product in a sterile container.

55. The process is carried out in a solution of about 150 mM NaCl, about 1 mM CaCl ２ 55. The process of any of claims 1 to 54, comprising the steps required to provide a final sterile AnxA5 protein product in a phosphate-free buffer (such as Bis-Tris or Tris buffer) of about pH 7.4 containing about 0.05% (w / w) polysorbate (such as Tween 80) or other non-ionic detergent, optionally wherein the concentration of the AnxA5 protein in the final sterile AnxA5 protein product is about 10 mg / mL.

56. 56. The process of any of claims 1 to 55, wherein the process provides a final, sterile AnxA5 protein product, wherein the NaCl concentration present maintains the AnxA5 protein in a predominantly monomeric form.

57. 57. The process of any of claims 1 to 56, wherein the process provides an overall yield of greater than 1 g of AnxA5 protein per liter of host cell culture, more preferably at least about 1.5 g / L, and even more preferably in the range of about 2 to about 4 g / L.

58. 58. The process of any preceding claim, wherein the process provides an overall recovery of AnxA5 protein of about 24% by weight of the AnxA5 protein present in the host cell culture.

59. 59. The process of any preceding claim, wherein the process provides a product containing less than 100, 90, 80, 70, 60, 50, 40, 30, 20 ng or less of host cell protein (other than the recombinantly expressed AnxA5 protein) per mg of AnxA5 protein.

60. 60. A process according to any preceding claim, wherein the process provides a product with an endotoxin content of less than 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 35, 20, 15, preferably less than 10, 5 or 1 EU per mg of AnxA5 protein; and / or preferably, the process provides a product in unit dosage form, the product containing less than 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 35, 20, 15, preferably less than 10, 5 or 1 EU per unit dose.

61. 61. A process according to any preceding claim, wherein the process provides a product comprising host cell nucleic acid levels of less than 1,000 pg / mg of AnxA5 protein, preferably less than 100 pg / mg of AnxA5 protein, more preferably less than 10 pg / mg of AnxA5 protein.

62. 62. A composition comprising AnxA5 protein, said composition being the direct or indirect product of (or obtainable directly or indirectly by) the process of any of claims 1 to 61.

63. 63. The composition of claim 62, wherein the composition is the direct or indirect product of (or is obtainable directly or indirectly by) the process of claim 50, or claim 51 when dependent on claim 50.

64. 64. The composition of claim 62 or 63, containing the AnxA5 protein at a concentration of at least about 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 50 100 mg / mL or more.

65. 65. The composition of any of claims 62 to 64, wherein the composition has been subjected to a sterile filtration process and / or is a sterile composition.

66. 66. The composition of claim 65, stored in a sterile container.

67. The composition contains about 150 mM NaCl, about 1 mM CaCl ２ 67. The composition of any of claims 62 to 66, comprising a sterile AnxA5 protein product in a phosphate-free buffer (such as Bis-Tris or Tris buffer) of about pH 7.4 containing about 0.05% (w / w) polysorbate (such as Tween 80) or other non-ionic detergent, optionally wherein the concentration of the AnxA5 protein in the final sterile AnxA5 protein product is about 10 mg / mL.

68. 68. The composition of any of claims 62-67, wherein the NaCl concentration present maintains the AnxA5 protein in a form that is predominantly monomeric.

69. A composition described in any of claims 62 to 68, comprising non-AnxA5 proteins such as host cell proteins at a level of less than 20 ng per mg of AnxA5 protein, optionally wherein the host cells are prokaryotic cells such as gram-positive or gram-negative cells, and in particular endotoxin-producing gram-negative bacterial cells, and further optionally wherein the host cell proteins are at detectable levels in the composition (despite being less than 20 ng per mg of AnxA5 protein).

70. 70. The composition of any of claims 62 to 69, comprising an endotoxin content of less than 100, 50, 20, 10, 5 or 1 EU per mg of AnxA5 protein, and optionally endotoxin is at a detectable level in the composition (despite being less than 100, 50, 20, 10, 5 or 1 EU per mg of AnxA5 protein).

71. 71. The composition of any of claims 62 to 70, wherein the product is in unit dosage form and contains less than 100, 50, 20, 10, 5 or 1 EU per unit dose, and optionally endotoxin is at a detectable level in said composition (despite being less than 100, 50, 20, 10, 5 or 1 EU per unit dose).

72. 72. The composition of any one of claims 62 to 71, comprising a nucleic acid level, such as a host cell nucleic acid level, of less than 1,000 pg, 100 pg, or 10 pg per mg of AnxA5 protein, optionally wherein the host cell is a prokaryotic cell, such as a Gram-positive or Gram-negative cell, and in particular an endotoxin-producing Gram-negative bacterial cell, and further optionally wherein the host cell nucleic acid is at a detectable level in the composition (despite being less than 1,000 pg, 100 pg, or 10 pg per mg of AnxA5 protein).

73. 73. The composition of any of claims 62 to 72, wherein the level of gluconoylated AnxA5 protein in the composition is within the range of 0.5 to 30%, or 0.5 to 20%, or 0.5 to 15%, or 0.5 to 10% of the total content of AnxA5 protein in the product.

74. A composition described in any of claims 62 to 73, wherein the level of gluconoylated AnxA5 protein in the composition is less than 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, or 1%, and preferably substantially 0%.

75. The composition of any of claims 62 to 74, wherein the AnxA5 protein does not contain a His tag and / or does not contain one or more RGD motifs.

76. The composition according to any one of claims 62 to 75, wherein the composition is a pharmaceutically and / or veterinarily acceptable composition.

77. A composition according to any one of claims 62 to 76 for use in medicine.

78. 80. A method of treating a human or animal subject in need of treatment comprising administering to said human or animal subject a composition according to any of claims 62 or 76.