Process of manufacture of annexin v

Abstract

To provide processes for the manufacture of protein comprising the sequence of Annexin A5 (AnxA5).SOLUTION: Provided is a process for recovery and / or purification of a recombinantly expressed intracellular protein comprising a sequence of Annexin A5 (AnxA5) from an endotoxin-producing host cell with a cell wall. The process comprises releasing the intracellular protein from the host cell, and is characterized in that the step of releasing the intracellular AnxA5 protein is conducted in presence of a homogenization buffer comprising non-ionic detergent. Therein, the process preferably does not include any centrifugation steps for the recovery and / or purification of the AnxA5 protein after the release thereof from the host cell, and / or the AnxA5 protein remains in solution throughout the process, except when temporarily bound to any chromatographic resins.SELECTED DRAWING: Figure 2

Description

[Technical field]

[0001] This application relates to a process for the production of a protein comprising the sequence of annexin A5 (AnxA5). More particularly, the process is for the recovery and / or purification of 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 having a culture volume of about 1000 L or more) to rapidly and conveniently produce pharmaceutical-grade AnxA5 protein products. [Background technology]

[0002] The listing or discussion herein of a document that is believed to be prior-published should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0003] Atherothrombosis, which forms from underlying atherosclerotic plaques, is the primary pathogenic mechanism behind the majority of clinically evident ischemic cardiovascular diseases, including acute coronary artery disease, cerebrovascular and peripheral arterial occlusion. As reviewed in Cederholm and Frostegard, 2007, Drug News Perspective., 20(5):321-6, annexin A5 (previously 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 mediated primarily by mechanical shielding of phospholipids, particularly phosphatidylserine, reducing their availability to the coagulation reaction. However, other interesting properties of annexin A5 that potentially contribute to its antithrombotic function have been reported, notably downregulation of surface-expressed tissue factor or interaction with additional ligands involved in homeostasis, such as sulfatide and heparin, as well as upregulation of urokinase-type plasminogen activator. The biological importance of annexin A5 as a member of an endogenous antithrombotic system in the body for the large vasculature and for the placental microcirculation has also been suggested.

[0004] Indeed, annexin A5 is known to have a wide range of utility in providing direct therapeutic effects in medicine, including, for example, the following uses of annexin A5: For the prevention of atherothrombosis and / or plaque rupture as described in WO2005 / 099744, the contents of which are incorporated herein by reference; For the treatment of vascular insufficiency, reduction of ischemic pain and / or treatment of vascular disease as described in WO2009 / 077764, the contents of which are incorporated herein by reference; For the prevention or treatment of restenosis as described in WO2009 / 103977, the contents of which are incorporated herein by reference; For use in inhibiting the activity of oxidized cardiolipin (oxCL) and for treating, preventing and / or reducing the risk of developing a cardiovascular disease, an autoimmune disease or an inflammatory condition, as described in WO2010 / 069605, the contents of which are incorporated herein by reference; and For the prevention and / or reduction of peri- or post-operative complications following surgical intervention, such as complications following vascular surgery, in particular peripheral vascular surgery, as described in WO2012 / 136819, the contents of which are incorporated herein by reference.

[0005] Thus, annexin A5 represents a protein of great therapeutic interest and potential. Accordingly, 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 and conveniently applied to commercial scale production (e.g., to harvest annexin A5 protein from recombinant host cell cultures having culture volumes of about 1000 L or more).

[0006] A particular challenge in recombinantly expressing annexin A5 in standard bacterial host cells such as E. coli is contamination with host cell-derived components, especially endotoxins. Endotoxins are lipopolysaccharides (LPS) formed of covalently linked lipids and polysaccharides composed of O-antigens, outer cores and inner cores. LPS is found in the outer membrane of Gram-negative bacteria and elicits a strong immune response in animals. Annexin A5 is characterized by strong binding to biological membranes containing negatively charged phospholipids, and thus 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 is no process available for producing therapeutic grade annexin A5 protein by an efficient and cost-effective process that can be scaled up and conveniently adapted for commercial-scale production (e.g., to harvest annexin A5 protein from recombinant host cell cultures having culture volumes of about 1000 L or more), much less in a manner that addresses 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 entitled "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 100 mL culture flasks, pelleting the cells, resuspending them in the presence of a homogenization / lysis buffer consisting of 50 mM Tris-HCl, 10 mM CaCl2, pH 7.2, and then disrupting the cells via sonication to release the Annexin A5 protein. Addition of CaCl2 caused binding of Annexin A5 to the cell membrane in the cell debris in a calcium-dependent manner, and the mixture was then subjected to a first purification centrifugation step for 20 minutes, after which the supernatant was discarded and the pellet containing the cell debris and the bound Annexin A5 was collected. Annexin A5 was released from the pellet using EDTA, followed by a second purification centrifugation step for 20 minutes and collection of Annexin A5 in the supernatant. This was then followed by overnight dialysis to change the buffer for Annexin A5 to Tris-HCl pH 8.0, followed by further steps of anion exchange on a DEAE-Sepharose column and elution of Annexin A5 using a salt gradient.

[0009] Applicant has found that Kumar's method has a number of limitations and shortcomings. First, it has only been demonstrated on a small scale using 100 mL cultures, and requires two separate centrifugation steps during the purification process. This cannot be scaled up in an efficient manner to commercial processes using high volume cultures (e.g., 1000 L or more). As discussed further below, centrifugation of such high volumes of fluid is extremely time-consuming and expensive. Yet, centrifugation is required as a preliminary capture step in Kumar's approach, which relies on using calcium-induced binding of annexin A5 to membranes in cell debris. Second, Applicant has found that Kumar's method leads to high losses of annexin A5 protein, for example, by disposing of unbound soluble annexin in the product supernatant of the first purification centrifugation step. Third, it is notable 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, the Kumar method is not capable of being scaled up for commercial production in an efficient and time-effective manner and leads to high losses (i.e., low yields) of Annexin A5 protein, resulting in low-grade protein purification that is not suitable for therapeutic use.

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

[0011] In 2014, a further method for purification of annexin A5 was proposed in "Production of recombinant human annexin V by fed-batch cultivation" by Marder et al., 2014, BMC Biotechnology, 14:33. Marder et al. report that the method is a fed-batch method for large-scale production of recombinant human annexin V, and it is proposed that this method may expand the commercial utility of recombinant human annexin A5, for example, for use in in vivo imaging studies.

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

[0013] The 1997 method by Kumar and the 2008 method by the Department of Laboratory Medicine at the University of Washington Medical Center, and the 2014 method by Marder clearly show that the art has developed and established approaches to the commercial production and purification of Annexin A5 products, but shortcomings in these methods have not been appreciated 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 adapted for scale-up or take into account industry standards or equipment available on a larger scale. The processes have inherent disadvantages that make them unsuitable for large-scale manufacturing. In particular, a very limiting feature of these prior art processes is the two or (in the case of the 2014 method by Marder et al.) three high G-force centrifugations that the processes require when Annexin A5 is alternatively in solution or as a precipitate. It can be reasonably estimated that applying only two centrifugation steps to the processing of one 1000L batch would result in a process that would require about 12 weeks of 12-hour shifts per day in any well-equipped biomanufacturing facility, resulting in unacceptably high production costs. See Comparative Example 1.

[0015] It is therefore an object of the present invention to provide methodological steps for efficient and cost-effective purification and recovery of annexin A5 for manufacturing processes operated on a commercial scale (e.g., recombinant host cell cultures having culture volumes of about 1000 L or more), 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 comprising the sequence of annexin A5 (AnxA5) 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 includes all of the developed process steps.

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

[0019] Thus, the present process can be applied to the processing of high volume (e.g., about 100 L, 500 L, 1,000 L, 5,000 L, 10,000 L, 50,000 L, 100,000 L or more) cultures of host cells in a very time-efficient manner without the bottlenecks caused by one or more purification centrifugation steps. For example, it may be preferred that the purification process be carried out in 5, 4, 3, 2 weeks or less, most typically in less than 1 week, per 1,000 L of host cell culture processed. Still further, the present process can be used to surprisingly provide improved yields and / or improved purity (including, for example, improved endotoxin removal) compared to the more time-consuming and less efficient prior art processes.

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

[0021] Applicants have also discovered (as discussed further below in Example 2) that, in contrast to conventional methods in which the loss of yield continues to increase with the successive addition of purification steps (as product is lost at each step), the combination of an anion exchange step and a heparin affinity chromatography step has the surprising benefit of achieving the high purity obtained with the heparin affinity chromatography step alone, but with substantially increased yield (i.e., recovery increases from about 30-40% to about 70-90%), which is the opposite of what would normally be expected from a combination of purification steps.

[0022] Thus, a second aspect of the invention provides a process for the recovery and / or purification of a protein comprising the sequence of annexin A5 (AnxA5) from a solution containing the AnxA5 protein and one or more impurities, the process comprising: subjecting the solution comprising 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 comprising the released AnxA5 protein; and 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.

[0023] Preferably, according to the process of the second aspect of the present invention, the affinity chromatography step may comprise binding of the AnxA5 protein to immobilized heparin, optionally which binding is 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 chelator such as EDTA.

[0024] In addition, as discussed in Example 3, Applicants have discovered that Tween 80 has a particularly advantageous effect on the heparin affinity chromatography step (compared to other non-ionic detergents, including other Tweens such as Tween 20). Incorporation of Tween 80, for example at around 0.1% (w / v), into the buffer used for the heparin affinity chromatography step can aid in elution of the AnxA5 protein in a single peak, reduce pressure, and prevent precipitation.

[0025] Thus, a third aspect of the present invention provides a process for the recovery and / or purification of a protein comprising the sequence of annexin A5 (AnxA5) from a solution comprising AnxA5 protein and one or more impurities, the method comprising subjecting a solution comprising 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 discussed 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 comprising released AnxA5 protein.

[0026] The fourth aspect of the present invention is based on the applicant's realization that calcium metal ion chelating agents (e.g., EDTA) can adversely affect the effectiveness of the anion exchange step. Free EDTA (or other chelating agents) can directly bind to the anion exchange functional groups, thereby reducing the capacity and even the separation achieved by the anion exchange step. On the other hand, attempts to remove calcium metal ion chelating agents before the anion exchange step waste time and therefore also increase costs. Thus, prior art methods involving slow dialysis steps for buffer exchange are inefficient. Furthermore, when incorporating calcium metal ion chelating agents into the AnxA5 product during the anion exchange step, they may be an important component to prevent calcium-mediated binding of AnxA5 protein to impurities, including endotoxins. Therefore, it would be convenient and effective to introduce an additive that blocks or reduces the binding of calcium ion chelating agents to the anion exchange resin, allowing the anion exchange step to be performed without the inconvenience and costs associated with dialysis and without blocking the beneficial effects of calcium metal ion chelating agents during the anion exchange step.

[0027] Applicants have realised that this may be achieved by incorporation of one or more additional selected metal ions into the AnxA5 protein product prior to anion exchange, the additional selected metal ions being 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. The selection of a suitable additional metal ion will depend on the nature of the calcium ion chelator and the nature of the anion exchange resin. For example, when EDTA is used as the calcium ion chelator, Mg 2+ Any ion is generally suitable for achieving the purposes of the present invention and may be added to the AnxA5 protein product prior to the anion exchange step.

[0028] Thus, a fourth aspect of the present invention relates to a process for the recovery and / or purification of a protein comprising the sequence of annexin A5 (AnxA5) from a composition comprising the AnxA5 protein and a calcium metal ion chelator, comprising: The process is characterized in that it comprises subjecting the composition to an anion exchange resin to perform an anion exchange step, whereby the AnxA5 protein is recovered and / or purified from the composition, The anion exchange step is further characterized in that it is carried out in the presence of an additional selected metal ion; This additional selected metal ion provides a process in which the calcium metal ion chelator is selected to have 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 invention provides a composition comprising an AnxA5 protein, the composition being a direct or indirect product of (or obtainable directly or indirectly by) a process according to any of the first, second, third or fourth aspects of the invention. Optionally, the composition is a pharma- ceutically acceptable and / or veterinarily acceptable composition.

[0030] A sixth aspect of the invention also provides a composition of the fifth aspect of the invention for use in medicine. Alternatively stated, the sixth aspect of the invention provides a method comprising administering to a human or animal in need of treatment a therapeutically effective amount of a composition of the fifth aspect of the invention.

[0031] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

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

[0033] These and further aspects of the invention will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. It should be understood, however, that the following description, while indicating various aspects and embodiments of the invention and numerous specific details thereof, is given by way of illustration and not by way of limitation. Many substitutions, modifications, additions and / or rearrangements may be made within the scope of the invention without departing from the spirit thereof, and the invention includes all such substitutions, modifications, additions and / or rearrangements. [Brief description of the drawings]

[0034] [Figure 1-1] The sequence shown is SEQ ID NO:1, which is the sequence of human annexin A5. [Figure 1-2] The sequence shown is SEQ ID NO:1, which is the sequence of human annexin A5. [Diagram 2] 1 shows a schematic flow diagram of an exemplary complete manufacturing process for Annexin A5. [Diagram 3] 1 provides a process flow diagram for an exemplary AX capture chromatography. [Figure 4] 1 shows a process flow diagram for an exemplary intermediate affinity chromatography. [Diagram 5] 1 shows a process flow diagram for an exemplary AX refining chromatography step. [Figure 6] 1 shows a process flow diagram for an exemplary ultra / diafiltration and formulation of Annexin A5. [Figure 7A] 7A shows the results of Example 3 demonstrating the effect of Tween 80 on heparin affinity chromatography purification of Annexin A5, of which FIG. 7A shows the results for Test 1 (without Tween 80) and FIG. 7B shows the results for Test 2 (with Tween 80). [Figure 7B] 7A shows the results of Example 3 demonstrating the effect of Tween 80 on heparin affinity chromatography purification of Annexin A5, of which FIG. 7A shows the results for Test 1 (without Tween 80) and FIG. 7B shows the results for Test 2 (with Tween 80). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] A. Annexin A5 Protein The present invention relates to methods for the purification and / or recovery of proteins comprising the sequence of annexin A5 (AnxA5), and to products and formulations thus produced, comprising the AnxA5 protein.

[0036] In one embodiment of the invention, the purified and / or recovered AnxA5 protein may comprise, consist essentially of, or consist of a protein having the sequence of human annexin A5 (SEQ ID NO:1 shown in Figure 1), with or without an N-terminal methionine.

[0037] In another embodiment, the purified and / or recovered AnxA5 protein may comprise, consist essentially of, or consist of a variant or mutant of a protein having the sequence of human annexin A5 (SEQ ID NO: 1 shown in FIG. 1), with or without an N-terminal methionine. For example, the variant or mutant may differ from SEQ ID NO: 1 at any one or more positions, for example, 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 an N-terminal methionine).

[0038] Thus, a variant or mutant of Annexin A5 may be a protein in which amino acid insertions, deletions or substitutions have been made at one or more positions, whether conservative or non-conservative. Preferably, the changes result in a protein whose basic properties are not significantly altered, which functions in a manner equivalent to Annexin A5. "Significantly" in this context means that the properties of the variant may still be different compared to those of the original protein, but are not unobvious.

[0039] Preferably, the isoelectric point (pI) of the variant or mutant is not altered or is not changed 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 compared to the unmodified protein.

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

[0041] By "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 made using protein engineering and site-directed mutagenesis techniques well known in the art.

[0043] In a further embodiment, the purified and / or recovered AnxA5 protein may be a dimer of a protein comprising, consisting essentially of, or consisting of a protein having the sequence of human annexin A5 (SEQ ID NO: 1 shown in Figure 1), with or without an N-terminal methionine, or a variant or mutant thereof as described above.

[0044] In a further embodiment, the purified and / or recovered AnxA5 protein may be a fusion protein, which comprises, consists essentially of, or consists 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 as shown in FIG. 1), with or without an N-terminal methionine, or a variant or mutant thereof as described above, or a dimer, (b) one or more protein sequences comprising, consists essentially of, or consists of a protein having the sequence of human annexin A5 (SEQ ID NO: 1 as shown in FIG. 1), with or without an N-terminal methionine, or a variant or mutant thereof as described above, or a dimer. For example, and without limitation, the fusion protein may have a general structure selected from the following: - in the case of a fusion of two amino acid sequences, for example H2N-(a)-(b)-COOH, or H2N-(b)-(a)-COOH, or - in the case of a fusion of three amino acid sequences, e.g. H2N-(a)-(b)-(a)-COOH, or H2N-(b)-(a)-(b)-COOH, or H2N-(a)-(b)-(b)-COOH, or H2N-(b)-(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-(b)-(a)-(a)-(a)-(a)-COOH, or H2N-(a)-(a)-(a)-(b)-(b)-COOH, or H2N-(a)-(a)-(b)-(a)-(b)-COOH, or H2N-(a)-(b)-(a)-(b)-COOH, or H2N-(b)-(a)-(a)-(b)-COOH, )-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)-(a)-(a)-COOH, or H2N-(b)-(b)-(a)-(b)-COOH, or H2N-(b)-(b)-(a)-(b)-COOH, or H2N-(b)-(a)-(b)-(b)-COOH, or H2N-(b)-(a)-(b)-(b)-COOH, or H2N-(a)-(b)-(b)-(b)-COOH, or - in the case of a fusion of five amino acid sequences, for example, H2N-(a)-(a)-(a)-(a)-(b)-COOH, or H2N-(a)-(a)-(a)-(b)-(a)-COOH, or H2N-(a)-(a)-(b)-(a)-(a)-(a)-COOH, or H2N-(a)-(b)-(a)-(a)-(a)-(a)-COOH, or H2N-(b)-(a)-(a)-(a)-(a)-(a)-COOH, or H2N-(a)-(a)-(a)-(a)-(a)-(b)-(b)-COOH, or H2N-(a)-(a)-(a)-(a)-(b)-(b)-COOH, or H2N-(a)-(a)-(a)-(a)-(b)-(a)-(b )-COOH, or H2N-(a)-(b)-(a)-(a)-(b)-COOH, or H2N-(b)-(a)-(a)-(a)-(b)-(a)-COOH, or H2N-(a)-(a)-(b)-(a)-(b)-(a)-COOH, or H2N-(a)-(b)-(a)-(b)-(a)-(a)-COOH, or H2N-(b)-(a)-(a)-(a)-(b)-(a)-COOH, or H2N-(a)-(b)-(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-(b)-(a)-(b)-(a)-(b)-COOH, or H2N-(b)-(a)-(b)-(a)-(b)-COOH, or H2N-(b)-(b)-(a)-(a)-(b)-COOH, or H2N-(b)-(b)-(a)-(a)-(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)-(a)-(a)-COOH, or H2N-(b)-(a)-(b)-(b)-(b)-(a)-(b)-COOH, or H2N-(b)-(b)-(a)-(b)-(b)-(a)-(b)-COOH, or H2N-(b)-(b)-(b)-(a)-(b)-COOH, or H2N-(b)-(b)-(b)-(a)-(b)-COOH, wherein (a) and (b) are as defined above in this paragraph. In the case of multiple fusion partner proteins 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 extending the half-life of the molecule in the patient's circulatory system and / or conferring additional functionality to the molecule, for example conferring additional therapeutic properties (e.g., anticoagulation, cell inhibition and / or killing, etc.). In the case of fusion proteins defined by (b) that include multiple protein sequences having the sequence of human annexin A5 (SEQ ID NO: 1 as shown in FIG. 1), with or without an N-terminal methionine, or a variant or mutant thereof as described above, or a dimer, the proteins may be the same or different.

[0045] In a further embodiment of the invention, the purified and / or recovered AnxA5 protein may be a protein comprising, consisting essentially of or consisting of a sequence of annexin A5 selected from the following, or a functional variant or mutant thereof: a) human annexin A5 (SEQ ID NO: 1), with or without the N-terminal methionine; b) the mammalian orthologue of human annexin A5; c) an allele or genetic variant of a) or b); d) a protein that is more than 50%, 60%, 70%, 75%, such as more than 80%, 85%, more than 90%, or even more preferably more than 95% or 99% identical to any of a), b) or c); e) a dimer of a), b), c), or d); or f) A fusion protein comprising one or more fusion partners fused to any of a), b), c), d) or e).

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

[0047] The percent identity between two amino acid sequences is determined as follows: First, the amino acid sequence is compared to, for example, SEQ 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, which is available from the U.S. government's National Center for Biotechnology Information website at ncbi.nlm.nih.gov. Instructions explaining how to use the Bl2seq program can be found in the readme file that accompanies BLASTZ. Bl2seq performs a comparison between two amino acid sequences using the BLASTP algorithm. To compare two amino acid sequences, the Bl2seq options are set as follows: -i is set to a file containing the first amino acid sequence to be compared (e.g., C:\seq1.txt), -j is set to a file containing the second amino acid sequence to be compared (e.g., C:\seq2.txt), -p is set to blastp, -o is set to any desired file name (e.g., C:\output.txt), and all other options are left 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 those regions of homology 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 in both sequences where identical nucleotides or amino acid residues are represented.

[0048] The percent identity 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 SEQ ID NO:1 (the length of the sequence shown in SEQ ID NO:1 is 320) and the number of matches is 288, then the sequence has a percent identity of 90 to the sequence shown in SEQ ID NO:1 (i.e., 288÷320×100=90).

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

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

[0051] A His tag is a polyhistidine amino acid motif in proteins, typically consisting of at least six histidine (His) residues, and often (but not always) located at the N- 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 a variety of 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 carboxymethylaspartic acid (Co-CMA) for cobalt, to which the polyhistidine tag binds with micromolar affinity. The resin is then typically washed with phosphate buffer to remove proteins that do not specifically interact with the cobalt or nickel ions. For Ni-based methods, the washing efficiency can be improved by the addition of 20 mM imidazole (proteins are typically eluted at 150-300 mM imidazole).

[0052] Although the His tag approach is convenient for purification, the presence of non-native polyhistidine motifs in therapeutic proteins, including AnxA5 proteins, is undesirable because it can lead to adverse patient reactions, such as immunological reactions. Alternatively, attempting to remove the His tag motif after protein production is cumbersome, time-consuming and expensive, and in fact, protein preparations with the His tag removed will typically retain one or more extraneous histidine residues.

[0053] As a result, preferably the AnxA5 protein does not contain a His tag and its recovery and purification is 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) a variant or mutant variant of a protein having the sequence of human annexin A5, 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 be used to enhance phagocytosis by using AnxA5 variants that bind to phosphatidylserine (PS) on apoptotic cells, activating phagocytes to engulf apoptotic cells instead of inhibiting phagocytosis.

[0055] B. Host cell culture The AnxA5 protein can be recombinantly expressed in a host cell culture. A preferred host cell culture expressing the AnxA5 protein according to the present invention is a culture at 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 5,000 L, about 6,000 L, about 7,000 L, about 8,000 L, about 9,000 L, about 10,000 L, about 11,000 L, about 12,000 L, about 13,000 L, about 14,000 L, about 15,000 L, about 16,000 L, about 17,000 L, about 18,000 L, about 19,000 L, about 21,000 L, about 22,000 L, about 23,000 L, about 24,000 L, about 25,000 L, about 26,000 L, about 27,000 L, about 28,000 L, about 29,000 L, about 30,000 L, about 31,000 L, about 32,000 L, about 33,000 L, about 34,000 L, about 35,000 L, about 36,000 L, about 37,000 L, about 38,000 L, about 39,000 L, about 40,000 L, about 40,000 L, about 41 In some embodiments, the culture may have a culture volume of about 100 L, about 6,000 L, about 7,0000 L, about 8,0000 L, about 9,0000 L, about 10,000 L, about 20,000 L, about 30,000 L, about 40,000 L, about 50,000 L, about 60,000 L, about 70,0000 L, about 80,0000 L, about 90,0000 L, about 100,000 L or higher. The term "about" in this context can include ±50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2% or 1% of the stated volume.

[0056] Methods for the recombinant expression of genes of interest are well known in the art.

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

[0058] Thus, a host cell may be transformed with a polynucleotide vector construct comprising a sequence encoding an AnxA5 protein.

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

[0060] Bacterial cells are preferred prokaryotic host cells in the context of the present invention. Bacterial host cells can be, for example, gram-positive or gram-negative host cells (although gram-neutral and gram-variable bacteria can 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 the context of at least the first aspect of the invention, and optionally in the context of all aspects of the invention, the host cell is an endotoxin-producing host cell having a cell wall, and thus is 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 (no. ATCC 31343) available from the American Type Culture Collection (ATCC), Rockville, MD, USA.

[0062] For the avoidance of doubt, the term "endotoxin-producing host cell having a cell wall" may be construed to exclude yeasts, such as Saccharomyces cerevisiae, and other eukaryotic cells.

[0063] Further particularly preferred endotoxin-producing strains of E. coli include strain BL21(DE3) (e.g., widely available commercially and as described in Marder et al., 2014, BMC Biotechnology, 14:33). However, the applicant has surprisingly found that annexin A5 expressed from BL21(DE3) exhibits unexpectedly high levels of undesired post-translational gluconoylation, such that approximately 40% of the annexin A5 protein is gluconoylated. This is much higher than the levels of gluconoylation of most other proteins recombinantly expressed in BL21(DE3), which typically exhibit levels of gluconoylation of only about 5-10%. It is therefore even more preferred that the endotoxin-producing strain of E. coli is a strain of BL21(DE3) that has been engineered to reduce the level of gluconoylation of the AnxA5 protein, for example by overexpressing phosphogluconolactonase (PGL), thereby suppressing post-translational 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%, as described in Aon et al. (Appl. Env. Microbiol., 2008, 74(4):950-958, the contents of which are incorporated herein by reference).

[0064] Typically, bacterial host cells for use in the present invention are walled bacterial host cells, and thus preferably exclude cells lacking a cell wall and (in the case of Gram-negative bacteria) an outer membrane, such as spheroplasts (such as those 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 do not have a cell wall. Spheroplasts are entirely unsuitable for commercial scale production due to their sensitivity and fragility, particularly due to the absence of a cell wall, which severely limits their ability to be productively grown in large volume cultures.

[0065] Optionally, the host cells are endotoxin-producing host cells that have a cell wall that cannot be lysed by osmotic shock and / or freeze / thaw treatments.

[0066] In a further alternative, the host cell culture is one in which the host cells are not 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. Particular antibiotics to avoid, in one embodiment, are antibiotics that result in the formation of spheroblasts, 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 generally 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 embryo cells NIH / 3T3 available from ATCC as CRL1658, and monkey kidney-derived COS-1 cells available from ATCC as CRL1650. A preferred insect cell is Sf9 cell, which can be transfected with a baculovirus expression vector.

[0068] Exemplary prokaryotic vector plasmids are 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 vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16A, pNH18A, 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 drive expression of cloned genes, with the highest expression levels 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 the glucocorticoid inducible promoter of the mouse mammary tumor virus long terminal repeat to drive expression of cloned genes.

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

[0071] Methods well known to those skilled in the art can be used to construct expression vectors containing the AnxA5 protein coding sequence and, for example, suitable transcriptional or translational control sequences. One such method involves ligation via homopolymeric tails. A homopolymeric poly dA (or poly dC) tail is added to the exposed 3'OH group on the DNA fragment to be cloned by terminal deoxynucleotidyl transferase. The fragment can then be annealed to the poly dT (or poly dG) tail added to the end of the linearized plasmid vector. The gaps left after annealing can be filled by DNA polymerase, and the free ends can be ligated by DNA ligase.

[0072] Another method involves ligation via sticky ends: compatible sticky ends can be generated on the DNA fragment and the vector by the action of suitable restriction enzymes. These ends rapidly anneal by complementary base pairing, and the remaining nicks can be joined by the action of DNA ligase.

[0073] Further methods use synthetic molecules called linkers and adapters. DNA fragments with blunt ends are generated by bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, which removes protruding 3' ends and fills in recessed 3' ends. Synthetic linkers, pieces of blunt-ended double-stranded DNA containing recognition sequences for defined restriction enzymes, can be ligated to the blunt-ended DNA fragments by T4 DNA ligase. They are then digested with the appropriate restriction enzyme to create sticky ends and ligated into an expression vector with compatible ends. Adapters are also chemically synthesized DNA fragments that contain one blunt end used for ligation, but also carry one implemented sticky 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 of modifying the DNA encoding AnxA5 protein is to use the polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491. In this method, the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers, which themselves become incorporated into the amplified DNA. The 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 appropriate cell hosts with a DNA construct containing a sequence encoding an AnxA5 protein is accomplished by well-known methods that typically depend on the type of vector used.

[0077] For 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 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 in transfecting such cells, such as calcium phosphate and DEAE-dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc. (Gaithersburg, MD 20877, USA).

[0078] Electroporation is also useful for transforming cells and is well known in the art for transforming yeast, bacterial 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 greatest number of transformants is consistently recovered following electroporation of DNA-cell mixtures suspended in 2.5x PEB using 6250V per cm at 25 μFD.

[0080] Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente (1990) Methods Enzymol. 194,182.

[0081] Physical methods may also be used to introduce DNA into animal and plant cells. For example, microinjection uses a very fine pipette to inject DNA molecules directly into the nucleus of the cell to be transformed. Another example involves bombardment of cells with high-velocity microprojectile particles, usually gold or tungsten particles coated with DNA.

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

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

[0084] Another method of identifying successfully transformed cells involves growing the 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 the 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, if the recombinant DNA is capable of directing the expression of a protein, successful transformation can be confirmed by well-known immunological methods. For example, cells successfully transformed with an expression vector will produce a protein that exhibits the appropriate antigenicity. Samples of cells suspected to be transformed are taken and assayed for the protein using a suitable antibody.

[0086] Thus, the transformed host cell itself can be cultured to provide a culture transformed host cell expressing the AnxA5 protein. The culture can be a monoclonal (clonally homogeneous) culture, or a culture derived from a monoclonal culture, in a nutrient medium.

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

[0088] Harvesting involves, for example, collecting the host cells (which are typically intact and typically retain substantially all (e.g., greater than 80%, 90%, 95% or 99% or 100%) of the AnxA5 protein within the cells) from the medium. This can generally be accomplished by centrifugation or filtration, where the cultured host cells are collected in the form of a biomass. In the case of centrifugation, the supernatant can be discarded and the cell pellet can be transferred directly or indirectly (e.g., after storage, such as by freezing) to a cell culture homogenization stage.

[0089] C. Cell Culture Homogenization 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 recombinantly expressed intracellular proteins comprising the sequence of Annexin A5 (AnxA5) from endotoxin-producing host cells having a cell wall, the process comprising releasing the intracellular proteins from the host cells, characterized in that the step of releasing the intracellular AnxA5 protein is carried out in the presence of a homogenization buffer comprising a non-ionic detergent.

[0090] Preferably, the non-ionic detergent is a polysorbate, more preferably a polysorbate selected from Tween 20 and Tween 80, most preferably Tween 80. Alternatively, although less preferred, other non-ionic detergents may be used, but they have an ultraviolet absorptivity λ similar to the protein absorption maximum, i.e., 275-280 nm. maxIt is preferable to avoid the use of non-ionic detergents having a λ (both in the cell culture homogenization step, and also in any other steps of the process) since they can interfere with the ability to monitor the presence of proteins by UV absorbance during the recovery process. In this context, "similar" means max can mean within 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2 nm, or 1 nm of the absorption maximum of the AnxA5 protein being purified. Thus, for example, a non-ionic detergent may have a λ max = 275 nm, and thus it may be preferred that Triton X-100 is not used in the cell culture homogenization step and / or any other step of the process of the invention.

[0091] Note that prior to cell homogenization to release intracellular AnxA5 protein, non-ionic detergent may be included in the homogenization buffer added to the cells (e.g., the homogenization buffer may be "pre-formed" with non-ionic detergent present), or the cells may be suspended in homogenization buffer that does not contain non-ionic detergent, and then the non-ionic detergent may be added to and mixed with the cells suspended in homogenization buffer.

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

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

[0094] It is preferred that no calcium ions or ionizable calcium compounds (such as CaCl2) are added or included in the homogenization buffer. Thus, it is preferred that the free calcium ion concentration in the homogenization buffer at the time of release of the intracellular AnxA5 protein from the host cell 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 chelator. 2+ In accordance with the optional subsequent step of using Mg 2+ It may be preferable to select a calcium ion chelator that does not bind strongly to Mg. Alternatively, it may be preferable to use an enzyme treatment, where such an enzyme accepts Mg as a cofactor. 2+ The optional subsequent step is to use Mg 2+ may be replaced with other steps not requiring 1. In that case, an optional calcium ion chelator such as EGTA or ethylenediaminetetraacetic acid (EDTA) may be included in the homogenization buffer.

[0096] Optionally, the concentration of free calcium ions in the homogenization buffer and / or the amount of calcium metal ion chelator is in an amount effective to reduce (such as by 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more) or prevent binding between annexin A5 and components of the cell membrane and / or wall of the host cell, e.g., compared to the level of binding that would be observed in the presence of a homogenization buffer consisting of 50 mM Tris-HCl, 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, 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 chelating agent, preferably the calcium metal ion chelating agent is 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 purification and separation of released AnxA5 protein from host cell debris. Nevertheless, a simple test using a centrifugation step can be performed on an aliquot of lysed cells to establish the tolerance to free calcium ions and / or an effective amount of calcium metal ion chelating agent in the homogenization buffer. After cell homogenization, an aliquot of lysed cells (e.g., 100 mL) is subjected to centrifugation (e.g., 38,900 g for 30 minutes), and then 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 for 30 min at 4° C. to release any bound AnxA5 protein, then centrifuged at 38,900 g for 30 min at 4° C. and the amount of bound AnxA5 protein released in the supernatant determined to give the level of "bound" AnxA5 protein. In this context, the percentage of AnxA5 binding to host cell plasma membrane and / or wall components=(level of "bound" AnxA5 protein / (level of "bound" AnxA5 protein+level of "free" AnxA5 protein))×100.

[0099] Preferably, the percentage of bound AnxA5 in the resulting biomass homogenate as determined by the methods described above 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] Although referred to herein as a "homogenization buffer", it is not essential that the solution is a pH buffer. Optionally, however, the homogenization buffer may further comprise additional components, including a buffer (e.g., Tris), and may optionally be pH adjusted as necessary, for example to around pH 6-8, more preferably in the range of pH 7-8.5, 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 step is to 2+ In some options, including enzymatic treatments with enzymes that require cofactors such as ribozymes, 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 aspect of the invention comprises, consists essentially of, 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 invention may comprise mixing biomass from a culture of host cells in a homogenization buffer at a concentration of about 1 g to 300 g biomass (wet weight) per liter of homogenization buffer, for example at a concentration of about 10 g to 200 g biomass per liter of homogenization buffer. Exemplary 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 resuspension ratio of about 100 g biomass per liter of homogenization buffer is particularly preferred. In this context, the term "about" is intended to include ±5 g / L, 4 g / L, 3 g / L, 2 g / L, or 1 g / L of the stated value.

[0104] Mixing is typically carried out at about room temperature, i.e., typically around 18° C. to 28° C., for example, about 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 so as to maintain it at or around a temperature selected from the above list (e.g., ±5, 4, 3, 2, or 1° C. thereto), although dynamic temperature control at this stage is not usually required.

[0105] Optionally, the homogenization buffer may also contain or be added to the homogenization buffer after mixing with the biomass but prior to host cell homogenization, one or more enzymes useful in enzymatic processing. For example, it may be suitable to include or add one or more nuclease enzymes after homogenization that aid in the degradation of nucleic acids (including DNA and / or RNA) from the host cells. This will reduce the viscosity of the homogenate produced thereafter, thereby aiding in downstream processing steps. Any suitable enzyme may be used. The enzyme may be, for example, a nuclease, such as nuclease A, preferably nuclease A from Serratia marescens. One such exemplary enzyme of interest is Benzonase nuclease, an endonuclease from Serratia marcescens, available from commercial sources including Merck / Novagen, Sigma Aldrich, etc., which can be used to degrade all forms of DNA and RNA while not having any proteolytic activity. It is effective over a wide range of conditions and possesses high specific activity. The enzyme completely digests nucleic acids up to 5′-monophosphate-terminated oligonucleotides 2-5 bases in length (below the hybridization limit), which is ideal for the removal of nucleic acids from recombinant proteins and allows compliance with FDA guidelines for nucleic acid contamination. Benzonase enzyme requires 1-2 mM Mg for activation. 2+and remains active in the presence of ionic and non-ionic detergents, reducing agents, the protease inhibitor PMSF (1 mM), the chelating agent EDTA (1 mM), and urea (relative activity depends on the specific conditions). One of skill in the art would be able to readily determine effective concentrations of such nuclease enzymes, but applicants have determined that Benzonase is effective when used prediluted at at least about 3.3 U per L of host cell culture or about 1.85 U per L of resuspended biomass. The term "about" in this context may include ±90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the stated unit number.

[0106] The step of releasing intracellular AnxA5 protein from host cells in homogenization buffer can involve any suitable technique for cell homogenization or lysis. For example, it can include lysing, disintegrating or homogenizing, sonicating or pressure treating the host cells so that the cell wall and cell membrane barrier of the host cells are broken, thereby releasing the intracellular AnxA5 protein. In a particular alternative of the first aspect of the present invention, this step does not include 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 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. In this context, the term "about" may include ±500, 400, 300, 200, 100, 50, 40, 30, 20, or 10 bar of the stated value.

[0108] Optionally, for example, in situations where no nuclease enzymes were added to degrade the nucleic acids (e.g., Mg 2+Mg in the homogenization buffer, for example by incorporating a chelating agent 2+ If the concentration of is too low), it may be beneficial to include multiple additional rounds of high-pressure homogenization (e.g., 2-4 times at pressures in the range of about 600-2500 bar) to degrade nucleic acids and reduce the viscosity of the homogenate.

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

[0110] Depending on the cell homogenization technique applied, the 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 undesired increase in temperature and / or to apply temperature control to prevent an undesired increase in temperature. However, for example, if the homogenate contains an enzyme treating agent such as Benzonase, it may be advantageous to use temperature increases to approach, and preferably within, the optimal temperature range of the enzyme. In the case of Benzonase, a temperature in the range of about 36-40° C. may be particularly suitable.

[0111] The cell homogenization procedure may be performed using metal ion cofactors (e.g., Mg 2+ ), and further, if the homogenization buffer excludes a calcium metal ion chelator, in one option a calcium ion chelator may be added after completion of the enzyme treatment, which may be done before or after the clarification step as discussed below.

[0112] After cell homogenization, the biomass homogenate typically 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 membrane components, host cell nucleic acids, and endotoxins.

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

[0114] D. Clarification of the Homogenate Optionally, and in a preferred embodiment, after production of the biomass homogenate, it is then subjected to a clarification step.

[0115] Thus, in a further embodiment of the first aspect of the invention, the process further comprises a step of clarifying the biomass homogenate, thereby producing a clarified product comprising the released AnxA5 protein. This step is carried out to reduce the content of nucleic acids and further to obtain a reduced particulate solution, which can be applied to subsequent purification steps such as capture chromatography, as further discussed below.

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

[0117] Preferably the filter is a depth filter and / or preferably the filter has a cut off of less than 4 μm, such as a cut off of less than 3 μm, 2 μm or 1 μm, most preferably a cut off in the range of 0.2-0.6 μm. For example the homogenate may be clarified by filtration using a 0.6-0.2 μm cut off depth filter, examples of such off-depth filters are commercially available such as those available as, for example, the cellulosic Cuno 60 SP depth filters. Other depth filters that have been found to provide good performance (i.e., good filtration without product loss), although not as good as the preferred cellulose Cuno 60 SP depth filter having a 0.6-0.2 μm cutoff, include cellulose+kieselguhr filters having a 0.5 μm cutoff (e.g., PR12 UP available from Begerow), polypropylene filters having a 1.2 μm cutoff (e.g., Sartopure PP2 available from Sartorius), cellulose filters having a 0.1 μm cutoff (e.g., Sartoclear S9 available from Sartorius), polypropylene filters having a 0.65 μm cutoff (e.g., Sartopure PP2 available from Sartorius), and cellulose filters having a 0.2-0.5 μm cutoff (e.g., EK 1P or EKM-P, both available from Pall, which are good but slow). Further testing has shown that filters with larger cutoffs (eg, greater than 4 μm) achieve only moderate particle removal and are therefore less preferred.

[0118] It has also been found by the applicants that positively charged cellulosic filters will further reduce the DNA content in the clarified homogenate and therefore may represent a particularly preferred class of filters for use in the clarification step.

[0119] Moreover, it has been found that cellulosic filters require a smaller filter area to provide effective clarification than do corresponding polypropylene filters, which may be a further reason why the cellulosic class of filters is particularly preferred for use in the clarification process.

[0120] The choice of filter area may also depend on the degree and nature of homogenization used. For example, the applicant has determined that when cell homogenization is performed by three homogenization cycles at about 600 bar, a filter area of ​​about 60 cm per L of homogenate is used. 2 is suitable, whereas if the cell homogenization is carried out by two homogenization cycles at about 800 bar, a filter area of ​​about 180 cm per liter of homogenate is preferred. 2 It has been found that a filter area of ​​10 to 500 cm per liter of homogenate to be clarified is suitable. Thus, the depth filter is optionally sized to 10 to 500 cm per liter of homogenate to be clarified. 2 , for example, 30 to 400 cm 2 / L, 40~250cm 2 / L, 50~200cm 2 / L or 60~180cm 2 / L, e.g., 50-100cm 2 / L, or 60~80cm 2 / L; or 120~240cm 2 / L, or 150~210cm 2 / L.

[0121] After clarification, the clarified product may be further conditioned by the addition of one or more additional additives in preparation for subsequent steps. For example, it may be suitable to condition the clarified product by the addition of one or both of: (a) a non-ionic detergent, such as polysorbate and most preferably Tween 80, and (b) a calcium metal ion chelator, such as EDTA (unless the clarified product already contains sufficient levels of chelator based on incorporation of the chelator in the homogenization buffer and / or by addition of the chelator after the enzyme treatment step).

[0122] In one exemplary embodiment, the clarified product is diluted approximately 2-fold with 1% non-ionic detergent (most preferably Tween 80) and a calcium ion chelator (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 that there is no need for any time-consuming dialysis steps on the clarified product prior to further chromatographic capture steps such as anion exchange capture discussed below. Thus, in an embodiment of the present invention, the clarified product is not subjected to dialysis prior to chromatographic capture of the AnxA5 protein.

[0124] E. Anion exchange capture In the first aspect of the invention, the process may further comprise the step of subjecting the released AnxA5 protein to an anion exchange resin to perform a first anion exchange capture step, thereby producing a first anion exchange product comprising the released AnxA5 protein.

[0125] Generally, protein capture from bacterial (e.g., E. coli) homogenates / lysates by anion exchange chromatography is not a first choice strategy because large amounts of host cell proteins (HCPs) and DNA bind to the capture resin, thereby adversely affecting the binding capacity of the target product and even taxing the resin performance. However, the applicant has determined that the above-described cell homogenization and clarification procedure according to the 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] Thus, in one embodiment, the clarified product comprising 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, thereby producing a first anion exchange product comprising released AnxA5 protein.

[0127] Optionally, 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, calcium ion chelator level and non-ionic detergent level. For example, the AnxA5 protein composition subjected to the anion exchange step may be formulated at a pH of about 6.9, optionally ±1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2 or 0.1 pH units (in one alternative, the preferred range is pH 6-8.5, more preferably pH 6.5-7.5, most preferably pH 6.9). The applicant has found that low pH values ​​around this range (e.g., at pH 6) tend to cause detectable host cell proteins in the eluted product, while at pH 8 and above, solubility is somewhat reduced. The AnxA5 protein composition subjected to the anion exchange step may optionally be adjusted to have a conductivity of about 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 in preparation for the subsequent step. As already discussed above, it may be suitable to condition the AnxA5 protein composition subjected to the anion exchange step prior to the first anion exchange step by the addition of one or both of (a) a non-ionic detergent, such as polysorbate and most preferably Tween 80, and (b) a calcium metal ion chelator, such as EDTA (unless the clarified product already contains a sufficient level of chelator based on the incorporation of the chelator in the homogenization buffer and / or by the addition of the chelator after the enzymatic treatment step). In one exemplary embodiment, the AnxA5 protein composition subjected to the anion exchange step is diluted approximately two-fold with 1% non-ionic detergent (most preferably Tween 80) and a calcium ion chelator (most preferably EDTA) is added to a final concentration of approximately 2 mM.

[0128] In this regard, the use of a non-ionic detergent and / or a calcium metal ion chelator to condition the AnxA5 protein composition to be 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 by the first anion exchange capture step, including cell wall components, cell membrane components, endotoxins, nucleic acids, etc. For example, the applicant has demonstrated highly efficient removal (around 99% reduction) of endotoxins by the first capture anion exchange step when carried out in the presence of a non-ionic detergent and a calcium metal ion chelator.

[0129] Although primarily discussed below in the context of the second anion exchange refinement step, in the option where a calcium metal ion chelator (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 (other than calcium) 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. One exemplary metal ion is Mg 2+ It is.

[0130] Typically, prior to contacting the anion exchange resin with the AnxA5 product, the anion exchange resin is equilibrated. Any suitable equilibration may be used. For example, the anion exchange resin may be equilibrated with a buffer (e.g., 20 mM Tris pH 7.4), a non-ionic 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 in an exemplary embodiment, 3 column volumes (CV) is a suitable volume.

[0131] Preferably, the first anion exchange capture step is performed in a positive mode with respect to the AnxA5 protein, such that the AnxA5 protein is temporarily bound to the anion exchanger during the anion exchange step, and a wash 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 comprising the released AnxA5 protein.

[0132] The applicant found that strong anion exchange resins provide acceptable capacity for AnxA5 protein capture, while weak anion exchange resins perform less acceptable, so strong anion exchange resins are preferred.Strong anion exchange resins are well known in the art, and examples include resins that contain quaternary ammonium functional groups, such as type I resins with trialkyl ammonium chloride or ammonium hydroxide, or type II resins with dialkyl 2-hydroxyethyl ammonium 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 not preferred, and examples include DEAE resins that contain diethylaminoethyl functional groups.Q Sepharose XL resins (e.g., provided by GE Healthcare) may be most preferred.

[0133] After loading onto the anion exchange resin, under positive mode, the AnxA5 protein binds temporarily to the resin, which can be washed to reduce / remove impurities. Any suitable washing conditions can be used. For example, the washing solution can comprise, consist essentially of, or consist of an aqueous solution of a buffer (e.g., 20 mM Tris pH 7.4), a non-ionic detergent (e.g., 0.1% polysorbate, preferably Tween 80), and a salt (e.g., 25 mM NaCl). Any suitable washing volume can 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 comprise, consist essentially of, or consist of a buffer (e.g., 20 mM Tris pH 7.4), a non-ionic detergent (e.g., 0.01-1% (w / v), more preferably 0.1% (w / v) polysorbate, preferably Tween 80), and an aqueous solution of salt at a higher concentration than the wash 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, applicants have found that in exemplary embodiments, 9 column volumes (CV) is a suitable volume for elution.

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

[0136] The anion exchange resin may then be regenerated and purified. Suitable methods for regeneration and purification are known in the art, and one such suitable protocol is discussed 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 cells. Preferably, the first anion exchange product contains more than 60%, 70% or 80% of the AnxA5 protein released from the host cells. In terms of comparison with the process of Marder et al. (see above), it is clear that the prior art processes exhibited large product losses 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 the pellet-bound annexin A5 is collected. The relative amounts of annexin A5 in the discarded supernatant and recovered from the pellet of Marder et al. (see above) are shown in Figure 3, columns 2 and 3, respectively. It is clear from the figures that about half of the released annexin A5 is discarded in the process of Marder et al. (see above), leading to a low-yield method.

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

[0139] Thus, in the first aspect of the invention, it is preferred that the first anion exchange product comprises more than 1 g, more than 2 g, more than 3 g, more than 4 g or about 5 g AnxA5 protein per litre 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, and without limitation, a 0.45-0.2 μM filtration step has been found to be suitable.

[0141] F. Affinity Chromatography Applicants have also discovered that, in contrast to conventional methods that tend to experience ever-increasing losses in yield with the sequential addition of purification steps (as product is lost at each step), the combination of an anion exchange step and a heparin affinity chromatography step has the surprising benefit of achieving the high purity obtained with the heparin affinity chromatography step alone, but with substantially increased yield (i.e., recovery increases from about 30-40% to about 70-90%) (see, e.g., Example 2 below). This large increase in yield associated with the addition of a purification step is the opposite of what would normally be expected from a combination of purification steps.

[0142] Thus, a second aspect of the invention provides a process for the recovery and / or purification of a protein comprising the sequence of annexin A5 (AnxA5) from a solution containing the AnxA5 protein and one or more impurities (which may or may not be the product of the clarification step described above), the process comprising: subjecting a solution comprising 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 steps discussed above) to an anion exchange resin to perform a first anion exchange step, thereby producing a first anion exchange product comprising AnxA5 protein; and 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.

[0143] Preferably, according to the process of the second aspect of the present invention, the affinity chromatography step may comprise binding of the AnxA5 protein to immobilized heparin, optionally which binding is 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 chelator such as EDTA.

[0144] In addition, as discussed in Example 3, Applicants have discovered that Tween 80 has a particularly advantageous effect on the heparin affinity chromatography step (compared to other non-ionic detergents, including other Tweens such as Tween 20). Incorporation of Tween 80, for example at around 0.1% (w / v), into the buffer used for the heparin affinity chromatography step can aid in elution of the AnxA5 protein in a single peak, reduce pressure, and prevent precipitation.

[0145] Thus, a third aspect of the present invention provides a process for the recovery and / or purification of a protein comprising the sequence of annexin A5 (AnxA5) from a solution comprising AnxA5 protein and one or more impurities, the method comprising subjecting a solution comprising AnxA5 protein and one or more impurities (which solution may or may not be the direct or indirect product of the cell homogenization, clarification and / or first anion exchange chromatography capture steps discussed above) 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 comprising released AnxA5 protein.

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

[0147] More generally, however, the first aspect of the invention may comprise subjecting the released AnxA5 protein to an affinity chromatography step, thereby producing a first affinity chromatography product comprising the released AnxA5 protein. It may be particularly preferred that the AnxA5 protein in the first anion exchange product, for example produced by the above-mentioned method, may be subjected directly or indirectly (e.g. after sterile filtration and / or addition of further components) to an affinity chromatography step.

[0148] Thus, in one embodiment of the first aspect of the invention (which may be combined with either or both of the features of the second and third aspects of the invention), the process comprises: (a) clarifying the biomass homogenate comprising the released AnxA5 protein by a clarification process as described above, thereby producing a clarified product comprising the released AnxA5 protein; (b) performing the first anion exchange step described above, 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 (directly or indirectly).

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

[0150] Thus, prior to the heparin affinity step, the AnxA5 product may be conditioned by the addition of any one or more of calcium ions (e.g., CaCl2), a non-ionic detergent (preferably Tween 80), and may optionally be buffered (e.g., Tris buffer at pH 7.4). Without limitation, applicants have demonstrated a beneficial effect when the filtered anion exchange product is diluted approximately 8-fold with a dilution buffer containing 20 mM Tris (pH 7.4), 0.1% Tween 80, and 2 mM CaCl2.

[0151] Thus, it may be preferred that the AnxA5 product is conditioned with polysorbate 80, more preferably the polysorbate 80 is at a final concentration of greater than about 0.01% up to 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%. The term "about" in this context refers to plus or minus 50%, 40%, 30%, 20%, 10%, or 5% of the stated value.

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

[0153] The conditions used in loading the affinity chromatography column (e.g., a heparin affinity chromatography column) allow the AnxA5 protein to bind to heparin, i.e., the affinity chromatography is typically performed in a positive mode with respect to the AnxA5 protein.

[0154] The affinity chromatography column (e.g., a heparin affinity chromatography column) can be loaded with the desired level of AnxA5 product. For example, loading may be performed at about 5 g per liter of column resin volume or more, e.g., about 10 g / L, about 15 g / L, about 20 g / L, about 25 g / L, about 30 g / L or more. The term "about" in this context refers to ±50%, 40%, 30%, 20%, 10%, or 5% of the stated value. In fact, the applicant has found that a loading of about 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 the context of such high loading amounts, the applicant has found that the presence of polysorbate 80 in the loading mixture is particularly beneficial in avoiding AnxA5 protein precipitation or insolubility. In the absence of the use of polysorbate 80, precipitation and increased back pressure are observed and the affinity chromatography step becomes less efficient. See Examples 3 and 4.

[0155] After loading of the AnxA5 protein, the column is typically washed one or more times to remove impurities. Any suitable washing protocol may be used. Applicant has found that a suitable washing protocol includes, without limitation, a two-step washing. For example, in the first washing step, 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 step may vary depending on the desired results and the exact nature of the washing solution used. Without limitation, Applicant has found that, for example, the exemplary washing buffer described above can be used successfully for a 15 CV wash. In the second washing step, the washing buffer may contain calcium in a lower amount than the first washing step, or preferably does not contain calcium (e.g., 20 mM Tris pH 7.4, 0.1% Tween 80). The volume of the second stage wash may vary depending on the desired results and the exact nature of the second stage wash solution used. Without limitation, Applicants have found, for example, that the exemplary wash buffers set forth above can be successfully used with a 2 CV wash.

[0156] Thereafter, 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, and without limitation, applicants have found that a suitable elution buffer is 20 mM Tris pH 7.4, 0.1% Tween 80, 10 mM EDTA, 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] In order 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, and without limitation, in the examples, the applicant has enabled concentrated elution by reducing the flow rate to 60 cm / h or less during elution. The complete elution peak can be collected, for example, starting from the rise of the UV signal at 0.05 AU to 0.05 AU in the descending peak, which corresponds to approximately 7 CV. Preferably, the elution profile shows a single peak, which is most ideally a sharp peak.

[0159] Thus, the first affinity chromatography product comprises the released AnxA5 protein and a calcium ion chelator such as EDTA or EGTA, optionally present at a concentration of about 0.1 mM to 500 mM, e.g., about 1 mM to about 100 mM, more typically about 2 mM to about 50 mM, more preferably about 5 mM to about 15 mM, and most preferably about 10 mM. In this context, the term "about" may include the meaning of ±50%, 40%, 30%, 20%, 10%, 5% or 1% of the stated concentration(s).

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

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

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

[0163] Suitable additives may include, for example, a diluent (e.g., water) to further dilute the AnxA5 protein in the affinity chromatography product, a buffer (e.g., Tris, e.g., at 35 mM and pH 8), a non-ionic detergent (e.g., polysorbate, more preferably Tween 80, such as at a concentration of about 0.1 w / v %), and / or one or more further additives in accordance with the fourth aspect of the invention discussed below.

[0164] In other words, the fourth aspect of the present invention is based on the applicant's realization that calcium metal ion chelating agents (e.g., EDTA) can adversely affect the effectiveness of the anion exchange step. Free EDTA (or other chelating agents) can directly bind to the anion exchange functional groups, thereby reducing the capacity and even the separation achieved by the anion exchange step. This is particularly problematic when anion exchange is performed on the product of the heparin affinity chromatography step, where the AnxA5 protein is bound to heparin in the presence of calcium ions, and then a calcium metal ion chelating agent (e.g., EDTA) is used to elute the bound AnxA5 protein. As a result, the eluted AnxA5 product of the heparin affinity chromatography step contains high levels of calcium ion chelating agents. Although it is generally desirable to be able to further purify the AnxA5 product in a further anion exchange step, the calcium ion chelating agent is a problematic component during that further anion exchange step. On the other hand, attempts to remove the calcium metal ion chelating agent before the anion exchange step are time-consuming and therefore also increase costs. Thus, for example, the prior art methods involving dialysis steps for buffer exchange are slow and inefficient, thus increasing production costs.Furthermore, incorporating calcium metal ion chelating agents into the AnxA5 product during the anion exchange step may be a key component to prevent calcium-mediated binding of AnxA5 protein to impurities, including endotoxins.Therefore, it would be advantageous and effective to introduce an additive that blocks or reduces the binding of calcium ion chelating agents to anion exchange resin, but still allows the anion exchange step to be performed without the inconvenience and expense associated with dialysis, and without blocking the beneficial effect of calcium metal ion chelating agents during the anion exchange step.

[0165] Applicants have realised that this may be achieved by incorporation of one or more additional selected metal ions into the AnxA5 protein product prior to anion exchange, the additional selected metal ions being 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. The selection of a suitable additional metal ion will depend on the nature of the calcium ion chelator and the nature of the anion exchange resin. For example, when EDTA is used as the calcium ion chelator, Mg 2+ Any ion is generally suitable for achieving the purposes of the present invention and may be added to the AnxA5 protein product prior to the anion exchange step.

[0166] Thus, a fourth aspect of the present invention relates to a process for the recovery and / or purification of a protein comprising the sequence of annexin A5 (AnxA5) from a composition comprising the AnxA5 protein and a calcium metal ion chelator, comprising: The process is characterized in that it comprises subjecting the composition to an anion exchange resin to perform an anion exchange step, whereby the AnxA5 protein is recovered and / or purified from the composition, The anion exchange step is further characterized in that it is carried out in the presence of an additional selected metal ion; This additional selected metal ion provides a process in which the calcium metal ion chelator is selected to have 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 ion is mixed with the composition comprising AnxA5 protein and a calcium metal ion chelator before the composition is subjected to the anion exchange resin. The solution (e.g., washing solution and / or elution buffer) used in the subsequent anion exchange step may or may not also contain the additional selected metal ion. Thus, in one embodiment of this aspect of the invention, performing the anion exchange step in the presence of the additional selected metal ion refers to adding the additional selected metal ion to the composition comprising AnxA5 protein and a calcium metal ion chelator before the composition is subjected to the anion exchange resin.

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

[0169] The calcium metal ion chelator may be present in the composition at and / or in a concentration in the range of about 0.1 mM to 500 mM, e.g., in the range of about 1 mM to about 100 mM, more typically in the range of about 2 mM to about 50 mM, e.g., in a concentration of about 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 more. In this context, the term "about" may include the meaning of ±0.5, 0.4, 0.3, 0.2, or 0.1 mM of the stated value. In the above context, the term "excess" may include the meaning that a sufficient amount of calcium metal ion chelator is present to remove any divalent ions that may contribute to binding of the AnxA5 protein to the immobilized heparin on the column in the preceding affinity chromatography step, thereby enabling the AnxA5 protein to be released from the column into solution and then used directly or indirectly in the anion exchange purification step.

[0170] In one exemplary embodiment of the fourth aspect of the invention, the selected metal ion is Mg 2+ ions.

[0171] It may be preferable that the selected metal ion is present in an amount effective to reduce (about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more) or prevent the interaction between the calcium ion chelator and the anion exchange resin during the anion exchange step, during the process of subjecting the composition to the anion exchange resin. For example, the selected metal ion may be present in an amount effective to reduce or prevent the interaction between the calcium ion chelator and the anion exchange resin during the anion exchange step, during loading of the composition 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 may be preferred that 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 a 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 (by about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more, etc.).

[0173] It may be preferred that the selected metal ion is present during the anion exchange step at a concentration of from about 1 to about 100 mM, for example, from about 2 to about 50 mM, from about 5 to about 25 mM, from about 10 to about 15 mM or about 12.5 mM.

[0174] Further, the calcium metal ion chelator is EDTA and the selected metal ion is Mg 2+ ions, more preferably Mg 2+It may be preferred that the molar ratio of ion to EDTA ranges from 0.5:1 to 2:1, most preferably at least 1:1 or greater than 1:1.

[0175] In the context of the fourth aspect of the present invention, in a further embodiment, the composition comprising AnxA5 protein and a calcium metal ion chelator and subjected to an anion exchange resin may be a direct or indirect product of a preceding process comprising subjecting AnxA5 protein to an affinity chromatography step and eluting the AnxA5 protein with a calcium ion chelator, thereby producing an affinity chromatography product that is a composition comprising AnxA5 protein and a calcium metal ion chelator. For example, the preceding affinity chromatography step may comprise binding of 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 chelator such as EDTA.

[0176] It may further be preferred that 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 application of the direct or indirect product to the anion exchange step.

[0177] In the context of the fourth aspect of the invention, in a further embodiment, the selected metal ion is added to the composition prior to 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 invention is treated with a further anion exchange step, such as the anion exchange step in the fourth aspect of the 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: AnxA5 protein concentration, pH, conductivity, level of calcium ion chelator and level of non-ionic detergent, or level of selected additional metal ions (in a fourth aspect of the invention).

[0180] Typically, prior to contact of the (second) anion exchange resin with the AnxA5 product in the refinement step, the anion exchange resin is equilibrated. Any suitable equilibration may be used. For example, the anion exchange resin may be equilibrated with a buffer (e.g., 20 mM Tris pH 7.4), a non-ionic 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 in an exemplary embodiment, 3 column volumes (CV) is a suitable volume.

[0181] Preferably, the (second) anion exchange refinement step is performed in positive mode with respect to the AnxA5 protein, such that the AnxA5 protein is temporarily bound to the anion exchanger during the anion exchange step, a wash 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 comprising 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 with trialkyl ammonium chloride or hydroxide, or Type II resins with dialkyl 2-hydroxyethyl ammonium chloride or hydroxide. Without limitation, an example of an anion exchange resin suitable for the second scouring step includes Source 15 Q. Source 15Q anion exchange resin can be defined as a polymeric strong anion exchanger, with quaternary ammonium ligands, a median cumulative volume distribution particle size (d) of about 15 μm. 50v ), a polystyrene / divinylbenzene matrix, and / or a pressure / flow specification of approximately 400 cm / h, 1000 kPa when evaluated in a FineLine 100 column with a bed height of 10 cm.

[0183] Further examples of anion exchange resins suitable for the second refinement step include, without limitation, Capto Q ImpRes, which may be defined as a strong anion exchanger, with a quaternary amine ligand, a fast flow agarose matrix, and a median particle size (d) of about 36-44 μm. 50v ), about 0.15~0.18mmol Cl - It may be further characterised as having an ionic capacity of 100 mg / ml of solvent, a binding capacity of greater than 55 mg BSA and greater than 48 mg β-lactoglobulin / ml of chromatography solvent, and / or a pressure / flow specification of at least about 300 kPa at 220 cm / h when evaluated as a 1 m diameter column with a bed height of 20 cm.

[0184] Without being bound by theory, the Applicant has found that it may be particularly advantageous to use Capto Q ImpRes anion exchange resin for the (second) anion exchange refinement step when the AnxA5 protein to be purified is derived from a recombinant source (e.g., E. coli BL21(DE3) that has been further engineered to overexpress PGL as described in Aon et al. (Appl. Env. Microbiol., 2008, 74(4):950-958, the contents of which are incorporated herein by reference), which abolishes or reduces the gluconoylation of the bacterially expressed AnxA5 protein. "Low" in this context then means that the level of gluconoylation is greater than that of the E. coli strain BL21(DE3) (e.g., widely available commercially and described in Marder et al., 2014, BMC "AnxA5 protein is gluconoylated at a level lower than the level of gluconoylation (such as less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of that level) of the AnxA5 protein expressed in a soluble form of glycerol (e.g., as described in Biotechnology, 14:33). For example, Capto Q for the (second) anion exchange refinement step. The level of gluconoylated AnxA5 protein in the product applied to the ImpRes anion exchange resin 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 applied product. Gluconylated variants of Anx5 can be measured and quantified, for example, by using ultra-performance liquid chromatography (UPLC) or high performance liquid chromatography (HPLC) chromatographic instruments using appropriate anion exchange or reverse phase columns. The various peaks can be further identified and characterized using mass spectrometry (MS).

[0185] The applicant has found that the use of Capto Q ImpRes anion exchange resin for the (second) anion exchange refinement step results in a much more efficient process (e.g., compared to the use of Source 15Q anion exchange resin for the (second) anion exchange refinement step) when there is no or low levels of gluconoylation of the AnxA5 protein to be purified, since 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 has a lower price. The quality and purity of the final product is 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 (having a 15 μm particle size) to Capto Q ImpRes anion exchange resin (having a 40 μm particle size) could provide a productivity increase that could exceed about five times (5×) by reducing the time required for the entire process operation (especially since the second anion exchange step used for purification is one of the most time-consuming steps) and could reduce the overall manufacturing costs by more than about 50%. The avoidance of resins with very small bead sizes (less than 30 μm) allows for high flow rates and allows the chromatography columns to be packed with higher resin bed heights without causing unacceptable back pressure. This increases productivity because for a given bed area (column diameter), more resin can be packed into the column and therefore more protein can be bound, while at the same time allowing for faster operation due to the higher flow rates.

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

[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 comprise, consist essentially of, or consist of a buffer (e.g., 20 mM Bis-Tris, pH 7), a salt (e.g., 180 mM NaCl, optionally ±100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or 5 mM) at a higher concentration than the wash solution, and optionally a non-ionic buffer (e.g., polysorbate, preferably Tween 80, such as at a level of about 0.1 w / v). Any suitable elution volume may be used, and without limitation, the applicant has found that in an exemplary embodiment, 33 column volumes (CV) with a linear gradient of increasing concentration of elution buffer from 0 to 100% is suitable for elution.

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

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

[0190] The refinement step is mainly carried out to reduce product-related impurities, such as separating various annexin A5 isoforms. In addition, the refinement step reaches the highest removal factor in the process for residual DNA and strongly reduces HCP. Endotoxins, which are already at low levels after the intermediate steps, are further reduced by about 99%, which (when used in combination with the preceding cell homogenization, nuclease treatment, clarification, capture anion exchange, filtration (such as sterile filtration steps), and heparin affinity steps) shifts endotoxin levels to about 0.0003% of the level in the clarified product before the first AX step.

[0191] Thus, in a further embodiment of the first, second, third and / or fourth aspect of the invention, there is provided a process for the recovery and / or purification of a recombinantly expressed protein comprising a sequence of intracellular annexin A5 (AnxA5) from a cell wall-bearing host cell or culture of the first aspect of the invention, preferably the process comprises the recovery and / or purification of recombinantly expressed intracellular AnxA5 protein from a culture of host cells, the culture being of at least about 100 L, about 200 L. , about 300L, about 400L, about 500L, about 600L, about 700L, about 800L, about 900L, about 1,000L, about 2,000L, about 3,000L, about 4,000L, about 5,000L, about 6,000L, about 7,0000L, about 8,0000L, about 9,0000L, about 10,000L, about 20,000L, about 30,000L, about 40,000L, about 50,000L, about 60,000L, about 70,0000L, about 80,0000L, about 90,0000L, about 100,000L or higher; (a) the process according to the first aspect of the invention comprises releasing intracellular proteins from host cells in the presence of a homogenization buffer comprising a non-ionic detergent, (b) optionally, the releasing step is according to any one or more of the embodiments of the first aspect of the invention described above in subsection C; (c) further optionally, the process includes clarifying the biomass homogenate according to any one or more of the embodiments described above in subsection D; (d) the process further comprises performing a first anion exchange step according to any one or more of the embodiments described above in subsection E, thereby subjecting the released AnxA5 protein directly or indirectly to an anion exchange resin to produce a first anion exchange product comprising the released AnxA5 protein; (e) the process further comprises subjecting the released AnxA5 protein to an affinity chromatography step directly or indirectly according to any of the first, second and / or third aspects of the invention as described above in subsection F, (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 AnxA5 protein and a calcium metal ion chelator, is 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 The process of any of the first, second, third or fourth aspects of the invention may further comprise, preferably at the end of the process, one or more further steps selected from the group consisting of concentration, buffer change, 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 additional steps used in the product formulation can be ultrafiltration / diafiltration (UF / DF), and optionally the product of the UF / DF step contains AnxA5 protein at a concentration of at least about 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 more.

[0194] In another example, one of the further steps used in the product formulation can be the addition of a non-ionic surfactant, preferably a polysorbate, and more preferably Tween 80. The non-ionic surfactant can 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 additional steps used in the product formulation may be a filtration step (such as a sterile filtration step), for example, using a 0.45-0.2 μm filter or a 0.22 μm filter.

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

[0197] The final concentration of AnxA5 protein in the filled container may be adjusted as necessary. Without being limited thereto, the applicant has given a final concentration of 10 mg / mL as an example. Suitable concentrations may be, for example, 1 to 125 mg / mL, 2 to 100 mg / mL, 5 to 50 mg / mL, 7 to 30 mg / mL, or about 10 to 20 mg / mL.

[0198] Optionally, the process of the invention may provide a final sterile AnxA5 protein product in a phosphate-free buffer (such as Bis-Tris or Tris buffer) of about pH 7.4 (e.g., ±0.5, 0.4, 0.3, 0.2, or 0.1 pH units) containing about 150 mM NaCl (e.g., ±50, 40, 30, 20 or 10 mM), about 1 mM CaCl2 (e.g., ±500, 400, 300, 200, 100, or 50 μM), about 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 non-ionic detergent. A pH of about 7.4 is a typical target pH for formulations intended for use in humans (especially for intravenous delivery) because it matches the pH in human blood and provides a stable AnxA5 protein with good solubility. Below pH 7, especially down to around pH 6, the AnxA5 protein can lose solubility and begin to precipitate.

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

[0200] In one embodiment, the process of the invention provides a therapeutically acceptable, final, sterile AnxA5 protein product with a total yield of AnxA5 protein greater than 1 g per L 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. In this context, the term "about" may include ±0.4, 0.3, 0.2 or 0.1 g / L of the stated value.

[0201] In another embodiment, the process of the invention provides a therapeutically acceptable final sterile AnxA5 protein product with a total recovery of at least about 24% by weight (e.g., ±10, 9, 8, 7, 6, 5, 4, 3, 2, 1%) of AnxA5 protein present in the host cell culture, or more, as can be determined, for example, by measuring soluble AnxA5 protein in the initial homogenate (which can be captured and measured by centrifuging an aliquot 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 the process (before filling the sterile container, if relevant), and typically after the final purification step described above, the AnxA5 protein can be chemically modified. For example, the AnxA5 protein can 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 peptidomimetic compound (in the present invention, the AnxA5 protein) is modified to have one or more polyethylene glycol (PEG) molecules covalently attached to the side chains of one or more amino acids or derivatives thereof. It is one of the most important molecular alteration structural chemistry techniques (MASC). Other MASC techniques may be used, such as to improve the pharmacological properties of a molecule, for example to extend the in vivo half-life of the molecule. The PEG-protein conjugate is formed by first activating the PEG moiety so that it reacts with and couples to the protein or peptidomimetic compound of the present invention. PEG moieties vary considerably in molecular weight and conformation, with early moieties (monofunctional PEG, or mPEG) being linear with molecular weights of 12 kDa or less and later moieties being of increased molecular weight. A recent innovation in PEG technology, PEG2, involves the coupling of 30 kDa (or less) mPEG to the lysine amino acid (although PEGylation can be extended to the addition of PEG to other amino acids), which is further reacted to form branched structures that behave like linear mPEG of much higher molecular weight (Kozlowski et al., 2001). Methods that can be used to covalently attach 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 and references cited therein.Advantages of PEGylation include reduced renal clearance, which for some products may result in more sustained adsorption and limited distribution after administration, leading to consistent 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 carry out one or more additional purification steps, for example, to reduce or remove unreacted components and / or to select a homogenous population of chemically modified AnxA5 protein for incorporation into the final product. Suitable techniques for purification of chemically modified proteins from the reaction process are known to those skilled in the art.

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

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

[0205] The pharmaceutical or veterinary composition may contain the AnxA5 protein in admixture with a pharma- ceutical or veterinary acceptable adjuvant, diluent or carrier, which is typically selected with respect to the intended route of administration and standard pharmaceutical practice. The composition 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, daily sub-dose or an appropriate fraction thereof, of the active ingredient.

[0206] The phrase "pharmaceutical or veterinary acceptable" includes reference to a composition that does not produce adverse, allergic or other untoward reactions when administered to animals or humans, as appropriate. The preparation of such pharmaceutical or veterinary compositions will be known to those of skill in the art in light of the present disclosure, and as set forth by way of example in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990. Moreover, it will be understood that for animal or human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biological Standards.

[0207] As used herein, a "pharmaceutically or veterinarily acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, salts, preservatives, drugs, drug stabilizers, excipients, disintegrants, such similar materials, and combinations thereof, as would be known to one of ordinary skill in the art. Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.

[0208] The pharmaceutical or veterinary compositions according to the invention are intended for parenteral, intravenous, intraarterial, intraperitoneal, intramuscular, intraocular, intracranial, intracerebral, intraosseous, intraventricular, intrathecal or subcutaneous administration, for administration from a drug-eluting stent, for administration by infusion techniques, or for topical administration (such as in a form suitable for the epidermis, e.g., a cream or ointment, inhalation, eye drops / eye drops, ear drops / ear drops, or through internal mucous membranes), and therefore may or may not be formulated in a manner suitable therefor. Sterile injectable solutions may be prepared by incorporating the required amount of active compound in an appropriate solvent with various other ingredients as listed above, followed by sterilization. The pharmaceutical compositions may best be used in the form of a sterile aqueous solution, which may contain other substances, e.g., sufficient salts or glucose to make the solution isotonic with blood. If necessary, the aqueous solution may be suitably buffered (preferably to a pH of 3 to 9). The preparation of suitable pharmaceutical formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to those skilled in the art.

[0209] Pharmaceutical or veterinary compositions according to the invention may alternatively be formulated in the form of a powder, such as a sterile powder, which may be a lyophilized powder.

[0210] A therapeutically effective amount of AnxA5 protein for administration to a patient, such as a human patient, can be from 0.01 to 1000 mg of AnxA5 protein per adult (e.g., about 0.001 to 20 mg / kg of patient body weight, e.g., 0.01 to 10 mg / kg, for example, greater than 0.1 mg / kg and up to 20, 10, 5, 4, 3 or 2 mg / kg, e.g., about 1 mg / kg), administered in single or divided doses, based on daily dosage levels.

[0211] In any event, the physician will determine the actual dosage that will be most suitable for any individual patient, which will vary with the age, weight and response of the particular patient. The above dosage amounts are given as examples of average practice. Of course, there can be individual instances in which higher or lower dosage ranges are merited, and such instances are within the scope of the present invention.

[0212] For veterinary use, the compounds of the invention will be administered in a suitably acceptable formulation in accordance with normal veterinary practice, with the veterinarian determining the dosage regimen and route of administration that will be most suitable for a particular animal.

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

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

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

[0216] In a further embodiment, the process of any of the aspects of the invention may provide a product comprising 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. The FDA and EMA require endotoxins to be less than 100 EU / dose (maximum allowed is 350 EU / dose), which corresponds to less than 10 EU / mg at a 10 mg dose. Within these parameters, a unit dosage form may contain about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg of AnxA5 protein or more (e.g., within the range of 0.1-1000 mg, or 1-100 mg). Endotoxin can be measured, for example, by using LAL-based techniques or other EMA and FDA approved methods.

[0217] In a further embodiment, the process of any of the aspects of the invention provides an amount of AnxA5 protein of less than 1,000 pg per mg of AnxA5 protein, preferably less than 500 pg per mg of AnxA5 protein, less than 400 pg per mg of AnxA5 protein, less than 300 pg per mg of AnxA5 protein, less than 200 pg per mg of AnxA5 protein, less than 100 pg per mg of AnxA5 protein, less than 50 pg per mg of AnxA5 protein, less than 40 pg per mg of AnxA5 protein, less than 30 pg per mg of AnxA5 protein, Products may be provided that include a host cell nucleic acid (e.g., DNA) level, such as a host cell nucleic acid (e.g., DNA) level of 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, for example, about 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 particularly preferred embodiments, the process of any of the aspects of the present invention may provide a product having any one or more attributes selected from the list consisting of: - AnxA5 protein concentration, typically around 8-12 g / L; - Host cell protein levels of less than 100 ng / mg, more preferably less than 20 ng / mg (as determined by ELISA); - Host cell DNA levels below 100 pg / mg, more preferably below 10 pg / mg - endotoxins less than or equal to 35EU / mg, more preferably less than 1EU / mg; - greater than 95% purity as determined by size exclusion chromatography; - Bioburden less than 1 cfu / mL (determined by Ph.Eur.2.6.12); - a clear, colorless appearance with no visible particles; and - The main band detected by Western blot 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" may then include the meaning 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 commercially and as described in Marder et al., 2014, BMC Biotechnology, 14:33), such as 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 may be within 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. Alternatively stated, the level of gluconoylated AnxA5 protein in the product can be below 40%, e.g., below 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, preferably substantially 0%. Gluconoylated variants of Anx5 can be measured and quantified, for example, by using UPLC or HPLC chromatography instrumentation using a suitable anion exchange or reverse phase column.

[0220] Thus, a fifth aspect of the invention provides a composition comprising an AnxA5 protein, which composition is a direct or indirect product of (or obtainable directly or indirectly by) a process according to any of the first, second, third or fourth aspects of the invention. Optionally, the composition is a pharma- ceutically and / or veterinarily acceptable composition.

[0221] J. Medical and Veterinary Uses A sixth aspect of the invention also provides a composition of the fifth aspect of the invention for use in medicine. Alternatively stated, the sixth aspect of the invention provides a method comprising administering to a human or animal in need of treatment a therapeutically effective amount of a composition of the fifth aspect of the invention.

[0222] In certain embodiments of the sixth aspect of the invention, the composition of the fifth aspect of the invention may be used for: (a) for the prevention or reduction of the risk of thrombosis (such as atherothrombosis) and / or plaque rupture, or for administration to patients in risk groups, including, but not limited to, systemic lupus erythematosus (SLE) patients and / or patients who have or have had (or are at risk of having) an upper respiratory tract or other infection (including pneumococcal infection) that may cause increased levels of antiphospholipid-associated antibodies, or for treating (either actively or prophylactically) or reducing the risk of thromboembolism, hemorrhagic or vasculitic stroke, myocardial infarction, angina 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 insufficiency, angina pectoris, ischemic heart disease, peripheral arterial disease, systolic hypertension, migraine headaches, type 2 diabetes and erectile dysfunction, for reducing ischemic pain and / or for the treatment of vascular disease rupture, e.g. as 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 hyperplasia), or vascular inflammation, such as those described in WO2009 / 103977, the contents of which are incorporated herein by reference; (d) for use in inhibiting the activity of oxidized cardiolipin (oxCL) and in treating the following diseases in a mammal: cardiovascular disease (CVD), type II diabetes, Alzheimer's disease, dementia in general, rheumatic diseases, atherosclerosis, hypertension, acute and / or chronic inflammatory conditions, myocardial infarction, acute coronary syndromes, stroke, transient ischemic attack (TIA), claudication, angina pectoris, type I diabetes, rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's syndrome, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, erythroderma, psoriatic arthritis, psoriatic spondylitis, ... for treating, preventing and / or reducing the risk of developing cardiovascular diseases, autoimmune diseases or inflammatory conditions, including but not limited to lupus varicella, 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, such as those described in WO2010 / 069605, the contents of which are incorporated herein by reference; and (e) For the prevention and / or reduction of peri- or post-operative complications following surgical intervention, such as complications following vascular surgery, in particular peripheral vascular surgery, e.g., 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 invention, the composition of the fifth aspect of the invention may be used in a prophylactic or therapeutic method of treating, preventing or reducing the risk of hematological disorders, including but not limited to sickle cell anemia.

[0224] In a further embodiment of the sixth aspect of the invention, the composition of the fifth aspect of the invention may be used in a prophylactic or therapeutic method of treating, preventing or reducing the risk of acute and chronic vascular inflammation, primary or secondary vasculitis, including but not limited to vasculitis with an autoimmune component and / or drug-induced vasculitis. Thus, the invention also provides a prophylactic or therapeutic method of treating, preventing or reducing the risk of vasculitis, Behcet'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), polymyalgia rheumatoid arteritis, and Takayasu's arteritis. Polymyalgia rheumatoid arteritis may be of particular interest.

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

[0226] In a further embodiment of the sixth aspect of the invention, the composition of the fifth aspect of the invention may be used in a prophylactic or therapeutic method to (i) prevent or reduce the rate of 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) Viruses that display phosphatidylserine (PS) and mediate cell infection and / or internalization via PS binding.

[0227] Thus, in a further embodiment, the invention provides a composition of the fifth aspect of the invention for use in a prophylactic or therapeutic method of preventing or reducing the rate of transmission of a viral infection in a subject, the viral infection being caused by a virus capable of causing haemorrhagic fever (VHF).

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

[0229] Stated yet another way, this embodiment provides a composition of the fifth aspect of the invention for use in the manufacture of a medicament for prophylaxis or therapy by preventing or reducing the rate of transmission of a viral infection in a subject, the viral infection being caused by a virus capable of causing hemorrhagic fever (VHF).

[0230] In another embodiment, the invention provides a composition of the fifth aspect of the invention for use in a prophylactic or therapeutic method of preventing or reducing the rate of transmission of a viral infection in a subject, the viral infection being caused by a virus that displays phosphatidylserine (PS) and mediates cell infection and / or internalization via PS binding.

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

[0232] Stated yet another way, this embodiment provides a composition of the fifth aspect of the invention for use in the manufacture of a medicament for prophylaxis or therapy by preventing or reducing the rate of transmission of a viral infection in a subject, the viral infection being caused by a virus that displays phosphatidylserine (PS) and mediates cell infection and / or internalization via PS binding.

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

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

[0235] Stated yet another way, this embodiment provides a composition of the fifth aspect of the invention for use in the manufacture of a medicament for prophylaxis or therapy by preventing or protecting against a viral infection in a subject, the viral infection being caused by a virus capable of causing hemorrhagic fever (VHF).

[0236] In another embodiment, the invention provides a composition of the fifth aspect of the invention for use in a prophylactic or therapeutic method of preventing or protecting against viral infection in a subject, the viral infection being caused by a virus that displays phosphatidylserine (PS) and mediates cell infection and / or internalization via PS binding.

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

[0238] Stated yet another way, this embodiment provides a composition of the fifth aspect of the invention for use in the manufacture of a medicament for prophylaxis or therapy by preventing or protecting against viral infection in a subject, the viral infection being caused by a virus that displays phosphatidylserine (PS) and mediates cell infection and / or internalization via PS binding.

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

[0241] Stated yet another way, this embodiment provides a composition of the fifth aspect of the invention for use in the manufacture of a medicament for the prophylaxis or therapy of treating a viral infection in a subject, the viral infection being caused by a virus capable of causing hemorrhagic fever (VHF).

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

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

[0244] Stated yet another way, this embodiment provides a composition of the fifth aspect of the invention for use in the manufacture of a medicament for prophylaxis or therapy by treating a viral infection in a subject, the viral infection being caused by a virus that displays phosphatidylserine (PS) and mediates cell infection and / or internalization via PS binding.

[0245] According to a further embodiment of the invention there is provided a composition of the fifth aspect of the invention for use in a method of treating a subject infected or suspected of being infected with a pathogen capable of causing hemorrhagic fever, such as a virus capable of causing hemorrhagic fever (VHF) or a bacterium capable of causing hemorrhagic fever (BHF).

[0246] Alternatively stated, this embodiment provides a method for treating a subject infected or suspected of being infected with a pathogen capable of causing hemorrhagic fever, such as a virus capable of causing hemorrhagic fever (VHF) or a bacterium capable of causing hemorrhagic fever (BHF), the method comprising administering to the subject a therapeutically effective amount of a composition of the fifth aspect of the invention.

[0247] Stated yet another way, this embodiment provides a composition of the fifth aspect of the invention for use in the manufacture of a medicament for the treatment of a subject infected or suspected of being infected with a pathogen capable of causing hemorrhagic fever, such as a virus capable of causing hemorrhagic fever (VHF) or a bacterium capable of causing hemorrhagic fever (BHF).

[0248] According to another embodiment of the invention there is provided a composition of the fifth aspect of the invention for use in a method of treating a subject who has been in contact with another subject infected or suspected of being infected with a pathogen capable of causing hemorrhagic fever, such as a virus capable of causing hemorrhagic fever (VHF) or a bacterium capable of causing hemorrhagic fever (BHF).

[0249] Alternatively stated, this embodiment provides a method for treating a subject who has been in contact with another subject infected or suspected of being infected with a pathogen capable of causing hemorrhagic fever, such as a virus capable of causing hemorrhagic fever (VHF) or a bacterium capable of causing hemorrhagic fever (BHF), the method comprising administering to the subject a therapeutically effective amount of a composition of the fifth aspect of the invention.

[0250] Stated yet another way, this embodiment provides a composition of the fifth aspect of the invention for use in the manufacture of a medicament for the treatment of a subject who has been in contact with another subject infected or suspected of being infected with a pathogen capable of causing hemorrhagic fever, such as a virus capable of causing hemorrhagic fever (VHF) or a bacterium capable of causing hemorrhagic fever (BHF).

[0251] According to a further embodiment of the invention there is provided a composition of the fifth aspect of the invention for use in a method of treating a subject who has come into contact with biological material present in or produced by another subject infected or suspected of being infected with a pathogen capable of causing hemorrhagic fever, such as a virus capable of causing hemorrhagic fever (VHF) or a bacterium capable of causing hemorrhagic fever (BHF).

[0252] Alternatively stated, this embodiment provides a method for treating a subject who has come into contact with biological material present in or produced by another subject infected or suspected of being infected with a pathogen capable of causing hemorrhagic fever, such as a virus capable of causing hemorrhagic fever (VHF) or a bacterium capable of causing hemorrhagic fever (BHF), the method comprising administering to the subject a therapeutically effective amount of a composition of the fifth aspect of the invention.

[0253] Stated yet another way, this embodiment provides a composition of the fifth aspect of the invention for use in the manufacture of a medicament for the treatment of a subject that has been in contact with biological material present in or produced by another subject infected or suspected of being infected with a pathogen capable of causing hemorrhagic fever, such as a virus capable of causing hemorrhagic fever (VHF) or a bacterium capable of causing hemorrhagic fever (BHF).

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

[0255] Viral hemorrhagic fever (VHF) is a diverse group of animal and human diseases that can be caused by at least five distinct families of RNA viruses: the Arenaviridae, Filoviridae, Bunyaviridae, Flaviviridae, and Rhabdoviridae families. All types of VHF can be characterized by fever and hemorrhagic lesions, and all can progress in many cases to high fever, shock, and death.

[0256] A subject suspected of being infected with a pathogen capable of causing hemorrhagic fever, such as a virus capable of causing hemorrhagic fever (VHF) or a bacterium capable of causing hemorrhagic fever (BHF), may be a subject who has been in contact with the disease in the past (e.g., through employment as a health care worker or due to infection of a household member) and / or may be a subject who exhibits one or more signs or symptoms of infection prior to a confirmatory diagnosis.

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

[0258] In a sixth aspect of the invention, the VHF may be Ebola and the subject may present with one or more symptoms of Ebola, such as early clinical symptoms such as excessive or profuse sweating, fever, muscle pain, general fatigue, and / or chills; and / or flu-like symptoms, optionally with gastrointestinal symptoms; maculopapular rash, petechiae, conjunctival hemorrhage, epistaxis, melena, hematemesis, shock and / or encephalopathy; leukopenia (e.g. associated with increased lymphoid cell apoptosis), thrombocytopenia, increased aminotransferase levels, 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 reference laboratory with advanced biocontainment capabilities. Laboratory findings vary somewhat between viruses, but generally include a decrease in total white blood cell count (especially lymphocytes), a decrease in platelet count, an increase in serum liver enzymes, and a decrease in blood clotting ability measured as an increase in both prothrombin (PT) and activated partial thromboplastin time (PTT). Hematocrit may be elevated. Serum urea and creatine may be elevated, depending on the hydration status of the patient. Bleeding time tends to be prolonged.

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

[0261] During acute disease, assays included 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 disease course, tests that can be used include a) IgM and IgG ELISA using authentic viral antigens, and in fatal cases, autopsy tissue can be used for a) antigen detection using immunostaining techniques, b) immunohistochemistry-assisted detection of Ebola antigens (Zaki et al, J Infect Dis, 1999;179(Suppl.1):S36e47., the contents of which are incorporated herein by reference in their entirety), and c) in situ hybridization techniques for detection of viral RNA.

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

[0264] An ELISA-based assay has been standardized by the CDC for the detection of Ebola virus-specific antibodies. This assay has been shown to have high sensitivity and to be capable of detecting antibodies in the serum of humans exposed to Ebola a decade ago. Cell-based plaque assays and end-point titration assays (TCID50) have also been developed to detect and quantitate filoviruses for use in preclinical studies (Shurtleff et al, Viruses 2012;4:3511e30, Smither et al, J Virol Methods 2013;193:565e71, the contents of which are incorporated herein by reference in their entirety).

[0265] For example, the VHF may be a virus of a family selected from Filoviridae, Arenaviridae, Bunyaviridae, Flaviviridae or Rhabdoviridae.

[0266] The Arenaviridae family includes the viruses responsible for Lassa fever, Lujo virus, Argentine, Bolivian, Brazilian, and Venezuelan hemorrhagic fever.

[0267] The Bunyaviridae family includes members of the genus Hantavirus, which causes hemorrhagic fever with renal syndrome (HFRS), Crimean-Congo hemorrhagic fever (CCHF) virus from the genus Nairovirus, Garissa virus and Ilesha virus from the genus Orthobunyavirus, and Rift Valley fever (RVF) virus from the genus Phlebovirus.

[0268] The Filoviridae family includes Ebola virus and 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 members of the Rhabdoviridae family responsible for two fatal and two non-fatal cases of hemorrhagic fever in the Bas-Congo region of the Republic of Congo was also reported, the latter occurring in health care workers who had been treating two other individuals, suggesting possible person-to-person transmission.

[0271] Thus, for example, in one embodiment of particular interest, the invention may be applied to viruses of the Filoviridae family, such as Ebola virus and Marburg virus, and in another embodiment of particular interest, the invention may be applied to viruses of the Flaviviridae family, such as Dengue virus.

[0272] The present invention therefore provides a composition of the fifth aspect of the invention for use in the above-mentioned prophylactic or therapeutic methods for (i) preventing or reducing the rate of transmission of Ebola infection, (ii) preventing or protecting against Ebola infection, or (iii) treating Ebola infection in a subject who is infected or suspected to be infected with the Ebola virus, or who has been in contact or is expected to be in contact with another subject infected or suspected to be infected with the Ebola virus, or who has been in contact or is expected to be in contact with biological material present in or produced by another subject infected or suspected to be infected with the Ebola virus.

[0273] Thus, the present invention provides a composition of the fifth aspect of the invention for use in the above-mentioned prophylactic or therapeutic methods for (i) preventing or reducing the rate of transmission of Marburg infection, (ii) preventing or protecting against Marburg infection, or (iii) treating Marburg infection in which a subject is infected or suspected to be infected with Marburg virus, or has had or is expected to have contact with another subject infected or suspected to be infected with Marburg virus, or has had or is expected to have contact with biological material present in or produced by another subject infected or suspected to be infected with Marburg virus.

[0274] The present invention therefore provides a composition of the fifth aspect of the invention for use in the above-mentioned prophylactic or therapeutic methods for (i) preventing or reducing the rate of transmission of dengue virus infection, (ii) preventing or protecting against dengue virus infection, or (iii) treating dengue virus infection, wherein a subject is infected or suspected to be infected with dengue virus, or has been in contact or is expected to be in contact with another subject infected or suspected to be infected with dengue virus, or has been in contact or is expected to be in contact with biological material present in or produced by another subject infected or suspected to be infected with dengue virus.

[0275] The present invention also provides a composition of the fifth aspect of the invention for use in the above-mentioned method for treating, delaying the onset and / or slowing the progression of infection by VHF or BHF in a subject.

[0276] The present invention also provides a composition of the fifth aspect of the invention for use in the above-described method for preventing, reducing, delaying the onset or slowing the progression of direct and / or indirect bacterial and / 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 to prevent, reduce, delay the onset or slow the progression of direct and / or indirect bacterial or viral damage to the immune system in a subject, for example in the context of Ebola infection. For example, the bacterial or viral damage may be selected from damage to the innate immune response, damage to the adaptive 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, or slow the progression of blood leakage (hemorrhage), hypotension, drop in blood pressure, shock, or death in a subject.

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

[0280] The invention provides a composition of the fifth aspect of the invention for use in a method of preventing, reducing, delaying the onset or slowing the progression of damage, activation, death and / or disruption of the integrity of the vascular endothelium or endothelial cells thereof 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 endothelial cells thereof may be determined, for example, by the extent of cellular or vascular epithelial leakage and / or by detection of one or more bleeding events in the subject, or the formation of oedema and / or dehydration.

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

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

[0283] A further embodiment of the invention provides a composition of the fifth aspect of the invention for use in a prophylactic or therapeutic method as described above with reference to the various embodiments of the invention, wherein the viral infection is caused by a virus that presents phosphatidylserine (PS) and mediates cell infection and / or internalization 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 internalization via PS binding may include encapsulated viruses that contain phosphatidylserine (PS) in the viral envelope, particularly in the outer layer. The presentation of PS by viruses can be determined by methods known in the art, for example, using ELISA studies that measure the binding of Annexin A5 to viruses. Suitable 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] A group of viruses of particular interest to the present invention includes those that mediate cell infection and / or internalization via binding to phosphatidylserine-mediated virus entry enhancing receptors (PVEER), discussed in Moller Tank, et al, 2013, J. Virol., 87(15), 8327-8341, one example of which is the T-cell immunoglobulin and mucin 1 (TIM-1) receptor. 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 one virus of particular interest that displays phosphatidylserine (PS) to mediate cell infection and / or internalization via PS binding to TIM-1. Moller Tank, et al, 2013, J. Virol., 87(15), 8327-8341.

[0287] The present invention recognises that annexin A5, and compositions of the fifth aspect of the invention, may be used to inhibit or block PS-mediated cell infection and / or internalisation of viruses, such as Ebola virus, via a PVEER, such as TIM-1, and thereby may be useful in prophylactic or therapeutic methods of (i) preventing or reducing the rate of transmission of a viral infection, (ii) preventing or protecting against transmission of a viral infection, or (iii) treating a viral infection in a subject, the viral infection being caused by viruses that present phosphatidylserine (PS) and mediate cell infection and / or internalisation via PS binding.

[0288] The virus that displays phosphatidylserine (PS) can be selected from the group consisting of, for example, Filoviridae family viruses (such as Ebola and Marburg), Flaviviridae family, Hepatitis A, alphavirus, baculovirus, and arenavirus.The virus can be infectious in humans or only in humans.The virus can be infectious in non-human animals, such as any one or more of the animals selected from dog, cat, cow, sheep, pig, goat, rodent, camel, domestic animal, and wild animal, or only in non-human animals.

[0289] PVEER, such as TIM-1, may be involved in the internalization of the virus into various cell types. In one embodiment, cell types of particular interest for protection and / or treatment 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 in particular activated CD4+ cells.

[0290] TIM-1, also known as HAVCR1 and KIM-1, was identified as a susceptibility gene for human asthma (McClntire 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 (SEQ ID NO: 2).

[0291] TIM-1 is a type I membrane protein with an IgV domain, an extracellular region containing 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 in which the CC' loop folds over the GFC β-strand, resulting in a unique cleft formed by the CC' and FG loops (Santiago et al, 2007, Immunity 26(3):299-310). The cleft constructed by the CC' and FG loops is the binding site for phosphatidylserine (Kobayashi et al, 2007, Immunity 27(6):927-40). Antibodies directed against the CC' / FG cleft of the TIM-1 IgV domain inhibit TIM-1 binding to phosphatidylserine and dendritic cells and exhibit therapeutic activity in vivo in a humanized mouse model of allergic asthma (Sonar et al, 2010, J. Clin. Invest. 120:2767-81).

[0292] A further embodiment of the present invention is based on the use of the composition of the fifth aspect of the present invention to prevent, inhibit or reduce the ability of the IgV domain of TIM-1 and other PVEER to bind to PS presented thereto. The AnxA5 protein in the composition 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 capable of competing with PVEER for binding to PS.

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

[0294] For example, this may be prophylactically or therapeutically useful in the context of presenting phosphatidylserine (PS) to inhibit, reduce or prevent infection of cells by viruses that mediate cell infection and / or internalization via PS.

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

[0296] Therefore, in another embodiment, the present invention provides a method for inhibiting or reducing the binding of TIM-1 or other PVEER to phosphatidylserine, comprising contacting a first cell expressing TIM-1 or other PVEER with an effective amount of the composition of the fifth aspect of the present invention to inhibit or reduce the binding of the first cell to a second cell containing phosphatidylserine on the cell surface or to a virus that displays phosphatidylserine (PS) on its surface.The method can be an in vivo method or an in vitro method.In the case of an in vivo method, it can be to treat or prevent a pathology that involves the binding of PS to TIM-1 or other PVEER.

[0297] In other words, this embodiment of the invention also provides a composition of the fifth aspect of the invention for use in a prophylactic or therapeutic method for inhibiting or reducing binding of TIM-1 or other PVEER to phosphatidylserine in a patient in need of treatment.

[0298] In another embodiment, the present invention provides a method for inhibiting or reducing the binding of PS to TIM-1 or other PVEER on dendritic cells, comprising contacting dendritic cells expressing TIM-1 or other PVEER with an effective amount of the composition of the fifth aspect of the present invention to inhibit or reduce the binding of PS to dendritic cells. The method may be an in vivo method or an ex vivo method. In the case of an in vivo method, it may be to treat or prevent a pathology involving the binding of PS to TIM-1 or other PVEER on dendritic cells.

[0299] In other words, this embodiment of the invention also provides a composition of the fifth aspect of the invention for use in a prophylactic or therapeutic method for inhibiting or reducing binding of PS to TIM-1 or other PVEER on dendritic cells in a patient in need of treatment.

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

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

[0302] Also disclosed is a method of treating or preventing asthma, the method comprising administering to a mammal having asthma a pharmaceutical composition comprising a composition of the fifth aspect of the invention.

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

[0304] Also disclosed is a method of treating or preventing an atopic disorder, the method comprising administering to a mammal having an atopic disorder a pharmaceutical composition comprising a therapeutically effective amount of the composition of the fifth aspect of the invention. The atopic disorder can be, for example, atopic dermatitis, contact dermatitis, urticaria, allergic rhinitis, angioedema, latex allergy, or an allergic lung disorder (e.g., asthma, allergic bronchial aspergillosis, or hypersensitivity pneumonitis).

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

[0306] The composition of the fifth aspect of the invention, as described herein, for use in treating or preventing a variety of TIM-1-associated disorders, including immunological disorders such as inflammatory disorders and autoimmune disorders, and other PVEER-associated disorders.

[0307] The term "treating" includes the meaning of administering a substance or composition described herein in an amount, manner, and / or mode effective to ameliorate a pathology, symptom, or parameter associated with a disorder, either to a statistically significant degree or to a degree detectable by one of skill in the art, or to prevent progression of the disorder or its worsening, including secondary damage caused by the disorder.

[0308] The composition of the fifth aspect of the invention can be administered to a subject at risk of, diagnosed with, or having one of these disorders in an amount and for such a time period that provides an overall therapeutic effect. The composition of the fifth aspect of the invention can be administered alone (monotherapy) or in combination with other agents (combination therapy), either mixed or by separate, simultaneous or sequential administration. In the case of combination therapy, the amount and time of administration can be such as to provide, for example, an additive or synergistic therapeutic effect. Furthermore, administration of the composition of the fifth aspect of the invention (with or without a second agent) can be used as a primary treatment, e.g., a first-line treatment, or as a second-line treatment, e.g., for subjects with an inadequate response to a previously administered therapy (i.e., a therapy other than a therapy with AnxA5 protein).

[0309] Diseases or conditions treatable with the compositions of the fifth aspect of the invention described herein include, for example, ischemia-reperfusion injury (e.g. organ ischemia-reperfusion injury, such as liver or renal ischemia-reperfusion injury), allergy, 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 invention described herein include, for example, autoimmune disorders.

[0311] Systematic lupus erythematosus (SLE, lupus) 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] Examples of other organ-specific or systemic autoimmune diseases suitable for treatment with the composition of the fifth aspect of the present invention described herein include myasthenia gravis, autoimmune hemolytic anemia, Chagas disease, Graves disease, idiopathic thrombocytopenic purpura (ITP), Wegener's 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 described herein.

[0313] Additional TIM-1-related diseases or conditions treatable with the composition of the fifth aspect of the 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 using Annexin A5 described herein. GVHD is initiated when donor T cells recognize host antigens as foreign. GVHD, which is often a fatal outcome of bone marrow transplantation (BMT) in human patients, can be acute or chronic. Acute and chronic forms of GVHD are examples of the development of antigen-specific Th1 and Th2 responses, respectively. Acute GVHD occurs within the first two months after BMT and is characterized by donor cytotoxic T cell-mediated damage to the skin, intestine, liver, and other organs. Chronic GVHD appears later (>100 days after BMT) and is characterized by overproduction of immunoglobulins (Ig), including autoantibodies, and damage to the skin, kidney, and other organs caused by Ig deposition. Nearly 90% of patients with acute GVHD go on to develop 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 cytotoxic activity is a hallmark of acute GVHD. The consequences of donor anti-host cytotoxic activity can be seen in a variety of ways. First, host lymphocytes are rapidly destroyed such that mice experiencing acute GVHD are severely immunosuppressed. Second, donor lymphocytes become engrafted and proliferate in the host spleen, and their cytotoxic activity can be measured directly ex vivo by utilizing cell lines expressing host antigens that can be recognized (as foreign) by the donor cells. Third, the disease becomes fatal as additional tissues and cell populations are destroyed.

[0314] Additional TIM-1-related diseases or conditions treatable with the compositions of the fifth aspect of the invention described herein include, for example, atopic disorders. Atopic disorders are characterized by the expression of cytokines, chemokines, and other molecules characteristic of Th2 responses, such as IL-4, IL-5, and IL-13 cytokines, among others, by immune system cells, including activated T cells and APCs. Such atopic disorders are therefore amenable to treatment with the compositions of the fifth aspect of the invention described herein. Atopic disorders include airway hyperresponsiveness 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 composition of the fifth aspect of the invention described herein include, for example, a number of immune or inflammatory disorders.Immune or inflammatory disorders include allergic rhinitis; autoimmune hemolytic anemia; acanthosis nigricans, Addison's disease; alopecia areata; alopecia universalis; amyloidosis; anaphylactoid purpura; anaphylactoid reactions; aplastic anemia; ankylosing spondylitis; cranial arteritis; giant cell arteritis; Takayasu's arteritis; temporal arteritis; ataxia telangiectasia; autoimmune oophoritis; autoimmune orchitis; autoimmune polyendocrine deficiency; Behcet's disease; Berger's disease; Buerger's disease; bronchitis; bullous pemphigoid; chronic mucocutaneous candidiasis; Kaplan's syndrome; post-myocardial infarction. syndrome;postpericardiotomy syndrome;carditis;celiac sprue;Chagas disease;Chediak-Higashi syndrome;Churg-Strauss disease;Cogan syndrome;cold agglutinin disease;CREST syndrome;Crohn's disease;cryoglobulinemia;idiopathic fibrosing alveolitis;dermatitis herpetiformis;dermatomyositis;diabetes mellitus;Diamond-Blackfan syndrome;DiGeorge syndrome;discoid lupus erythematosus;eosinophilic fasciitis;episcleritis;erythema elevatus perstans;erythema marginatum;erythema multiforme;erythema nodosum;familial Mediterranean fever;Felty's syndrome;pulmonary fibrosis;anaphylactoid Glomerulonephritis;Autoimmune glomerulonephritis;Poststreptococcal glomerulonephritis;Posttransplant glomerulonephritis;Membranous glomerulopathy;Goodpasture's syndrome;Immune-mediated granulocytopenia;Granuloma annulare;Allergic granulomatosis;Granulomatous myositis;Graves' disease;Hashimoto's thyroiditis;Hemolytic disease of the newborn;Idiopathic hemochromatosis;Henoch-Schonlein purpura;Chronic active and chronic progressive hepatitis;Histiocytosis X;Hypereosinophilic syndrome;Idiopathic thrombocytopenic purpura;Jobs' syndrome;Juvenile dermatomyositis;Juvenile rheumatoid arthritis (juvenile chronic arthritis);Kawasaki disease; Keratitis;Keratoconjunctivitis sicca;Landry-Guillain-Barré syndrome;Lepromatous leprosy;Leffler's syndrome;Lupus;Lyell's syndrome;Lyme disease;Lymphomatoid granulomatosis;Systemic mastocytosis;Mixed connective tissue disease;Mononeuritis multiplex;Muckle-Wells syndrome;Mucocutaneous lymph node syndrome;Mucocutaneous lymph node syndrome;Multicentric reticulohistiocytosis;Multiple sclerosis;Myasthenia gravis;Mycosis fungoides;Systemic necrotizing vasculitis;Nephrotic syndrome;Overlap syndrome;Panniculitis;Paroxysmal cold hemoglobinuria;Paroxysmal nocturnal hemoglobinuria;Pemphigoid;Pemphigus;Pemphigus erythematosus;Pemphigus foliaceus;Pemphigus vulgaris;Pigeon breeder's disease;Polyarteritis nodosa;Polymyalgia rheumatica;Polymyositis;Idiopathic polyneuropathy;Portuguese familial polyneuropathy;Preeclampsia / eclampsia;Primary biliary cirrhosis;Progressive systemic sclerosis (scleroderma);Psoriasis;Psoriatic arthritis;Pulmonary 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 ophthalmia;Systemic lupus erythematosus;Transplant rejection rejection); ulcerative colitis; undifferentiated connective tissue disease; chronic urticaria; cold urticaria; uveitis; vitiligo; Weber-Christian disease; Wegener's granulomatosis; or Wiskott-Aldrich syndrome;

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

[0317] The following examples are included to demonstrate certain embodiments of the invention. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to work well in carrying out the invention, and therefore may be considered to constitute preferred modes for carrying out the invention. However, those skilled in the art should, in light of this disclosure, understand that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results, without departing from the spirit and scope of the invention.

[0318] Comparative Example 1 The process by Marder et al., 2014, BMC Biotechnology, 14:33, reports the processing of a 1 L culture, which consists of two 38,900 g centrifugations of 30 min duration, where the first centrifugation step precipitates Annexin A5 bound to the cell debris, and the second centrifugation step precipitates the cell debris while keeping Annexin A5 in solution.

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

[0320] Based on the selection of the best centrifuges currently available, equipped with the best rotors for maximum throughput, they can accommodate 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 the Thermo Scientific™ Sorvall™ LYNX ultra-high speed centrifuge, or the JLA-16.250 rotor, fixed angle, aluminum, biosafety lid, 6 x 250 mL, 38,400 x g for use in the Beckmancoulters Avanti JXN-26. If such a high-end high-performance centrifuge was used, it would take about 45 minutes to load the centrifuge, start it, accelerate to the required speed, spend 30 minutes at maximum G-force, then carefully break the pellet until it comes to rest so as not to disturb it, and then empty the rotor.

[0321] Thus, the best centrifuges currently available allow processing of 1.5 liters per 45 minutes.

[0322] Marder et al. (in the section entitled "Purification") report that 3 g wet weight of cells are suspended in 30 mL of buffer prior to sonication and centrifugation. Thus, the cell wet weight (WCW) concentration used by Marder et al. during centrifugation was (3 grams in 30 mL buffer) = 9.1% WCW.

[0323] Marder et al. also reported (page 2, under the heading "Bioreactor cultivation") that the biomass concentration was 27.48 g(DCW)L -1 The authors report the mean (SD = 1.96) of the mean cell dry weight (DCW) concentration in the Marder fermentor was therefore 27.48 grams / L = 2.748%. It is known that 1 gram DCW = approximately 4 grams cell wet weight (WCW). Thus, in the cell concentration fermentor there was a WCW concentration of 2.748 x 4 = 11.0%. If this is scaled up to a 1000L culture volume and conservatively assuming a 5% cell loss while harvesting from the 1000L, the WCW in the 1000L tank using the Marder method would be 1000 x 11% x 0.95 = 104.5 kg WCW.

[0324] The Marder centrifugation method used a 9.1% WCW concentration during the centrifugation step. Therefore, 104.5 kg WCW from a 1000 L culture would need to be diluted to a 9.1% WCW concentration, which would require a total centrifugation volume of 1148 L.

[0325] Assuming generously that the biomanufacturing facility has two high-end, high-performance centrifuges, one of which can be used to pellet the annexin along with the cell debris (first centrifuge), while the other can run in parallel with the second centrifuge when the annexin is in solution and the cell debris is being pelleted, the following is done:

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

[0327] Assuming that the biomanufacturing facility operated 12 hours per day, processing of WCW from a 1000 L vessel using the method of Marder et al. would take 48 days of work, or 10 weeks of centrifugation alone (assuming 5 work days per week).

[0328] Overall, approximately another two weeks would be required for fermentation, downstream processing, and other operations. Assuming that the biomanufacturing facility is fully occupied with that one process, and therefore no other production can take place in the same facility during that time, this would take 12 weeks in the manufacturing plant to prepare and process the cells from the 1000L culture. This is despite the generous assumption that two centrifuges are available. If only one centrifuge is used, production would be 22 weeks per 1000L batch.

[0329] In contrast, as discussed below, the method of the present invention can process a 1000 L culture in only 2 weeks, i.e., approximately 6-fold more rapidly (and also provides a much higher quality product, in much higher yields, than the Marder et al. process).

[0330] The production cost is directly proportional to the production time since the manufacturing plant is occupied during which no other production can take place in the same plant.

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

[0332] Furthermore, the purity of the protein from Marder's process would not be suitable for human use. Despite careful centrifugation, only one anion exchange chromatography step is used, which falls far short of the requirements to reach sufficient purity, both with respect to in-process related impurities (especially endotoxins) and product-related variants. Marder does not present any data on endotoxin levels or other impurities, further suggesting a lack of suitability for pharmaceutical use.

[0333] In addition, the very slow centrifugation step of the Marder process requires the Annexin A5 protein to be exposed to an unstable environment for a long period of time, which is likely to result in product degradation or denaturation, a further drawback of long manufacturing runs with negative impacts on product quality.

[0334] Example 1 TIFF2025066728000002.tif240170TIFF2025066728000003.tif54170

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

[0336] The manufacturing process is scaled up from laboratory scale equivalent to a 3 L fermentation volume to large scale equivalent to a 100 L 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 for Annexin A5. As a final refinement step, high solubility anion exchange chromatography is used. The refinement step allows for the separation of product-related impurities. Formulation is performed by ultra-diafiltration using 10 kD NMWCO cassettes.

[0338] This example describes and evaluates planned adaptations / changes in the manufacturing process, determines the operational parameters and necessary measures to ensure successful transfer, and defines sufficient criteria to determine the success. Successful transfer is demonstrated by the performance of downstream process operations that implement the adaptations to the laboratory-scale procedures on scale-up resulting in DS of comparable yield and quality.

[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] FIG. 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 by the 3L and 100L processes. Table 1: Comparison of raw materials used by the 3L and 100L processes TIFF2025066728000004.tif239170TIFF2025066728000005.tif31170

[0343] Table 2 compares and evaluates the consumables used by the 3L and 100L processes. The 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 TIFF2025066728000006.tif152170

[0344] Review: • All consumables used are single use or product specific materials. Bags used for buffer storage and as intermediate product containers are from Sartorius with a PE / EVOH layer (CX5-14 film) throughout the process, i.e. bags validated by the manufacturer for sterility, low endotoxins and leakable and extractable materials. All other consumables used, including product contact materials such as tubing, connectors, sample collection systems or sample containers, are fit for purpose at each step of the process. This typically includes USP Class VI certification, sterility and / or low endotoxins where applicable. Platinum cured silicone tubing is used throughout the DSP, except for the C-Flex tubing which is integrated with the bag. All consumables used are free of animal-derived components or have TSE certification available.

[0345] Table 3 compares the equipment used by the 3 L and 100 L processes for the manufacture of Annexin A5. Table 3: List of equipment used TIFF2025066728000007.tif233170

[0346] Media, buffers and solutions are as shown in Table 4. Buffer specifications apply only for conductivity and are based on test buffer preparation. Buffers are prepared prior to the process, tested according to their specifications, microfiltered (0.2 μm filter) and stored (holding time of 3 months or less at room temperature). Buffers are sampled at the time of use (for reference; degradation: endotoxins, bioburden). Table 4: List of media and solutions. TIFF2025066728000008.tif235170TIFF2025066728000009.tif167170

[0347] A scalable fed-batch fermentation process was developed and scaled up in a production unit. 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 allow 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] A successful test run was performed on a DSP matching 3 L fermentation volume, demonstrating sufficient process performance for all process steps.

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

[0350] Process Comparison In the next section, the lab-scale downstream process (DSP) is detailed based on a trial run performed at a process scale equivalent to a 3 L fermentation volume. In general, apart from the loading of the first capture step, the chromatographic steps are performed at lab-scale using an Aekta Explorer system. At large scale, all chromatographic steps are carried out on a Bioprocess system. The scale-up factor of the DSP is 33 (3 L USP From 100L USP to).

[0351] 1.1.1 Biomass resuspension, benzonase treatment and cell disruption After fermentation, the biomass is harvested by centrifugation and stored at -20°C. Downstream processing begins with thawing of the biomass and resuspension in homogenization buffer 1. Prior to 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 Benzonase, pre-diluted in 1000 cc (1000 mL) is added to the resuspended cells. The resuspension ratio is set at 1 g biomass / 10 mL. Homogenization is performed for 3 cycles at 600 bar to achieve a high degree of homogeneity that is beneficial for the following capture step. No active cooling is required within the homogenization, since an elevated temperature of up to 40 °C is desired to allow optimal digestion by benzonase of the nucleic acids. At small scale, a temperature range of 36-40 °C was obtained.

[0352] A process flow diagram for biomass resuspension, benzonase treatment and cell disruption is shown below. TIFF2025066728000010.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 reduced particle solution amenable to capture chromatography. The depth filter is pre-washed with water according to the manufacturer's instructions.

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

[0355] This conditioned pool is applied to AX capture chromatography offline via a peristaltic pump. The AX capture column is equilibrated with 2 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 and then transferred to the chromatography system for a further 5 CV wash. Annexin A5 elution is performed by step elution using increasing salt (20 mM Tris pH 7.4; 0.1% Tween 80; 300 mM NaCl) over 9 CV. Fractionation is performed by UV spectrometry in 0.1 absorbance unit (AU) increments. 280nm The signal is defined as rising to 0.2 AU at the descending peak. All eluted peaks are further processed.

[0357] A two-step CIP procedure is performed to regenerate and clean the column (step 1: 3 CV of 2M NaCl, 100 cm / h; upflow / step 2: 3 CV of 1M NaOH; incubation >15 h, 40 cm / h upflow). The column is finally stored in 20 mM NaOH.

[0358] FIG. 3 provides a process flow diagram for AX capture chromatography.

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

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

[0361] The filtered AX pool was then diluted 8-fold (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 slow compared to ionic interactions. Contact time is critical and therefore chromatography is performed at 100 cm / h or less.

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

[0363] Elution is performed by step 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 bind to heparin only in the presence of calcium. To allow concentrated elution, the flow rate was reduced to 60 cm / h or less during elution. The complete elution peak was collected, for example, starting from the rise of the UV signal at 0.05 AU and up to 0.05 AU in the descending peak, which corresponds to approximately 7 CV. The elution profile shows a single sharp peak.

[0364] A two-step CIP procedure is performed to regenerate and clean the column (step 1: 3 CV of 2M NaCl, 100 cm / h; upflow / step 2: 3 CV of 0.1M NaOH; incubation >15 h, 40 cm / h upflow). The column is finally stored in 1M NaCl in 25% EtOH.

[0365] FIG. 1 shows a process flow diagram for intermediate affinity chromatography.

[0366] The intermediate step is the most potent purification step in the process scheme. Annexin A5 binds calcium ions. In this calcium-bound state, the product can form a highly specific bond with heparin. Only correctly folded Annexin A5 forms capable of complexing with calcium can bind heparin. The affinity chromatographic step can thereby discriminate between correctly folded and misfolded products. In addition, the intermediate step reaches a high removal factor when the highly specific interaction is combined with a specific elution mode by chelating calcium with EDTA. Thus, a strong reduction of endotoxins (even approximately 99% reduction) and HCPs is observed in combination with a moderate reduction of DNA content.

[0367] The combined endotoxin reducing effects of the first AX capture step (approximately 97%) and the intermediate affinity chromatography step (approximately 99%) provide an annexin A5 product in which endotoxin levels are reduced to approximately 0.03% of the levels in the clarified product before the first AX step.

[0368] 1.1.4 Refining AX Chromatography The resulting AF elution pool (300 mL / L USP ) is diluted 2-fold (35 mM Tris pH 8; 0.1% Tween 80; 12.5 mM MgCl2) and filtered (Sartopore2 0.45-0.2 μm) before scouring chromatography. The dilution reduces the conductivity of the AX load, but also complexes the free EDTA molecules with Mg ions. The free EDTA would otherwise bind to the column, thereby primarily reducing the capacity of this step, but also reducing the resolution.

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

[0370] A post-loading wash is performed with Buffer A (20 mM Bis-Tris pH 7; 25 mM NaCl) for 3 CV. Elution is performed with a linear gradient to 100% B (20 mM Bis-Tris pH 7; 180 mM NaCl) in 33 CV. 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 major peak is collected up to the valley between peak 1 and peak 2 (corresponding to approximately 7 CV).

[0372] A two-step CIP procedure is performed to regenerate and clean the column (step 1: 3 CV of 2M NaCl, 100 cm / h; upflow / step 2: 3 CV of 1M NaOH; incubation >15 h, 40 cm / h upflow). The column is finally stored in 25 mM NaCl.

[0373] Recent results from small scale experiments suggested a positive effect of Tween 80. A process carried out with 0.1% Tween 80 increased the product yield after intermediate processing by approximately 30%. To ensure adequate solubility, the loading in the refinery step is limited to 16 g / L resin. The yield improvement also has an impact on the scale-up scenario. The calculated column dimensions in the refinery step were designed for two cycles. 100 L USP When processing the full amount from scale, the increased yield requires a scenario of 4-5 cycles.

[0374] FIG. 2 shows the process flow diagram for the AX refinement chromatography step.

[0375] The refinement step is mainly carried out to reduce product-related impurities, such as separating various annexin A5 isoforms. In addition, the refinement step reaches the highest removal factor in the process for residual DNA and strongly reduces HCP. Endotoxins, which are already at low levels after the intermediate step, are further reduced by about 99%, which shifts endotoxin levels to about 0.0003% of the level in the clarified product before the first AX step.

[0376] 1.1.5 Ultra / Diafiltration and Formulation of Annexin A5 The AX pool is transferred directly to UF / DF to increase the product concentration and perform the buffer change. Following the buffer change, Tween 80 is added to a final concentration of 0.05% and the drug substance is sterile filtered.

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

[0378] FIG. 3 shows a process flow diagram for ultra / diafiltration and formulation of Annexin A5.

[0379] Two targets and acceptance criteria The following targets and acceptance criteria are defined to evaluate the performance and scale-up of this process against small scale DSP runs: Targets are defined based on IPC / bulk analysis of commissioning runs. Critical process steps were identified and critical process parameters were characterized to enhance confidence in process performance.

[0380] 2.1 Key process parameters Table 5 shows the key process parameters. Achievement of target ranges during the process indicates successful process scale-up. Table 5: Process parameters and their respective target values ​​for the process steps TIFF2025066728000011.tif159170

[0381] 2.2 Critical process parameters A high degree of homogeneity of the suspension prior to capture chromatography is important. To achieve this, the use of three homogenization cycles is preferred. Moreover, a temperature increase within the homogenization is preferred to obtain a lysate temperature in the range of 37° C. This is important for the filtration step and the activity of benzonase, which has a direct impact on the capture performance.

[0382] Pooling from the refining step is also important as this step is used to separate product related impurities. The UF / DF step is run under moderate conditions with respect to the TMP to minimize the formation of coating layers. Table 6: Critical process parameters TIFF2025066728000012.tif58170

[0383] 2.3 In-process control Table 7 shows the in-process controls. Achieving the target ranges during this process indicates successful process scaling. The target ranges are set based on observations in previous small scale runs, only driven by the implemented changes. TIFF2025066728000013.tif234170TIFF2025066728000014.tif246170TIFF2025066728000015.tif244170TIFF2025066728000016.tif238170 TIFF2025066728000017.tif232170TIFF2025066728000018.tif224170TIFF2025066728000019.tif238170TIFF2025066728000020.tif111170

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

[0385] After commissioning with a scaled-up 200 L in a GMP pilot plant, the process delivered a product with 1.8 pg host cell DNA per mg AnxA5 protein, 16.6 ng host cell protein per mg AnxA5 protein, and 0.1 EU per mg AnxA5 protein.

[0386] Throughout the manufacturing process, the annexin A5 protein is maintained in solution in its active form when not transiently bound to the chromatography resin.

[0387] When applied to a 1,000 L culture, the overall process time in a manufacturing plant would be 1 week for fermentation, harvest, cell disruption and treatment prior to chromatography, and 1 week in succession for downstream processing. The entire process in GMP would be 2 weeks. This is scale independent, perfectly aligned with industry standards, and would be amenable to any CMO or pharmaceutical manufacturer.

[0388] As noted above, by comparison, the Marder et al. process would take around 12 weeks to process a 1,000 L culture.

[0389] Moreover, the yield in the process of the present invention is 2-3 times higher per batch than that of Marder et al., which means that the manufacturing cost per gram of annexin A5 drug substance is (6-8 x 2-3 =) 12-24 times higher in the Marder process.

[0390] Moreover, in addition to providing a faster and higher yield purification process than the Marder et al. 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 the Marder process would not be suitable for human use. Despite careful centrifugation, only one anion exchange chromatography step is used in the Marder process, which is far below the requirements to reach sufficient purity, both in terms of in-process related impurities (especially endotoxins) and product related variants. Marder does not provide any data on endotoxin levels or other impurities, further suggesting a lack of suitability for pharmaceutical use.

[0391] In contrast, the process of the present application provides a highly pure Annexin A5 protein product having the following recited attributes: -Typically, concentrations are around 8-12g / L. - Host cell protein levels below 100 ng / mg, more typically below 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 35EU / mg, more typically less than 1EU / mg; - greater than 95% purity as determined by size exclusion chromatography; - Bioburden less than 1 cfu / mL (determined by Ph.Eur.2.6.12); - a clear, colorless appearance with no visible particles; and - The main band detected by Western blot analysis corresponds to the Annexin A5 reference.

[0392] The process was repeated using Capto Q ImpRes instead of Source 15Q for the second purification step. This provided an even more efficient process since Capto Q ImpRes anion exchange resin has a higher binding capacity, can withstand high flow rates without back pressure, can be packed at higher bed heights, and has a lower cost. The quality and purity of the final product was maintained.

[0393] Compared to Source 15Q, Capto Q ImpRes resin has the following: More than double the capacity in grams / liter resin ● Withstands more than twice the flow rate at the same back pressure can be packed at higher bed heights, typically about 35-60% higher, which provides higher capacity for any given column bed area; and Costs less than half the purchase price per litre 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 with respect to 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 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 pre-filtered lysate; + 10 mM CaCl2 Buffer A: 50 mM Tris pH 7.4, 5 mM CaCl2 Buffer A B: 50 mM Tris pH 7.4, 40 mM EGTA, 50 mM NaCl CIP: 3M NaCl Table 7: Comparison of AX and heparin affinity chromatography capture TIFF2025066728000021.tif64170

[0397] These results demonstrate that performing AX capture prior to affinity chromatography does not significantly improve purity, but has a significant impact on the yield of the heparin step. Moreover, the expensive affinity resin has a long life when used as an intermediate step. Yield is very important.

[0398] Capture by AX can be viewed as a conditioning step that allows efficient use of the highly specific affinity step by heparin chromatography.

[0399] Example 3 A partially purified annexin A5 product was obtained using a method similar to that of Example 1 up to the first anion exchange chromatography capture step.

[0400] Briefly, recombinant E. coli expressing Annexin A5 were resuspended in homogenization buffer (50 mM Tris, 1 mM MgCl, 1% Tween 20 pH 7.5) with 3200 U of Bezonase and homogenized by 3 cycles at 600 bar pressure. The temperature after homogenization was measured to be 36°C. A clarification step using Cuno 60 SP 0.6-0.2 μm was performed and the clarified solution was diluted 1:2 in 1% Tween 20 by addition of 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), washed with Buffer A (20 mM Tris, 25 mM NaCl, 0.1% Tween 20, pH 7.4) and then eluted 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 a variety of conditions were tested.

[0402] The heparin affinity chromatography conditions used were as follows: TIFF2025066728000022.tif65170

[0403] Sterile filtration of the capture elution pool was performed with Sartopore 2 0.45-0.2 μm. This pool was diluted 8-fold with (1050 mL) Buffer A from the 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 Tween 80, 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% Tween 20, pH 7.4 Buffer B 20mM Tris, 10mM EDTA, 25mM NaCl, 0.1% Tween20, pH7.4 Test 2: Buffer A 20 mM Tris, 25 mM NaCl, 2 mM 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 FIG. 7A, and the results for test 2 are shown in FIG. 7B.

[0405] The results show that, in contrast to the eluate that separated under standard conditions (Test 1), the addition of Tween 80 shifted the eluate towards a single peak. Tween 80 appears to stabilize Annexin A5. A possible explanation for this change in elution behavior is that the theoretical precipitation on the column is prevented. Two main positive effects can be described regarding the use of Tween 80 in the heparin affinity chromatography step: - Reduced pressure: The pressure on the column, which increased from 0.5 to 2-3 bar at loading, was clearly reduced to 0.5 bar. This is especially beneficial for large scale. Some precipitation seen after long incubation could be the reason for this pressure increase. - Prevention of precipitation: A second positive effect is seen in the elution. During fractionation of the eluate, the highly concentrated main peak elution fraction has a tendency to precipitate. Assuming that this effect is related to the very high concentration of Annexin A5 in the main peak, after pooling of the elution fractions no precipitation is observed anymore and, moreover, the precipitation seems 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 Tween 80 also prevented the formation of precipitates in the main peak elution fraction. Based on these results, the additional addition of Tween 80 in all intermediate chromatography buffers appears to be advantageous as it has a stabilizing effect on Annexin A5.

[0406] Example 4 A partially purified annexin A5 product was obtained using a method similar to that of Example 1 up to the first anion exchange chromatography capture step.

[0407] 1.25 mL of the product of the anion exchange step was then mixed with 8.75 mL of the various forms of test dilution buffer. The mixtures were 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. TIFF2025066728000023.tif192170

[0409] These results demonstrated the benefit of adding Tween 80 (i.e., polysorbate 80) in avoiding product precipitation in the sample, which is important in the context of conditioning the AnxA5 product prior to application to a chromatographic column, such as an affinity chromatography column, to reduce precipitation and prevent increased backpressure when pumping through the column.

Claims

1. A process for the recovery and / or purification of a protein comprising 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 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 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, the AnxA5 protein remains in solution throughout the entire process, including any preceding or subsequent steps, except when transiently bound to any chromatographic resin.

2. The affinity chromatography step comprises binding of the AnxA5 protein to immobilized heparin, the binding being promoted by the presence of calcium ions; Optionally, the AnxA5 protein is eluted from the immobilized heparin using an elution buffer containing a calcium ion chelator, such as EDTA. The process of claim 1.

3. The method includes subjecting the first anion exchange product directly or indirectly to a heparin affinity chromatography step, which is carried out in the presence of Tween 80 (preferably in the presence of 0.1% Tween 80), thereby producing a first affinity chromatography product comprising released AnxA5 protein; 3. The process of claim 1 or 2, wherein the AnxA5 protein preferably remains in solution throughout the process, including any preceding or subsequent steps, except when temporarily bound to any chromatographic resin.

4. The process is 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, The process comprises releasing intracellular proteins from the host cell prior to carrying out the steps of the process of any one of claims 1 to 3, The step of releasing 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.

5. The process described in claim 4, wherein the non-ionic detergent is a polysorbate, preferably a polysorbate selected from Tween 20 and Tween 80, most preferably Tween 80.

6. A process described in claim 4 or 5, wherein the step of releasing 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 binding between annexin A5 and endotoxin.

7. A process described in any one of claims 4 to 6, wherein the step of releasing intracellular AnxA5 protein is carried out in the presence of a homogenization buffer containing 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. and / or wherein 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 The process according to any one of claims 4 to 7, wherein the homogenization buffer contains or is modified to contain a calcium metal ion chelator after releasing the intracellular AnxA5 protein.

9. The process described in claim 8, wherein the calcium metal ion chelating agent is selected from EDTA or a salt thereof, EGTA or a salt thereof, and most preferably EDTA.

10. A process described in claim 8 or 9, wherein the level of free calcium ions and / or the amount of calcium metal ion chelator is effective to reduce or prevent binding between annexin A5 and components of the cell wall of the host cell.

11. The process of any one of claims 8 to 10, wherein the homogenization buffer contains or is adjusted to contain (before or after the release of the AnxA5 protein) 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 calcium metal ion chelator, preferably the calcium metal ion chelator is EDTA.

12. A process according to any one of claims 8 to 11, wherein the homogenization buffer comprises a non-ionic detergent according to any one of the processes of claims 5, 6 or 7.

13. The process of claim 12, wherein the calcium metal ion chelating agent is EDTA and / or the non-ionic detergent is Tween 80.

14. (a) The process comprises recovering and / or purifying recombinantly expressed intracellular AnxA5 protein from a culture of host cells, the culture having a volume of at least 100 L, 500 L, 1,000 L, 5,000 L, or 10,000 L. (b) the process comprises mixing biomass from a culture of host cells in a homogenization buffer at a concentration of about 10 g of biomass per mL of homogenization buffer; and / or (c) releasing intracellular AnxA5 protein from host cells in the homogenization buffer comprises lysing, disintegrating, homogenizing, sonicating or pressure-treating host cells so that the cell wall and cell membrane barrier of the host cells are disrupted, thereby releasing the intracellular AnxA5 protein, optionally this process does not include the use of osmotic shock and / or freeze-thaw processes, and optionally the releasing intracellular AnxA5 protein from 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; The process according to any one of claims 4 to 13.

15. The step of releasing intracellular AnxA5 protein produces a biomass homogenate containing the released AnxA5 protein, and optionally, (a) 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; and / or (b) clarifying the biomass homogenate, thereby producing a clarified product comprising the released AnxA5 protein, e.g., (i) the step of clarifying the biomass homogenate comprises treatment of the homogenate with a nuclease, such as nuclease A, preferably nuclease A from Serratia marescens, optionally the nuclease being included in the homogenization buffer prior to release of the intracellular AnxA5 protein; and / or (ii) clarifying the biomass homogenate (preferably following the nuclease treatment according to (b)(i) of this claim) 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), the filter effluent being a clarified product containing the released AnxA5 protein; The process according to any one of claims 4 to 14.

16. The process described in claim 15, wherein the clarified product containing the released AnxA5 protein produced by the method described in (b)(ii) of claim 15 is subjected to a first anion exchange step as described in claim 1, thereby producing a first anion exchange product containing the released AnxA5 protein.

17. A process described in any one of claims 1 to 16, 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.

18. A process according to any one of claims 1 to 17, 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 to obtain a final non-ionic detergent concentration of, for example, 0.01 to 1% (w / v), more preferably about 0.1% (w / v).

19. A process described in any of claims 1 to 18, wherein AnxA5 protein binds during the anion exchange step, and a first anion exchange product comprising 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.

20. A process described in any one of claims 1 to 19, wherein the AnxA5 protein in the first anion exchange product is subjected to an affinity chromatography step.

21. The process of any one of claims 1 to 20, wherein the first affinity chromatography product comprises 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.

22. The process, comprising subjecting the composition to an anion exchange resin to perform an anion exchange step, whereby AnxA5 protein is recovered and / or purified from the composition; further characterized in that the anion exchange step is carried out in the presence of additional selected metal ions; 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; 22. The process of any one of claims 1 to 21, wherein the AnxA5 protein preferably remains in solution throughout the process, including any preceding or subsequent steps, except when temporarily bound to any chromatographic resin. (a) the calcium metal ion chelating agent is selected from EDTA or a salt thereof, EGTA or a salt thereof, and most preferably EDTA; (b) the calcium metal ion chelator is present in the composition at a concentration of greater than about 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM or more and / or at a concentration of about or at least 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 15 mM, 20 mM or more; (c) the selected metal ion is a divalent cation, such as Mg 2+ ion; (d) 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 an anion exchange step; (e) 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 a 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; (f) 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 a composition comprising AnxA5 protein and a calcium metal ion chelator prior to subjecting the composition to the anion exchange resin); and / or (g) the calcium metal ion chelator is EDTA and the selected metal ion is Mg 2+ ion, preferably the molar ratio of Mg 2+ ion to EDTA is in the range of 0.5:1 to 2:1, most preferably at least 1:1 or greater; 23. The process of claim 22.

24. A composition comprising AnxA5 protein and a calcium metal ion chelator, the composition being subjected to an anion exchange resin, comprising:

24. A process according to claim 22 or 23, which is a direct or indirect product of a preceding process, comprising subjecting 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 AnxA5 protein and a calcium metal ion chelator.

25. The process described in claim 24, wherein the preceding affinity chromatography step involves binding of AnxA5 protein to immobilized heparin, and optionally, the binding is facilitated by the presence of calcium ions.

26. The process of claim 25, wherein the AnxA5 protein is eluted from the immobilized heparin using an elution buffer containing a calcium ion chelator such as EDTA or EGTA.

27. ​​A process described in any one of claims 24 to 26, wherein there is no dialysis step between the preceding affinity chromatography step and the anion exchange step prior to application of the direct or indirect product to the anion exchange step, and / or no removal of calcium ion chelating agent from the product of the preceding affinity chromatography step is performed.

28. The process of any one of claims 22 to 27, wherein the selected metal ion is added to the composition before or during the anion exchange step.

29. 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, or a culture thereof having a volume of at least 100 L, comprising: The process is (a) performing a step of releasing intracellular proteins from endotoxin-producing host cells in the presence of a homogenization buffer containing a non-ionic detergent; (b) subjecting the released AnxA5 protein directly or indirectly to an anion exchange resin to perform a first anion exchange step, thereby producing a first anion exchange product comprising the released AnxA5 protein; (c) subjecting the released AnxA5 protein of 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; and (d) the first affinity chromatography product is a composition comprising AnxA5 protein and a calcium metal ion chelator; (e) the direct or indirect product of the affinity chromatography step, comprising AnxA5 protein and a calcium metal ion chelator, is subjected to an anion exchange step according to any one of claims 22 to 28; Preferably, none of steps (a) to (e) 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.

30. A process as described in any one of claims 1 to 29, preferably comprising, at the end of the process as described in any one of claims 1 to 29, 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.

31. (a) one of the further steps is diafiltration, and optionally, the product of the diafiltration step contains 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 mg / mL, or 100 mg / mL. (b) filtration uses a 0.45-0.2 μm filter or a 0.22 μm filter, preferably a sterile filtration process; and / or (c) Sterile filtration is the final purification step before storing the AnxA5 protein-containing product in a sterile container; 31. The process of claim 30.

32. (a) The process includes the steps required to provide a final sterilized AnxA5 protein product in a phosphate-free buffer (such as Bis-Tris or Tris buffer) at about pH 7.4 containing about 150 mM NaCl, about 1 mM CaCl 2 , about 0.05% (w / w) polysorbate (such as Tween 80) or other non-ionic detergent, and optionally, the concentration of AnxA5 protein in the final sterilized AnxA5 protein product is about 10 mg / mL. (b) the process provides a final, sterile AnxA5 protein product in which the NaCl concentration present maintains the AnxA5 protein in a predominantly monomeric form; (c) the process provides an overall yield of greater than 1 g of AnxA5 protein per L 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; (d) the process provides an overall recovery of AnxA5 protein of about 24% by weight of said AnxA5 protein present in the host cell culture. (e) 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 recombinantly expressed AnxA5 protein) per mg of AnxA5 protein; (f) the process provides a product comprising 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, and / or (g) the process provides a product containing host cell nucleic acid levels of less than 1,000 pg / mg of AnxA5 protein, preferably less than 100 pg / mg of AnxA5 protein, and more preferably less than 10 pg / mg of AnxA5 protein. The process of any one of claims 1 to 31.

33. A composition comprising AnxA5 protein, which is the direct or indirect product of (or is obtainable directly or indirectly by) the process described in any one of claims 1 to 32.

34. A composition comprising a recombinant AnxA5 protein, (a) the composition has been subjected to a sterile filtration process and / or is a sterile composition; (b) the composition comprises an endotoxin content of less than 100 EU per mg of AnxA5 protein; (c) the composition comprises a nucleic acid level of less than 1000 pg per mg of AnxA5 protein; and (d) the AnxA5 protein contains a His-tag; composition.

35. The composition described in claim 34, wherein the nucleic acid level is a host cell nucleic acid level.

36. (a) containing 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 mg / mL, or 100 mg / mL; (b) the composition is stored in a sterile container; (c) the composition comprises a sterile AnxA5 protein product in a phosphate-free buffer (such as Bis-Tris or Tris buffer) at about pH 7.4 containing about 150 mM NaCl, about 1 mM CaCl 2 , about 0.05% (w / w) polysorbate (such as Tween 80) or other non-ionic detergent, optionally wherein the concentration of said AnxA5 protein in the final sterile AnxA5 protein product is about 10 mg / mL; (d) the NaCl concentration maintains the AnxA5 protein in a predominantly monomeric form; (e) comprising non-AnxA5 proteins, such as host cell proteins, at a level of less than 20 ng per mg of AnxA5 protein, optionally the host cells are prokaryotic cells, such as gram-positive or gram-negative cells, and in particular may be endotoxin-producing gram-negative bacterial cells, and further optionally the host cell proteins are at detectable levels in the composition (despite being less than 20 ng per mg of AnxA5 protein); (f) the composition contains detectable levels of endotoxin (albeit less than 100 EU per AnxA5 protein) in the composition; (g) the composition is a product in unit dosage form and contains less than 100 EU per unit dose, and optionally endotoxin is at a detectable level in the composition (despite being less than 100 EU per unit dose); (h) the composition comprises a detectable level of nucleic acid in the composition (albeit at less than 1000 pg per mg of AnxA5 protein), optionally the nucleic acid level is a host cell nucleic acid level, e.g., the host cell is a prokaryotic host cell, such as a gram-positive or gram-negative cell, particularly the host cell is an endotoxin-producing gram-negative bacterial cell; (i) the level of gluconoylated AnxA5 protein in the composition is in the range of 0.5 to 30% of the total content of AnxA5 protein in the product; (j) the level of gluconoylated AnxA5 protein in the composition is less than 40% of the product; (k) the AnxA5 protein does not contain one or more RGD motifs; (l) The AnxA5 protein is chemically modified; (m) the AnxA5 protein is a fusion protein comprising the AnxA5 protein and a fusion partner; and / or (n) the composition is a pharmaceutically acceptable and / or veterinarily acceptable composition; 36. The composition of claim 34 or 35.

37. The composition described in claim 36, wherein the chemically modified AnxA5 protein (j) is a PEGylated AnxA5 protein.

38. The composition of any one of claims 34 to 37, in the form of a pharmaceutical or veterinary composition suitable for parenteral, intravenous, intra-arterial, intraperitoneal, intramuscular, intraocular, intracranial, intracerebral, intraosseous, intraventricular, intrathecal, subcutaneous or topical administration.

39. A composition described in any one of claims 34 to 38, wherein the composition is suitable for topical administration and is in the form of a cream, ointment, or eye drops.

40. A composition described in any one of claims 34 to 39, wherein the AnxA5 protein in the composition is present in the form of a pharmaceutical or veterinary composition comprising the AnxA5 protein in admixture with a pharmaceutically or veterinarily acceptable carrier, wherein the carrier is a coating.

41. A composition according to any one of claims 34 to 40, wherein the composition is in the form of an immediate release, delayed release or controlled release formulation.

42. A composition according to any one of claims 34 to 41 in the form of a pharmaceutical or veterinary composition that is a drug-eluting stent.

43. A composition according to any one of claims 34 to 42 for use in medicine.

44. - For treating, preventing, and / or reducing the risk of developing cardiovascular disease, autoimmune disease, or inflammatory conditions; - to prevent or reduce the risk of thrombosis, - for treating, preventing or reducing the risk of peripheral arterial occlusive disease, - for treating, preventing, or reducing the risk of hematological disorders, including but not limited to sickle cell anemia; - for treating, preventing or reducing the risk of pulmonary fibrosis, - for treating, preventing and / or reducing the risk of developing atherosclerosis, acute coronary syndrome, stroke, claudication, angina pectoris, ischemic heart disease, peripheral arterial disease, systolic hypertension, thromboembolism, hemorrhagic or vasculitic stroke, myocardial infarction, unstable angina pectoris, intermittent claudication, unstable angina, other forms of severe angina, or transient ischemic attack (TIA), - for treating, preventing or reducing the risk of vascular insufficiency, - to reduce ischemic pain, - for preventing or treating restenosis, e.g. neointima formation or hyperplasia, - for treating, preventing, or reducing the risk of ischemia-reperfusion injury, organ ischemia-reperfusion injury (such as liver or kidney ischemia-reperfusion injury), transplant rejection, or graft-versus-host disease (GVHD), including acute and / or chronic GVHD, or for treating a subject after transplantation; for the prevention and / or reduction of peri- or post-operative complications after surgical interventions, such as complications after vascular surgery, for example after peripheral vascular surgery, - for the prevention or treatment of vascular inflammation or for the treatment, prevention or risk reduction of acute and chronic vascular inflammation or carditis, - for treating, preventing and / or reducing the risk of developing acute and / or chronic inflammatory conditions and / or inflammatory bowel disease, - for treating, preventing and / or reducing the risk of developing rheumatic diseases; arthritis, including osteoarthritis, rheumatoid arthritis or psoriatic arthritis; or - for treating, preventing and / or reducing the risk of developing Alzheimer's disease, dementia in general, multiple sclerosis or myasthenia gravis, The composition according to any one of claims 34 to 43.

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