RU2779098C2 - Manufacturing method - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to biotechnology, in particular to methods for isolation and purification of recombinantly expressed intracellular protein annexin A5 (hereinafter – AnxA5) from a host cell producing endotoxin with a cellular wall, and to its use. Methods include release of intracellular protein AnxA5 from the host cell in the presence of buffer for homogenization containing non-ionic detergent selected from Twin 20 and Twin 80. Then, a composition containing protein AnxA5 and chelator of metal calcium ions AnxA5 is exposed to anion-exchange resin in the presence of additional selected metal ions. At the same time, protein AnxA5 remains in the solution at all stages of methods except for cases, when it temporarily binds to any chromatographic resins.

EFFECT: inventions exclude the need for use of any stages of purifying centrifugation for protein AnxA5 after isolation from a host cell, allow for minimization of a degree of binding endotoxin and AnxA5 at initial stages of isolation, and contribute to the production of a purified product of annexin A5 on an industrial scale, suitable for therapeutic use.

98 cl, 8 dwg, 11 tbl, 4 ex

Description

Technical field

This application relates to methods for making a protein containing the annexin A5 (AnxA5) sequence. More specifically, said method consists in isolating and/or purifying the AnxA5 protein, especially from a recombinant host cell, such as a bacterial host cell. The methods described herein are highly efficient and cost effective and can be applied on an industrial scale (eg, with recombinant host cell cultures having a culture volume of about 1000 L or more) to quickly and conveniently produce a pharmaceutical grade AnxA5 protein product.

State of the art

The listing or discussion of a clearly previously published document in this specification should not necessarily be construed as an endorsement that said document is part of the state of the art or is in the public domain.

Atherothrombosis resulting from the formation of atherosclerotic plaques is a key pathogenetic mechanism underlying the majority of clinically significant cardiovascular ischemic diseases, including acute coronary insufficiency and occlusion of cerebrovascular and peripheral arteries. As discussed in Cederholm and Frostegård, 2007, Drug News Perspect., 20(5): 321-6, annexin A5 (formerly known as annexin V), a member of the annexin superfamily, is a protein with potent and unique antithrombotic properties. It is assumed that the antithrombotic effect exhibited by annexin A5 is mediated mainly by the mechanically protective effect of phospholipids, in particular phosphatidylserine, thereby reducing their availability for coagulation reactions. However, other interesting properties of annexin A5 have been reported that potentially contribute to its antithrombotic function, especially as a result of suppression of surface expressed tissue factor, or interaction with additional ligands involved in hemostasis, such as sulfatide and heparin, and also as a result of increased activity of plasminogen activator. urokinase type. In addition, the biological significance of annexin A5 as part of the endogenous antithrombotic system in vivo for the large vascular bed, as well as for the placental microcirculation, has been suggested.

Indeed, annexin A5 is known to have a wide range of applications in medicine, providing direct therapeutic effects. Examples include the use of annexin A5:

to prevent atherothrombosis and/or plaque rupture, as described in WO 2005/099744 (the contents of which are incorporated herein by reference);

for the treatment of vascular dysfunction, reduction of the intensity of ischemic pain and/or treatment of vascular diseases accompanied by a violation of the integrity of the vessels, as described in WO 2009/077764 (the contents of which are incorporated herein by reference);

for the prevention or treatment of restenosis, as described in WO 2009/103977 (the contents of which are incorporated herein by reference);

for use in inhibiting the activity of oxidized cardiolipin (oxCL), as well as for treating, preventing and/or reducing the risk of developing a cardiovascular disease, an autoimmune disease or an inflammatory pathological condition, as described in WO 2010/069605 (the contents of which are incorporated herein via link) as well as

to prevent and/or reduce peri- or postoperative complications after surgery, such as complications after vascular surgery, especially peripheral, as described in WO 2012/136819 (the contents of which are incorporated herein by reference).

In this regard, annexin A5 is a protein with high therapeutic interest and potential. Thus, there is an urgent need to develop an efficient method for producing therapeutic grade annexin A5 protein, using an efficient and cost-effective process that can be implemented on an industrial scale and conveniently used for commercial production (for example, to obtain annexin A5 protein from recombinant cultures). host cells having a culture volume of about 1000 L or more).

Of particular difficulty are cases where the recombinantly expressed annexin A5 in standard bacterial host cells, such as E. coli, is contaminated with host cell components, and in particular with endotoxin. Endotoxin is a lipopolysaccharide (LPS), which is formed by a lipid and a polysaccharide consisting of an O-antigen, an outer core and an inner core connected by a covalent bond; LPS is found in the outer membrane of Gram-negative bacteria and elicits strong immune responses in animals. Annexin A5 is characterized by strong binding to biological membranes containing negatively charged phospholipids and therefore has a particularly high affinity for endotoxin. This further complicates the production of annexin A5 from endotoxin-producing hosts on a large industrial scale.

To date, there is no method for producing therapeutic grade annexin A5 protein that is an efficient and cost-effective process that can be commercially implemented and conveniently applied to commercial production (for example, to obtain annexin A5 protein from cultures of recombinant host cells, having a culture volume of about 1000 L or more), not to mention the problem of endotoxin contamination.

In 1991, Kumar reported the development of a method for the manufacture and purification of annexin A5 (college bachelor's thesis on "Expression, Purification, and Large-Scale Fabrication of Recombinant Human Annexin-V Protein", defended at the Department of Chemistry and Technology College as part of Arkansas Tech University, Fayetteville, Arkansas, available online at: https://uarkive.uark.edu/xmlui/handle/10826/981). Kumar's method involved expression of annexin A5 in recombinant E. coli host cells in 100 ml culture flasks, granulation of cells, resuspension of cells in the presence of a homogenization/lysis buffer consisting of 50 mM Tris-HCl, 10 mM CaCl ₂ at pH 7.2 , followed by disruption of the cells by sonication to release the annexin A5 protein. The addition of CaCl ₂ caused the binding of annexin A5 to cell membranes in the debris in a calcium-dependent manner; then the mixture was subjected to the first stage of cleansing centrifugation for 20 minutes, after which the supernatant was removed and the pellet containing cell debris and associated annexin A5 was recovered. Annexin A5 was released from the pellet using EDTA, followed by a second purification centrifugation step for 20 minutes and collected annexin A5 in the supernatant. Dialysis was then performed overnight to change the annexin A5 buffer to Tris-HCl at pH 8.0, followed by an anion exchange step on a DEAE-sepharose column and eluting annexin A5 using a salt gradient.

It has become clear to the Applicant that there are many limitations and drawbacks to the method proposed by Kumar. First, this method has only been demonstrated on a small scale using 100 ml cultures and requires two separate centrifugation steps during the purification process. Kumar's method is not applicable to industrial scales that effectively utilize a large volume culture (eg, 1000 liters or more). As discussed below, centrifuging such large volumes of liquid would be extremely laborious and costly. At the same time, the approach proposed by Kumar, which relies on the use of calcium-induced binding of annexin A5 to membranes in cell debris as a pre-uptake step, necessitates centrifugation. Secondly, Applicants have realized that the Kumar method results in high losses of annexin A5 protein, eg removal of soluble annexin A5 that does not bind in the product supernatant in the first purification centrifugation step. Thirdly, it is not possible to completely remove endotoxin to a level acceptable for therapeutic use using the Kumar method, and it should be noted that no analysis of endotoxin levels in the final product is carried out. Thus, the Kumar method cannot be used efficiently and with minimal time to obtain the annexin A5 protein on an industrial scale, since it causes high losses (i.e., low yield) and leads to a low degree of protein purification, which is unsuitable for therapeutic use. applications.

In 2008, the Department of Laboratory Medicine, Washington State University Medical Center, Tait Research Laboratory staff published a paper titled "Production of Recombinant Annexin V from Plasmid pET12a-PAPI". The said work is available online at: https://depts.washington.edu/labweb/Faculty/Tait/108.pdf. The described method is very similar to the methodology proposed by Kumar. This method included the expression of annexin A5 in recombinant E. coli host cells in 1 L cultures, cell granulation, cell resuspension in the presence of a homogenization/lysis buffer consisting of 50 mM Tris-HCl, 10 mM CaCl ₂ at pH 7.2, followed by disruption of the cells by sonication to release the annexin A5 protein. The addition of CaCl ₂ caused the binding of annexin A5 to cell membranes in the debris in a calcium-dependent manner; then the mixture was subjected to the first stage of cleansing centrifugation for 20 minutes, after which the supernatant was removed and the pellet containing cell debris and associated annexin A5 was recovered. Annexin A5 was released from the pellet using EDTA, followed by a second purification centrifugation step for 20 minutes and collected annexin A5 in the supernatant. Dialysis was then performed to change the annexin A5 buffer to Tris-HCl at pH 8.0, followed by an anion exchange step on a Mono Q column and eluting annexin A5 using a salt gradient. It became clear to the applicant that in this method there are many limitations and disadvantages that are characteristic of the Kumar method.

In 2014, another method for purifying annexin A5 was proposed in Marder et al., 2014, BMC Biotechnology, 14:33 titled "Production of recombinant human annexin V by fed-batch culture". Marder et al. (Marder et al.), reports that his method is a fed-batch culture principle to produce recombinant human annexin V on a large scale, and suggests that his method could expand the commercial application of recombinant annexin A5, for example, in imaging studies in vivo.

Once again, it should be noted that the method of Marder et al. very similar to the Kumar method proposed in 1991, and to the method developed by the Department of Laboratory Medicine at the University of Washington Medical Center in 2008. Marder et al. expresses annexin A5 in recombinant E. coli host cells in 1 L cultures contained in 2 L reservoirs. As discussed (under "Purification" in the description of the methods of Marder et al.), harvested cells were resuspended in the presence of homogenization/lysis buffer (buffer A) consisting of 50 mM Tris-HCl, 10 mM CaCl ₂ at pH 7.2, c subsequent disruption of the cells by sonication to release the annexin A5 protein. The addition of CaCl ₂ caused the binding of annexin A5 to cell membranes in the debris in a calcium-dependent manner; then the mixture was subjected to the first stage of cleansing centrifugation for 30 minutes, after which the supernatant was removed and the pellet containing cell debris and associated annexin A5 was recovered. Annexin A5 was released from the pellet using EDTA, followed by a second cleansing centrifugation step for 30 minutes and collected annexin A5 in the supernatant. A dialysis step was then performed to change the annexin A5 buffer to Tris-HCl at pH 8.0, followed by a third purifying centrifugation step for 20 minutes to remove residual pellet, and an anion exchange step on a Mono Q column and eluting annexin A5 using saline. gradient. Again, it became clear to the applicant that in this method there are many limitations and disadvantages that are characteristic of the Kumar method.

Kumar's method, proposed in 1997, and the method developed at the Department of Laboratory Medicine at the University of Washington Medical Center in 2008, as well as the method of Marder et al., 2014, clearly show that the approach to manufacturing and purification of products containing annexin A5 for commercial purposes, although the limitations and disadvantages of these methods have been underestimated in the art, with no publicly available alternative.

All of these prior art methods for isolating Anexin A5 have only been demonstrated on a laboratory scale and cannot be adapted to large scales or take into account industry standards or equipment used for larger scales. These methods have inherent disadvantages, which makes them unsuitable for large-scale production. In particular, two or (in the case of the method of Marder et al. 2014) three high gravity centrifugations are significantly limiting characteristics of these prior art methods, which require Annexin A5 to be alternately in solution or in the form of sediment. It can be objectively estimated that a manufacturing method using only two centrifugation steps to process a single batch of 1000 L would take about 12 weeks with a 12 hour daily shift in any well equipped bio-industrial plant, which would result in an unacceptably high manufacturing cost. See Comparative Example 1

Accordingly, it is an object of this invention to provide methodological steps for purifying and isolating annexin A5 that are efficient and cost-effective for an industrial scale manufacturing process (e.g., recombinant host cell cultures with a culture volume of about 1000 liters or more) and, besides moreover, overcoming the drawbacks of product loss and low purity (including endotoxin contamination) associated with prior art methods.

In addition, the purpose of this invention is to provide pharmaceutical products containing annexin A5, which are obtained using the methods of this invention.

Brief description of the essence of the invention

Applicant has made numerous changes and improvements to the method for making protein containing the annexin A5 (AnxA5) sequence and has developed several highly efficient purification steps that can be used independently and/or used in combination to optimize existing methods. Most preferably, the method for manufacturing annexin A5 contains all of the developed method steps.

In particular, applicant's developments provide a highly efficient method for isolating the AnxA5 protein, in which the AnxA5 protein preferably remains in solution at all stages of the method (except when it is temporarily associated with chromatographic resins). That is, Applicant's developments provide a method for isolating the AnxA5 protein, which can preferably be performed without the need for any purification centrifugation steps for the AnxA5 protein after said AnxA5 protein has been isolated from the host cell. The present invention is of great industrial benefit because high gravity centrifugation to collect precipitates is a complex, slow and costly process in large scale biopharmaceutical plants. Furthermore, this means that all methods of the invention can be performed without exploiting the ability of annexin A5 to attach to membranes (e.g., host cell membranes and/or liposomes), which can often result in the purification of annexin A5 along with undesirable contaminants. such as endotoxin.

Accordingly, this method can be used to process large volumes (for example, about 100 L, 500 L, 1000 L, 5000 L, 10,000 L, 50,000 L, 100,000 L or more) of host cell cultures with minimal time and without restrictions. due to one or more cleansing centrifugation steps. For example, it may be preferred that the purification process be carried out in 5, 4, 3, 2 weeks or less, and typically less than 1 week per 1000 L of host cell culture. In addition, it has surprisingly been found that this method can be used to produce more product and/or higher purity (including, for example, improved endotoxin removal) compared to slower and less efficient prior art methods.

Accordingly, the first aspect of the present invention provides an optimized step for releasing the protein from the host cell. More specifically, in a first aspect, a method is provided for isolating and/or purifying a recombinantly expressed intracellular protein comprising an annexin A5 (AnxA5) sequence from a cell wall endotoxin-producing host cell, said method comprising releasing the intracellular protein from the host cell. , and is characterized in that the step of releasing the intracellular AnxA5 protein is carried out in the presence of a homogenization buffer containing a non-ionic detergent. Preferably the non-ionic detergent is a polysorbate, more preferably a polysorbate selected from Tween 20 and Tween 80, and most preferably Tween 80.

The Applicant has also found (as discussed further in Example 2 below) that, in contrast to conventional methods in which successive addition of purification steps results in an ever-increasing loss of product (because there is product loss at each step), the combination of an anion exchange step and an affinity chromatography step with heparin has the surprising advantage of achieving a high degree of purity, which is also obtained using only one stage of heparin affinity chromatography, but with a significantly increased amount of product obtained (i.e. recovery increases from about 30-40% to about 70 -90%). This is the exact opposite of what you would normally expect from a combination of purification steps.

Accordingly, in a second aspect of the present invention, there is provided a method for isolating and/or purifying a protein containing an annexin A5 (AnxA5) sequence from a solution containing an AnxA5 protein and one or more contaminants, the method comprising

exposing the solution containing the AnxA5 protein and one or more impurities to an anion exchange resin to carry out the first anion exchange step, and thereby obtaining a first anion exchange product that contains the released AnxA5 protein; as well as

subjecting the first anion exchange product, directly or indirectly, to an affinity chromatography step, and thereby obtaining a first affinity chromatography product that contains the released AnxA5 protein.

Preferably, according to the method of the second aspect of the present invention, the affinity chromatography step may include binding of the AnxA5 protein to the immobilized heparin, wherein the binding is optionally stimulated by the addition of calcium ions, and furthermore, the AnxA5 protein is optionally eluted from the immobilized heparin with an elution buffer containing a chelator. calcium ions such as EDTA.

In addition, as described in Example 3, Applicant has found that Tween 80 is particularly beneficial in the heparin affinity chromatography step (compared to other non-ionic detergents, including other Tweens such as Tween 20). Incorporating Tween 80, eg, about 0.1% (w/v), in the buffers used in the heparin affinity chromatography step can help elute the AnxA5 protein in a single peak, reduce pressure, and prevent precipitation.

Accordingly, in a third aspect of the present invention, there is provided a method for isolating and/or purifying a protein comprising an annexin A5 (AnxA5) sequence from a solution containing an AnxA5 protein and one or more contaminants, the method comprising subjecting a solution comprising an AnxA5 protein and one or more more impurities (which may or may not be a direct or indirect product of the first anion exchange capture chromatography step as discussed herein), a heparin affinity chromatography step in the presence of Tween 80 (preferably in the presence of about 0.1% w/v . Tween 80), resulting in a first affinity chromatography product that contains the released AnxA5 protein.

The fourth aspect of the present invention is based on the Applicant's realization that calcium metal ion chelators (eg EDTA) can adversely affect the efficiency of the anion exchange steps. Free EDTA (or other chelator) can directly bind to functional anion exchange groups and thereby reduce the capacity as well as the separation achieved in the anion exchange step. On the other hand, attempts to remove the calcium metal ion chelator prior to the anion exchange step are time consuming and hence costly. Therefore, prior art methods involving slow dialysis steps to exchange the buffer are ineffective. In addition, the incorporation of a calcium ion chelator into the AnxA5 product during the anion exchange step may be an important component to prevent calcium-dependent binding of the AnxA5 protein to impurities, including endotoxin. Therefore, it would be convenient and effective to introduce an additive that blocks or reduces the binding of the calcium ion chelator to the anion exchange resin, which would allow the anion exchange step to be carried out without the trouble and expense associated with dialysis and without preventing the positive effect of the calcium ion chelator in the anion exchange step.

Applicant has realized that this can be achieved by including in the AnxA5 protein product prior to anion exchange one or more types of additional selected metal ions, wherein the additional selected metal ions are selected such that the calcium metal ion chelator has a binding affinity for the selected metal ions that is is higher than the binding affinity for the anion exchange resin, but lower than its binding affinity for calcium ions. The choice of appropriate additional metal ions will depend on the nature of the calcium ion chelator and the nature of the anion exchange resin. For example, in the case of using EDTA as a calcium ion chelator, Mg ^{2+ ions} are generally useful for the purpose of this invention and can be added to the AnxA5 protein product prior to the anion exchange step.

Accordingly, in a fourth aspect of the present invention, there is provided a method for isolating and/or purifying a protein containing the annexin A5 (AnxA5) sequence from a composition that contains the AnxA5 protein and a calcium metal ion chelator, characterized in that the method comprises exposing the composition to an anion exchange resin to carry out an anion exchange step and thus isolating and/or purifying the AnxA5 protein from the composition and

additionally characterized by the fact that the anion exchange step is carried out in the presence of additional selected metal ions,

wherein the additional selected metal ions are selected such that the calcium metal ion chelator has a binding affinity for the selected metal ions that is higher than the binding affinity for the anion exchange resin but lower than its binding affinity for calcium ions.

In a fifth aspect of the present invention, there is provided a composition comprising an AnxA5 protein, wherein the composition is a direct or indirect product (resulting from a direct or indirect) process according to any one of the first, second, third or fourth aspects of the present invention. Optionally, said composition is a pharmaceutically acceptable and/or veterinarily acceptable composition.

The sixth aspect of the present invention also provides a composition according to the fifth aspect of the present invention for use in medicine. In other words, in a sixth aspect of the present invention, there is provided a method comprising administering to a human or animal in need thereof a therapeutically effective amount of the composition of the fifth aspect of the present invention.

It is contemplated that any method or composition described herein can be implemented in relation to any other method or composition described herein.

In this context, the word in the singular, when used in conjunction with the term "comprising" in the claims and/or description, may mean "one", but it also corresponds to the meaning of "one or more", "at least one" and "one or more than one."

These and additional aspects of the present invention may be better appreciated and understood when considered in conjunction with the following description and accompanying drawings. However, it should be understood that the following description, indicating various aspects and embodiments of the present invention and numerous specific details thereof, is provided by way of illustration and not limitation. Many substitutions, modifications, additions and/or rearrangements may be made within the scope of the present invention without departing from the spirit thereof, and the present invention includes all such substitutions, modifications, additions and/or rearrangements.

Description of graphic materials

In FIG. 1 depicts the sequence of SEQ ID NO: 1 which is the sequence of human annexin A5.

In FIG. 2 is a flowchart of a complete process flow for annexin A5 as an example.

In FIG. 3 is a flowchart of an example AX capture chromatography process.

In FIG. 4 is a flowchart of an example intermediate affinity chromatography process.

In FIG. 5 is a block diagram of the final chromatographic purification step of AX as an example.

In FIG. 6 is a flowchart of the ultra/diafiltration process and annexin A5 formulation as an example.

In FIG. 7 shows the results of Example 3, which demonstrates the effect of Tween 80 on the purification of annexin A5 by heparin affinity chromatography, while FIG. 7A shows the results of Test 1 (without Tween 80) and FIG. 7B shows the results of test 2 (with Tween 80).

Detailed Description of the Invention

A. Annexin A5 protein

This invention relates to methods for purifying and/or isolating a protein containing the sequence of annexin A5 (AnxA5) and thus produced products and formulations containing the AnxA5 protein.

According to one embodiment of the present invention, the AnxA5 protein that is purified and/or recovered may comprise, consist essentially of, or consist of proteins having the human annexin A5 sequence (SEQ ID NO: 1 as shown in FIG. 1), either with or without N-terminal methionine.

In another embodiment of the present invention, the AnxA5 protein that is purified and/or recovered may comprise, consist essentially of, or consist of a variant or mutant of a protein having the human annexin A5 sequence (SEQ ID NO: 1 as shown in FIG. 1 ), either with or without an N-terminal methionine. For example, a variant or mutant may differ from SEQ ID NO: 1 (either with or without an N-terminal methionine) in any one or more positions equal to or less than 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.

Thus, an Annexin A5 variant or mutant may be a protein in which amino acid insertions, deletions or substitutions, either conservative or non-conservative, have been made at one or more positions. Preferably, the modifications result in a protein without significant changes in its basic properties of functioning in a manner equivalent to annexin A5. The term "significantly" in this context means that a person skilled in the art would say that the properties of the variant may still be different, but not obscure compared to the original proteins.

Preferably the isoelectric point (pI) of the variant or mutant does not change compared to the unmodified protein or is not modified 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.

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

The term "conservative substitutions" refers to combinations such as Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser, Thr; Lys, Arg; and Phe, Tyr.

Variants and mutants can be generated using protein engineering and site-directed mutagenesis techniques that are well known in the art.

In a further embodiment of the present invention, the AnxA5 protein that is purified and/or recovered may be a dimer of a protein that contains, consists essentially of, or consists of a protein having the sequence of human annexin A5 (SEQ ID NO: 1, as shown in 1), either with or without N-terminal methionine, or with a variant or mutant thereof, as described above.

In a further embodiment of the present invention, the AnxA5 protein that is purified and/or recovered may be a fusion protein that contains, consists essentially of, or consists of: (a) one or more protein sequences containing the sequence of a fusion partner, which(s) is/are merged(s); (b) one or more protein sequences that contains, consists essentially of, or consists of a protein having the sequence of human annexin A5 (SEQ ID NO: 1 as shown in FIG. 1) or with an N-terminal methionine, either without it, or with its variant, or mutant, or dimer, as described above. For example, without limitation, a fusion protein may have a general structure selected from:

- in the case of a fusion of two amino acid sequences, for example: H ₂ N-(a)-(b)-COOH; or H ₂ N-(b)-(a)-COOH; or

- in the case of a fusion of three amino acid sequences, for example: H ₂ N-(a)-(b)-(a)-COOH; or H ₂ N-(b)-(a)-(b)-COOH; or H ₂ N-(a)-(b)-(b)-COOH; or H ₂ N-(b)-(b)-(a)-COOH; or H ₂ N-(a)-(a)-(b)-COOH; or H ₂ N-(b)-(a)-(a)-COOH; or

- in the case of a fusion of four amino acid sequences, for example: H ₂ N-(a)-(a)-(a)-(b)-COOH; or H ₂ N-(a)-(a)-(b)-(a)-COOH; or H ₂ N-(a)-(b)-(a)-(a)-COOH; or H ₂ N-(b)-(a)-(a)-(a)-COOH; or H ₂ N-(a)-(a)-(b)-(b)-COOH; or H ₂ N-(a)-(b)-(a)-(b)-COOH; or H ₂ N-(b)-(a)-(a)-(b)-COOH; or H ₂ N-(a)-(b)-(b)-(a)-COOH; or H ₂ N-(b)-(a)-(b)-(a)-COOH; or H ₂ N-(b)-(b)-(a)-(a)-COOH; or H ₂ N-(b)-(b)-(b)-(a)-COOH; or H ₂ N-(b)-(b)-(a)-(b)-COOH; or H ₂ N-(b)-(a)-(b)-(b)-COOH; or H ₂ N-(a)-(b)-(b)-(b)-COOH; or

- in the case of a fusion of five amino acid sequences, for example: or H2N-(a)-(a)-(a)-(a)-(b)-COOH; or H ₂ N-(a)-(a)-(a)-(b)-(a)-COOH; or H ₂ N-(a)-(a)-(b)-(a)-(a)-COOH; or H ₂ N-(a)-(b)-(a)-(a)-(a)-COOH; or H ₂ N-(b)-(a)-(a)-(a)-(a)-COOH; or H ₂ N-(a)-(a)-(a)-(b)-(b)-COOH; or H ₂ N-(a)-(a)-(b)-(a)-(b)-COOH; or H ₂ N-(a)-(b)-(a)-(a)-(b)-COOH; or H ₂ N-(b)-(a)-(a)-(a)-(b)-COOH; or H ₂ N-(a)-(a)-(b)-(b)-(a)-COOH; or H ₂ N-(a)-(b)-(a)-(b)-(a)-COOH; or H ₂ N-(b)-(a)-(a)-(b)-(a)-COOH; or H ₂ N-(a)-(b)-(b)-(a)-(a)-COOH; or H ₂ N-(b)-(a)-(b)-(a)-(a)-COOH; or H ₂ N-(b)-(b)-(a)-(a)-(a)-COOH; or H ₂ N-(a)-(a)-(b)-(b)-(b)-COOH; or H ₂ N-(a)-(b)-(a)-(b)-(b)-COOH; or H ₂ N-(b)-(a)-(a)-(b)-(b)-COOH; or H ₂ N-(a)-(b)-(b)-(a)-(b)-COOH; or H ₂ N-(b)-(a)-(b)-(a)-(b)-COOH; or H ₂ N-(b)-(b)-(a)-(a)-(b)-COOH; or H ₂ N-(a)-(b)-(b)-(b)-(a)-COOH; or H ₂ N-(b)-(a)-(b)-(b)-(a)-COOH; or H ₂ N-(b)-(b)-(b)-(a)-(a)-COOH; or H ₂ N-(a)-(b)-(b)-(b)-(b)-COOH; or H ₂ N-(b)-(a)-(b)-(b)-(b)-COOH; or H ₂ N-(b)-(b)-(a)-(b)-(b)-COOH; or H ₂ N-(b)-(b)-(b)-(a)-(b)-COOH; or H ₂ N-(b)-(b)-(b)-(b)-(a)-COOH,

where (a) and (b) are as defined above in this paragraph. In the case of multiple fusion partner proteins as defined in (a), the multiple fusion partners may be the same or different. Any merger partner of interest may apply. For example, the fusion partner polypeptide sequence(s) may be useful for extending the half-life of the molecule from the patient's circulatory system and/or adding additional functionality to the molecule, such as adding additional therapeutic properties (eg, anticoagulant properties, inhibition and/or cell destruction, etc.). .d.). In the case of fusion proteins containing a plurality of protein sequences having the human annexin A5 sequence (SEQ ID NO: 1 as shown in FIG. 1), either with or without an N-terminal methionine, or with a variant or mutant or dimer thereof, as described above, and as defined in paragraph (b), these proteins may be the same or different.

According to a further embodiment of the present invention, the AnxA5 protein that is purified and/or recovered may be a protein that contains, consists essentially of, or consists of the sequence of Annexin A5 or a functional variant or mutant thereof selected from:

a) human annexin A5 (SEQ ID NO: 1), with or without N-terminal methionine;

b) a mammalian ortholog of human annexin A5;

c) an allelic or genetic variant of a) or b);

d) a protein that is more than 50%, 60%, 70%, 75% identical, such as more than 80%, 85%, more than 90%, or even more preferably more than 95% or 99% identical any of the elements in a), b) or c);

e) a dimer of an element according to any of a), b), c) or d); or

f) a fusion protein containing one or more fusion partners fused to any element of a), b), c), d) or e).

In specific embodiments of this invention, the AnxA5 protein is a functional variant or mutant of annexin A5 that is more than 50%, 60%, 70%, 75% identical, e.g., more than 80%, 85%, more than 90% identical. % or even more preferably more than 95% or 99% human annexin A5, SEQ ID NO: 1, with or without N-terminal methionine.

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 offline version of BLASTZ containing BLASTN version 2.0.14 and BLASTP version 2.0.14. This offline version of BLASTZ can be found on the US National Center for Biotechnology website at ncbi.nlm.nih.gov. Instructions for using 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 the file containing the first amino acid sequence to be compared (eg C:\seq1.txt); - j is set to a file containing the second amino acid sequence to be compared (eg C:\seq2.txt); -p set to blastp; -o set to any desired file name (for example, C:\output.txt); all other parameters are left by default. For example, to generate an output file containing a comparison between two amino acid sequences, the following command could be used: C:\Bl2seq -i c:\seq1.txt -j c:\seq2.txt -p blastp -o c:\output.txt. If two sequences being compared share homology, then the specified output file will represent these areas of homology as aligned sequences. If the two sequences being compared do not share homology, then the specified output file will not represent aligned sequences. After alignment, the number of matches is determined by counting the number of positions in which an identical nucleotide or amino acid residue is present in both sequences.

The percent identity is determined by dividing the number of matches by the length of the sequence indicated in the identified sequence, and then multiplying the resulting value by 100. For example, if the sequence is compared with the sequence indicated in SEQ ID NO: 1 (the length of the sequence indicated in SEQ ID NO: 1 , is 320) and the number of matches is 288, then the sequence has a percent identity of 90 (i.e. 288 ÷ 320 * 100 = 90) with the sequence specified in SEQ ID NO: 1.

The AnxA5 protein may be a dimer of annexin A5 (such as diannexin) or a functional variant or mutant thereof. DiannexinA5 is described in WO 02/067857.

Preferably, the AnxA5 protein does not include a His tag, and no His tag sequence affinity step is used to isolate and purify it.

A His tag is a polyhistidine amino acid motif in proteins that typically consists of at least six histidine (His) residues and is often (though not necessarily) located at the N- or C-terminus of the protein. Polyhistidine tags are often used for the affinity purification of polyhistidine-tagged recombinant proteins expressed in Escherichia coli and other prokaryotic expression systems by incubation with an affinity resin containing bound divalent nickel or cobalt ions, which are commercially available in various forms. These resins are typically sepharose/agarose functionalized with a chelator such as iminodiacetic acid (Ni-IDA) and nitrilotriacetic acid (Ni-NTA) for nickel and carboxymethyl aspartate (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 ion. When using Ni-based methods, washing efficiency can be improved by adding 20 mM imidazole (typically proteins are eluted with 150-300 mM imidazole).

While the His-tag approach is convenient for purification, the presence of non-native polyhistidine motifs in therapeutic proteins, including the AnxA5 protein, is undesirable as it may lead to adverse effects in patients such as immunological reactions. In addition, attempts to remove His tag motifs after protein production are cumbersome, time consuming and costly, and in practice protein preparations from which His tags have been removed typically retain one or more external histidine residues.

Therefore, it is preferable that the AnxA5 protein does not include a His tag, and the step of affinity binding to the His tag sequence is not used for its isolation and purification.

The AnxA5 protein of the invention may or may not be (preferably not) a variant or mutant of a protein having the sequence of human annexin A5 that is modified to contain one or more, for example up to twenty, RGD (arginine- glycine-aspartate) as described in WO 2010/140886, the contents of which are incorporated herein by reference. As described in WO 2010/140886, the addition of one or more RGD motifs can be done to enhance phagocytosis using AnxA5 variants that bind to phosphatidylserine (PS) on apoptotic cells and activate phagocytes to engulf the apoptotic cell instead of inhibiting phagocytosis.

B. Host Cell Culture

The AnxA5 protein can be recombinantly expressed in cultured host cells. Preferred cultures of host cells that express the AnxA5 protein of the present invention are those that are commercially available for production of the AnxA5 protein, such as cultures with a culture volume of about 100 L, about 200 L, about 300 L, about 400 L, approx. 500 l, approx. 600 l, approx. 700 l, approx. 800 l, approx. 900 l, approx. 1000 l, approx. 2000 l, approx. 3000 l, approx. 4000 l, approx. l, approx. 9000 l, approx. 10,000 l, approx. 20,000 l, approx. 30,000 l, approx. 40,000 l, approx. 50,000 l, approx. about 100,000 liters or more. The term "about" in this context may include the value ± 50%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2% or 1% of the specified volume.

Methods for recombinant expression of a gene of interest are well known in the art.

Typically, the nucleotide sequence encoding the AnxA5 protein is recombinantly expressed in host cell culture. For example, the AnxA5 protein coding sequence can be introduced into a host cell by transforming the host cell with a plasmid or other vector containing the AnxA5 protein coding sequence and optionally the AnxA5 protein coding sequence can be integrated into chromosomes (or plastomes). ) host cell or maintained on a replicable extrachromosomal vector.

Accordingly, the host cell can be transformed with a polynucleotide vector construct containing the sequence encoding the AnxA5 protein.

The host cell can be either prokaryotic or eukaryotic.

In the context of this invention, bacterial cells are the preferred prokaryotic host cells. The 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.

At least in the context of the first aspect of the present invention, and optionally in the context of all aspects of the present invention, the host cell is an endotoxin-producing host cell with a cell wall and, therefore, is usually a Gram-negative bacteria such as E. coli, for example, E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and RR1, available from the American Type Culture Collection (ATCC) Rockville, MD, USA (ATCC No 31343).

For the avoidance of ambiguity, the term "endotoxin-producing cell-walled host cell" can be explained in a way that excludes yeasts such as Saccharomyces cerevisiae and other eukaryotic cells.

Another particularly preferred endotoxin-producing E. coli strain is BL21 (DE3) (eg, which is widely available on the market and is described in Marder et al., 2014, BMC Biotechnology, 14:33). However, Applicant found that annexin A5 expressed from strain BL21 (DE3) unexpectedly exhibited unexpectedly high levels of unwanted post-translational gluconoylation, resulting in about 40% of the annexin A5 protein being gluconoylated. This is well above the level of gluconoylation for most other recombinantly expressed proteins in BL21 (DE3), which typically show levels of about 5-10% gluconoylation. Therefore, it is more preferable that the endotoxin-producing E. coli strain is BL21 (DE3), which allows to reduce the level of gluconoylation of the AnxA5 protein, for example, by overexpressing phosphogluconolactonase (PGL), as described in Aon et al. (Appl. Env. Microbiol., 2008, 74(4): 950-958; the contents of which are incorporated herein by reference) and thereby suppress post-translational gluconoylation of the recombinantly expressed protein and thus reduce the generation of gluconoylated variants of the AnxA5 protein to below 40%, such as below 30%, 20%, 10%, 9%. 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% and preferably substantially up to 0%.

Typically, a bacterial host cell for use in the present invention is a walled bacterial host cell and therefore preferably excludes cells lacking a cell wall and also (in the case of Gram-negative bacteria) an outer membrane such as a spheroplast (as described in Liu et al, 2006, J. Exp. Microbiol., 9: 81-85, the contents of which are incorporated herein by reference). Spheroplasts in the context of this application are not endotoxin-producing host cells and do not have a cell wall. Spheroplasts are completely unsuitable for industrial production, especially due to their sensitivity and fragility, given the lack of a cell wall, which severely limits their ability to be grown productively in large volumes.

Optionally, the host cell is an endotoxin-producing host cell with a cell wall that cannot be lysed by osmotic shock and/or freeze/thaw.

In yet another embodiment, the host cell culture is one in which the host cell is not or has not been cultured in the presence of an antibiotic to which it is not resistant, and optionally in the absence of any antibiotic. Specific antibiotics to be avoided in one embodiment of the present invention are antibiotics that lead to the formation of spheroblasts, such as ampicillin.

Eukaryotic host cells may include yeast and mammalian cells, preferably vertebrate cells such as mouse, rat, monkey or human fibroblast cell lines. Yeast host cells include YPH499, YPH500, and YPH501, which are commercially 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 NIH/3T3 mouse embryonic cells available from ATCC as CRL 1658, and COS-1 cells from monkeys available from ATCC as CRL 1650. Preferred insect cells are Sf9 cells which can be transfected with baculovirus expression vectors.

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).

An exemplary mammalian cell vector plasmid is pSVL, available from Pharmacia (Piscataway, NJ, USA). This vector uses the SV40 late promoter to stimulate the expression of cloned genes, with the highest level of expression 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 mouse mammary tumor virus long terminal repeat promoter to stimulate expression of the cloned gene.

Suitable yeast plasmid vectors include pRS403-406 and pRS413-416, which are commercially available from Stratagene Cloning Systems (La Jolla, CA 92037, USA). Plasmids pRS403, pRS404, pRS405 and pRS406 are yeast integration plasmids (YIps) and include the yeast selectable markers HIS3, TRP1, LEU2 as well as URA3. Plasmids pRS413-416 are yeast centromeric plasmids (YCps).

Methods well known to those skilled in the art can be used to construct expression vectors containing the sequence encoding the AnxA5 protein and, for example, suitable transcriptional or translational controls. One such method involves ligation via homopolymer tails. Homopolymer polydA (or polydC) tails are added to expose the 3'-OH groups to the DNA fragment to be cloned by terminal deoxynucleotidyltransferases. The fragment then becomes capable of annealing to polydT (or polydG) tails added to the ends of the linearized plasmid vector. The gaps left after annealing can be filled with DNA polymerase and free ends joined with DNA ligase.

Another method involves ligation with sticky ends. Compatible "sticky ends" can be generated on the DNA fragment and vector by the action of suitable restriction enzymes. These ends will quickly anneal through complementary base pairing and the remaining single strand breaks can be closed by the action of DNA ligase.

Another method uses synthetic molecules called linkers and adapters. Blunt-ended DNA fragments are generated by bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, which remove the overhanging 3' ends and fill in the recessed 3' ends. Synthetic linkers, blunt-ended double-stranded DNA fragments that contain recognition sequences for certain restriction enzymes, can be ligated to blunt-ended DNA fragments using T4 DNA ligase. They are then cleaved with the appropriate restriction enzymes to create "sticky ends" and ligated to the expression vector with compatible ends. Adapters are also chemically synthesized DNA fragments that contain one blunt end used for ligation, but also have one preformed "sticky end".

Synthetic linkers containing multiple restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, Connecticut, USA.

A desirable method for modifying the DNA encoding the AnxA5 protein is by using the polymerase chain reaction as described 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 are themselves inserted into the amplified DNA. These specific primers may contain restriction endonuclease recognition sites that can be used for cloning into expression vectors using methods known in the art.

Transformation of appropriate host cells using a DNA construct containing the sequence encoding the AnxA5 protein is carried out using well-known methods, which usually depend on the type of vector used.

For the transformation of prokaryotic host cells, see, for example, Cohen et al (1972) Proc. Natl. Acad. sci. USA 69, 2110 and Sambrook et al (2001) Molecular Cloning, A Laboratory Manual, 3rd ^Ed . Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Yeast cell transformation 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 can also be used. As regards vertebrate cells, reagents suitable for transfection of such cells, for example calcium phosphate and DEAE-dextran or liposomal formulations, are available from Stratagene Cloning Systems or Life Technologies Inc., Gaithersburg, MD, 20877, USA.

Electroporation is also suitable for cell transformation and is well known in the art for transformation of yeast cells, bacterial cells and vertebrate cells.

For example, many bacterial species can be transformed using the methods described in Luchansky et al (1988) Mol. microbiol. 2, 637-646, which is incorporated herein by reference. The largest number of transformants are consistently recovered after electroporation of a mixture of DNA cells suspended in 2.5X PEB using 6250 V/cm at 25 μFD.

Methods for transforming yeast by electroporation are described in Becker & Guarente (1990) Methods Enzymol. 194, 182.

Physical methods can 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 cells to be transformed. Another example is the bombardment of cells with high speed microparticles, usually gold or tungsten particles, which are coated with DNA.

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

The marker gene can be used to identify transformants, but it is desirable to determine which cells contain recombinant DNA molecules and which contain self-ligated vector molecules. This can be achieved using a cloning vector, wherein the introduction of a DNA fragment destroys the integrity of one of the genes present on the molecule. Therefore, recombinants can be identified based on the loss of function of this gene.

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

In addition to direct analysis for the presence of recombinant DNA, successful transformation can be confirmed by well-known immunological methods when the recombinant DNA is able to direct protein expression. For example, cells successfully transformed with an expression vector produce proteins that exhibit appropriate antigenicity. Samples of cells with expected transformation are collected and analyzed for protein using suitable antibodies.

Thus, the transformed host cells themselves can be cultured to produce a transformed culture of host cells expressing the AnxA5 protein. The culture may be a monoclonal (clonal homogeneous) culture or a culture derived from a monoclonal culture in a nutrient medium.

A culture of transformed host cells expressing the AnxA5 protein is grown under suitable growth conditions until the desired cell density is reached, which is typically chosen to balance the time and cost associated with the culture phase, after which the cells are typically harvested. . The optimal cell collection time can be determined empirically for any given culture.

For example, harvesting cells may include harvesting host cells (which are typically intact and which typically retain substantially all (e.g., more than 80%, 90%, 95%, 99%, or 100%) of the AnxA5 protein intracellularly ) from the culture medium. Typically, this can be achieved by centrifugation or filtration to collect cultured host cells as biomass. In the case of centrifugation, the supernatant can be recovered and the cell pellet can be transferred directly or indirectly (eg after storage, eg freezing) for use in the cell culture homogenization step.

C. Cell culture homogenization

As discussed above, the first aspect of the present invention provides an optimized step for releasing the protein from the host cell. More specifically, in a first aspect, a method is provided for isolating and/or purifying a recombinantly expressed intracellular protein comprising an annexin A5 (AnxA5) sequence from a cell wall endotoxin-producing host cell, said method comprising releasing the intracellular protein from the host cell. , and is characterized in that the step of releasing the intracellular AnxA5 protein is carried out in the presence of a homogenization buffer containing a non-ionic detergent.

Preferably the non-ionic detergent is a polysorbate, more preferably a polysorbate selected from Tween 20 and Tween 80, and most preferably Tween 80. Alternatively, although less preferred, another non-ionic detergent may be used, although preferably avoided (as in the cell culture homogenization step, and in any other steps of the process) using non-ionic detergents that have a UV absorbance λ _max that is similar to the maximum absorbance of proteins in the range between 275 and 280 nm, since this can interfere with the ability of UV absorbance to control the presence of protein in the process. selection. In this context, the term "analogous" may include λ _max that is within the 10 nm, 9 nm, 8 nm, 7 nm, 6 nm, 5 nm, 4 nm, 3 nm, 2 nm, or 1 nm protein absorption maxima AnxA5 being purified. Thus, for example, it may be preferable that the non-ionic detergent is not Triton X-100, which has λ _max = 275 nm, and therefore it may be preferable that Triton X-100 is not used in the cell culture homogenization step and or in any other step. method according to this invention.

It should be noted that the non-ionic detergent may be included in the homogenization buffer that is added to the cells (eg, the homogenization buffer may be "preformed" with the non-ionic detergent present); or cells can be suspended in homogenization buffer without non-ionic detergent, and then non-ionic detergent can be added and mixed with suspended cells in homogenization buffer before cells are homogenized to release intracellular AnxA5 protein.

Preferably, the step of releasing the intracellular AnxA5 protein is carried out in the presence of a homogenization buffer containing an amount of a non-ionic detergent that effectively reduces (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more ) or prevents binding of annexin A5 to endotoxin. Endotoxin levels can be measured using methods well known and accepted in the art, for example, using the horseshoe crab amoebocyte lysate (LAL) test.

For example, the homogenization buffer may contain from 0.01 to 10% (w/w) non-ionic detergent, for example, from 0.02 to 5% (w/w), from 0.05 to 2% (w/w). ./wt.) or about 1% (wt./wt.) non-ionic detergent. The term "about" in this context may include ±0.5%, 0.4%, 0.3%, 0.2%, or 0.1% (w/w).

It is preferable not to add or include any calcium ions or ionizable calcium compounds (such as CaCl ₂ ) in the homogenization buffer. Accordingly, it is preferable that the concentration of free calcium in the homogenization buffer at the time of release of the intracellular AnxA5 protein from the host cell is below 10 mM, preferably below 5 mM, 4 mM, 3 mM, 2 mM or 1 mM, more preferably below 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 essentially zero.

In one embodiment of the present invention, the homogenization buffer may contain a calcium metal ion chelator. It may be preferable, in view of optional downstream steps involving enzymatic treatment in which such enzymes use Mg ²⁺ as a cofactor, to select a calcium ion chelator that does not bind Mg ²⁺ strongly, such as ethylene glycol tetraacetic acid (EGTA). Alternatively, optional subsequent steps involving enzymatic treatment in which such enzymes use Mg ²⁺ as a cofactor can be replaced with other steps not requiring Mg ²⁺ . In this case, any calcium ion chelator such as EGTA or ethylenediaminetetraacetic acid (EDTA) can be included in the homogenization buffer.

Optionally, the concentration of free calcium ions and/or the amount of calcium ion chelator in the homogenizing buffer is (or are) an amount effective to reduce (e.g., 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more) or preventing binding between annexin A5 and components of the cell membrane and/or host cell wall, for example, 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 CaCl ₂ at pH 7.2.

For example, the homogenization buffer according to the first aspect of the present invention may contain 0.01 to 500 mM, such as 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, and preferably the calcium metal ion chelator is EDTA or EGTA.

As discussed above, the cell culture homogenization step according to the first aspect of the present invention does not include a centrifugation step to purify and separate the released AnxA5 protein from host cell debris. However, in order to establish tolerance to free calcium ions and/or an effective amount of calcium ion chelator in homogenization buffer, a simple test can be performed using a centrifugation step on an aliquot of lysed cells. After cell homogenization, an aliquot (eg, 100 ml) of lysed cells is subjected to centrifugation (eg, at 38,900 g for 30 minutes), and then the supernatant and pellet are separated. To obtain the level of "free" AnxA5 protein, the amount of AnxA5 protein in the supernatant is determined. The pellet is resuspended in 50 mM Tris-HCl, 20 mM EDTA, pH 7.2, with stirring for 30 minutes at 4°C to release any bound AnxA5 protein, and then centrifuged at 38,900 g for 30 minutes at 4°C , and to obtain the level of "bound" AnxA5 protein, the amount of bound AnxA5 protein that is released into the supernatant is determined. In this context, the percentage of AnxA5 binding to host cell membrane and/or wall components = (level of "bound" AnxA5 protein / (level of "bound" AnxA5 protein + "free" AnxA5 protein)) x 100.

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

Although the term is referred to herein as "homogenization buffer", it is not essential for the solution to be a pH buffer. However, optionally, the homogenization buffer may further contain additional components, including buffers (e.g., Tris), and may optionally be adjusted as needed, e.g., to about pH 6-8, more preferably in the pH range of 7-8.5, e.g. 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 of the present invention, a pH of 7.4 may be selected for use.

As discussed above, in some embodiments in which the subsequent steps include enzymatic treatment with enzymes requiring cofactors such as Mg ²⁺ , it may also be advantageous to include the cofactor in the homogenization buffer.

In one exemplary embodiment of the present invention, the homogenization buffer for use according to the first aspect of the present invention comprises, consists essentially of, or consists of an aqueous solution of 50 mM Tris, pH 7.4, 1 mM MgCl ₂ and 1% (w/w .) Twin 80.

The method according to the first aspect of the present invention may include the step of mixing the biomass from the culture of host cells in homogenization buffer at a concentration of 1 to 300 g of biomass (wet weight) per liter of homogenization buffer, for example, at a concentration of 10 to 200 g of biomass per liter buffer for homogenization. Typical concentrations may be around 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. Particularly preferred is a resuspension ratio of about 100 g of biomass per liter of homogenization buffer. 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.

As a rule, mixing is carried out at room temperature, i.e., usually about 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 is preferable to control the temperature during the mixing process to maintain it at or near (for example, ± 5, 4, 3, 2, or 1°C) a temperature selected from the list above, although active temperature control is usually not required at this stage.

Optionally, the homogenization buffer may also include one or more enzymes suitable for enzymatic treatment, which can also be added to the buffer after mixing with the biomass, but before homogenizing the host cells. For example, it may be useful to include or add one or more nuclease enzymes to promote cleavage of nucleic acids (including DNA and/or RNA) in host cells after homogenization. This will reduce the viscosity of the subsequently obtained homogenate and thus aid in subsequent processing steps. For this purpose, any suitable enzyme can 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 which is available from numerous commercial sources including Merck/Novagen, Sigma Aldrich and the like, which can be used to cleave all forms of DNA and RNA in the absence of proteolytic activity. . The specified enzyme is effective in a wide range of conditions and has a high specific activity. The enzyme completely cleaves nucleic acids to 2 to 5 base 5'-monophosphate terminal oligonucleotides (below the hybridization limit), which is ideal for removing nucleic acids from recombinant proteins and meeting FDA recommendations regarding nucleic acid contamination. Activation of the benzonase enzyme requires 1-2 mM Mg ²⁺ , while the enzyme remains active in the presence of ionic and non-ionic detergents, reducing agents, PMSF protease inhibitor (1 mM), EDTA chelator (1 mM) and urea (relative activity depends on specific conditions ). One skilled in the art will readily be able to determine the effective concentration of such nuclease enzymes, although Applicant has found that benzonase is effective at least when prediluted to a concentration of about 3.3 U per liter of host cell culture, or about 1.85 U per liter of resuspended biomass. The term "about" in this context may include ± 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the specified number of units.

The step of releasing the intracellular AnxA5 protein from the host cell in homogenization buffer can be based on any approach to cell homogenization or lysis. For example, said step may include lysing, disrupting, or otherwise homogenizing, sonicating, or applying pressure to the host cell, whereby the host cell's cell wall and cell membrane barrier are destroyed and, as a result, the intracellular AnxA5 protein is released. In some embodiments of the first aspect of the present invention, this step does not include exposure to osmotic shock and/or a freeze-thaw step.

In one preferred embodiment of the first aspect of the present invention, the step of releasing the intracellular AnxA5 protein from the host cell comprises high pressure homogenization, e.g., one or more high pressure homogenization cycles in the range of about 400 bar to about 2500 bar, preferably three homogenization cycles. approx. 600 bar or two homogenization cycles approx. 800 bar. In this context, the term "about" may include ± 500, 400, 300, 200, 100, 50, 40, 30, 20 or 10 bar of the specified value.

Optionally, for example, in situations where nuclease enzymes were not added to cleave nucleic acids (for example, when Mg ²⁺ concentrations in the homogenization buffer are too low, for example, due to the inclusion of a Mg ²⁺ chelator), it may be useful to carry out several additional cycles of high pressure homogenization (eg, 2 to 4 cycles at a pressure in the range of about 600 to 2500 bar) to cleave the nucleic acids and reduce the viscosity of the homogenate.

Accordingly, after the cells are homogenized by the step of releasing the intracellular AnxA5 protein, a biomass homogenate containing the released AnxA5 protein can be obtained. Preferably the biomass homogenate is homogeneous; this term should be understood that at least 80%, preferably at least 90%, more preferably at least 95%, 96%, 97%, 98%, 99% or essentially 100% of the biomass cells are disintegrated .

Depending on the applied principle of cell homogenization, the method may cause an increase in the temperature of the homogenate. It may be preferable to apply the method of homogenizing cells in such a way as to prevent undesirable temperature rise, and/or to control the temperature for this purpose. However, for example, in the case where the homogenate is treated with an enzyme such as benzonase, then it may be advantageous to carry out an increase in temperature to approach the optimum temperature range of the enzyme, and preferably within it. In the case of using benzonase, a temperature range of 36 to 40° C. may be particularly suitable.

In the event that the cell homogenization process includes enzymatic treatment with an enzyme that requires a metal ion cofactor (for example, Mg ²⁺ ), and, in addition, in the case when the homogenization buffer excludes a calcium metal ion chelator, then in one of the options a calcium ion chelator can be added after the enzymatic treatment is completed. This may occur before or after the clarification step, as described below.

After cell homogenization, the biomass homogenate typically additionally contains one or more (usually all) impurities selected from the group consisting of host cell proteins, host cell wall components, host cell membrane components, cell nucleic acid- host and endotoxin.

Preferably, the viscosity of the homogenate is low enough to aid in subsequent processing steps.

D. Homogenate clarification

Optionally, and in a preferred embodiment of the present invention, once the biomass homogenate has been obtained, it is treated in a clarification step.

Accordingly, in a further embodiment of the first aspect of the present invention, the method further comprises the step of clarifying the biomass homogenate, resulting in a clarified product containing the released AnxA5 protein. This step is carried out to reduce the content of nucleic acids and, in addition, to obtain a solution with a reduced number of particles, which can be used in subsequent purification steps, such as capture chromatography, as described below.

Any suitable purification step or combination of steps can be carried out. For example, the clarification method may include (preferably after nuclease treatment) the step of passing the biomass homogenate containing the released AnxA5 protein through a filter, such as a cellulose or polypropylene filter, in which the filtrate is a clarified product containing the released AnxA5 protein.

Preferably the filter is a depth filter and/or preferably the filter has a cutoff of less than 4 µm, such as a cutoff of less than 3 µm, 2 µm or 1 µm, and most preferably a cutoff of between 0.2 and 0.6 µm. For example, the homogenate can be purified by filtration using a depth filter with a cutoff of 0.6-0.2 µm; examples of such a filter are commercially available, such as available cellulose-based Cuno 60 SP depth filters. Examples of other depth filters that provide adequate performance (i.e. proper filtration without product loss), albeit somewhat less than the preferred 0.6-0.2 µm Cuno 60 SP cellulose depth filters, include a cellulose + diatomaceous earth filter with a cut-off of 0.5 µm (for example, the PR12 UP filter, which is available from Begerow); a 1.2 µm polypropylene filter (eg Sartopure PP2 filter available from Sartorius); a cellulose filter with a cut-off of 0.1 µm (for example, the Sartoclear S9 filter, which is available from Sartorius); a 0.65 µm polypropylene filter (eg Sartopure PP2 filter available from Sartorius); and a 0.2-0.5 µm cut-off cellulose filter (eg K 1P or EKM-P filters, both available from Pall, with good performance but low filtration speed). Further testing has shown that filters with large cut-offs (eg > 4 µm) only achieve moderate particle removal and are therefore less preferred.

The Applicant has also found that positively charged cellulose based filters further reduce the DNA content of the clarified homogenate and may therefore represent a particularly preferred class of filters for use in the clarification step.

In addition, cellulose-based filters have been found to require a smaller filter area to provide effective clarification compared to the corresponding polypropylene filters. This may be another reason for the particular preference for the cellulose-based filter class for use in the clarification step.

The choice of filter area may also depend on the degree and nature of the applied homogenization. For example, the Applicant has found that in the case of cell homogenization with three homogenization cycles of about 600 bar, the filter area is about 60 cm ² per 1 l of homogenate, while in the case of cell homogenization with two homogenization cycles of about 800 bar, the filter area is about 180 cm ² per 1 liter of homogenate. Accordingly, the depth filter may optionally be selected with an area of 10 to 500 cm ² per liter of clarified homogenate, for example 30 to 400 cm ² /l, 40 to 250 cm ² /l, 50 to 200 cm ² /l or from 60 to 180 cm ² /l, for example, from 50-100 cm ² /l, or 60-80 cm ² /l; or from 120-240 cm ² /l, or 150-210 cm ² /l.

After clarification, the purified product can be further prepared by adding one or more additional additives for use in subsequent steps. For example, for conditioning the clarified product, 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 proper levels of chelator after it has been incorporated into the homogenization buffer and/or added after the enzymatic treatment step.

In one exemplary embodiment of the present invention, the clarified product is diluted approximately 2-fold with a 1% non-ionic detergent (most preferably Tween 80) and a calcium ion chelator (most preferably EDTA) is added to a final concentration of about 2 mM.

Another preferred feature of the process of this invention is that no labor intensive dialysis steps are required for the clarified product prior to additional chromatographic capture steps such as anion exchange capture, as discussed below. Accordingly, in an embodiment of the present invention, the clarified product is not dialyzed prior to chromatographic capture of the AnxA5 protein.

E. Anion exchange capture

The method according to the first aspect of the present invention may further include the step of exposing the released AnxA5 protein to an anion exchange resin to perform the first anion exchange capture step and thereby obtain a first anion exchange product that contains the released AnxA5 protein.

Typically, protein capture by anion exchange chromatography from a bacterial (e.g., E. coli) homogenate/lysate is not the first choice strategy, as high amounts of host cell proteins (HCPs) and DNA bind to the capture resin, affecting the binding abilities of the target. product and even confirming the effectiveness of the resin. However, the Applicant has determined that the cell homogenization and clarification procedures described above according to the first aspect of the present invention are effective in obtaining an AnxA5 protein product that can be effectively further purified by anion exchange chromatography.

Accordingly, in one embodiment of the present invention, the clarified product containing the released AnxA5 protein obtained from the homogenate/lysate purification and/or nucleic acid cleavage step as discussed above is subjected to a first anion exchange capture step, resulting in a first anion exchange product that contains released AnxA5 protein.

Optionally, before the first anion exchange step, one or more environmental parameters of the released AnxA5 protein are adjusted, selected from the group consisting of pH, conductivity, calcium ion chelator level, and nonionic detergent level. For example, the AnxA5 protein composition that is subjected to the anion exchange step can 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 embodiment, the preferred range is pH 6-8.5, more preferably 6.5-7.5, most preferably 6.9. Applicant has found that low values pHs in this range (e.g., pH 6) tend to result in detectable host cell proteins in the eluted product, while the degree of separation decreases somewhat at or above pH 8. An AnxA5 protein composition that undergoes an anion exchange step may optionally be adjusted with a specific 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 purified product can be further prepared by adding one or more additional additives for use in subsequent stages.As discussed above, for conditioning the AnxA5 protein composition to be subjected to the anion exchange step, prior to the first anion exchange step, it may be appropriate to add 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 proper levels of chelator after it has been incorporated into the homogenization buffer and/or added after the enzymatic processing step. In one exemplary embodiment of the present invention, the AnxA5 protein composition that is subjected to the anion exchange step is diluted approximately 2-fold with a 1% non-ionic detergent (most preferably Tween 80) and a calcium ion chelator (most preferably EDTA) is added to a final concentration of about 2 mM.

In this context, to enhance the separation of the AnxA5 protein from host cell contaminants (including cell wall components, cell membrane components, endotoxin, nucleic acids, etc.) in the first anion exchange capture step, the use of a non-ionic detergent and/or chelator can be very beneficial. calcium metal ions to condition the AnxA5 protein composition which is subjected to said first anion exchange capture step. For example, Applicant has demonstrated highly efficient endotoxin removal (about 99% reduction) in the first anion exchange capture step, carried out in the presence of a non-ionic detergent and a calcium metal ion chelator.

While the discussion below is primarily in the context of a second anion exchange purification step, for an embodiment in which a calcium ion chelator (e.g., EDTA) is present in the AnxA5 protein composition that is subjected to the first anion exchange scavenging step, for said first anion exchange step to function properly, It may also be useful to include one or more types of additional selected metal ions (not calcium), wherein the additional selected metal ions are selected such that the calcium ion chelator has a binding affinity for the selected metal ions that is higher than the binding affinity for the anion exchange resin, but lower than its binding affinity for calcium ions. One example of a metal ion is Mg ²⁺ .

Typically, the anion exchange resin is equilibrated prior to contacting the anion exchange resin and the AnxA5 product. To this end, you can use any suitable method of balancing. For example, the anion exchange resin can be equilibrated with a buffer (eg, 20 mM Tris, pH 7.4), a non-ionic detergent (eg, 0.1% polysorbate, preferably Tween 80), and salt (eg, 25 mM NaCl). Any suitable equilibrium volume may be used; without limitation, Applicant has found 3 column volumes (CV) as a usable volume in the exemplary embodiment of the present invention.

Preferably, the first anion exchange capture step is performed in a positive mode with respect to the AnxA5 protein, and therefore the AnxA5 protein is temporarily bound to the anion exchanger during the anion exchange step, with the wash solution typically passed through a column to remove contaminants from the bound AnxA5 protein, and then the product the first anion exchange, which contains the released AnxA5 protein, is obtained by applying the elution buffer to the anion exchange resin to release the bound AnxA5 protein.

Strong anion exchange resins are preferred because Applicant has found that they provide acceptable entrapment capabilities for the AnxA5 protein, while performance with weak anion exchange resins has been less acceptable. Strong anion exchange resins are well known in the art and examples include quaternary ammonium functional resins such as type I resin containing trialkylammonium chloride or hydroxide, or type II resins containing dialkyl 2-hydroxyethylammonium chloride or hydroxide (e.g., Q Sepharose XL from GE Healthcare, Capto Q from GE Healthcare, Unosphere Q from Biorad, or Eshmuno Q from Merck). Weak anion exchange resins are not preferred; examples thereof include DEAE resin with a diethylaminoethyl functional group. Q Sepharose XL resin (eg, available from GE Healthcare) may be most preferred.

After loading onto an anion exchange resin, in positive mode, the AnxA5 protein temporarily binds to the resin and can be washed to reduce/remove impurities. Any suitable washing conditions may be used. For example, the wash solution may contain, consist essentially of, or consist of an aqueous solution of a buffer (for example, 20 mM Tris, pH 7.4), a non-ionic detergent (for example, 0.1% polysorbate, preferably Tween 80) and salt (for example, 25 mM NaCl). Any suitable rinsing volume may be used; without limitation, Applicant has found 10 column volumes (CV) as a usable volume in the exemplary embodiment of the present invention.

The bound AnxA5 protein is then released from the anion exchange resin using an elution buffer. For this purpose, any suitable elution buffer may be used. For example, the elution buffer may contain, 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., 0.01 to 1% (w/v) , more preferably 0.1% (w/v) polysorbate, preferably Tween 80) and salt at a concentration higher than the wash solution (e.g. 300 mM NaCl, optional ± 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 or 5 mM). Any suitable elution volume may be used; without limitation, Applicant has found 9 column volumes (CV) as a suitable elution volume in the exemplary embodiment of the present invention.

Accordingly, the AnxA5 protein is captured in the elution buffer, resulting in a first anion exchange product.

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

Typically, the first anion exchange product that contains the released AnxA5 protein contains substantially more than 50% of the AnxA5 protein that is released from the host cells. Preferably, the product of the first anion exchange contains more than 60%, 70% or 80% of the AnxA5 protein, which was obtained from host cells. Compared to the method of Marder et al. (above), it is clear that prior art processes exhibit large product losses of about 50% or more. For example, the method of Marder et al., (above) includes an initial cleansing centrifugation step in which the supernatant is removed and annexin A5 associated with the pellet is collected. The relative amounts of annexin A5 in the removed supernatant and annexin A5 isolated from the pellet are shown in FIG. 3, lanes 2 and 3, respectively, after Marder et al. (above). This figure shows that in the method of Marder et al. (above) about half of the released annexin A5 is removed, which makes this method as low-yielding.

As an additional comparison parameter, Applicant found that the illustrated example provided a yield of about 5 g of AnxA5 protein per liter of culture at the end of the first anion exchange capture chromatography step, which is much greater than the yield reported by Marder et al. (above). (see "Conclusions", second paragraph), amounting to 0.953 g of purified annexin A5 per liter of culture.

Accordingly, according to the first aspect of the present invention, it is preferred that the first anion exchange product contains more than 1 g, more than 2 g, more than 3 g, more than 4 g, or about 5 g of AnxA5 protein per liter of culture.

Optionally, the first anion exchange product is subjected, directly or indirectly, to a filtration step (eg, a sterilizing filtration step). It has been found that, for example, without limitation, a filtration step of 0.45-0.2 μM is suitable.

F. Affinity Chromatography

The Applicant has also found (for example, see Example 2 below) that, in contrast to well-known methods, in which the successive addition of purification steps results in an ever-increasing loss of product obtained (since there is a loss of product at each step), the combination of an anion exchange step and an affinity chromatography step with heparin has the surprising advantage of achieving a high degree of purity, which is also obtained using only one stage of heparin affinity chromatography, but with a significantly increased amount of product obtained (i.e. recovery increases from about 30-40% to about 70 -90%). The significant increase in product yield associated with the addition of a purification step is in direct contrast to what would normally be expected from a combination of purification steps.

Accordingly, in a second aspect of the present invention, there is provided a method for isolating and/or purifying a protein containing the annexin A5 (AnxA5) sequence from a solution containing the AnxA5 protein and one or more impurities (which may or may not be the product of the clarification step as described above), the method comprising exposing a solution containing the AnxA5 protein and one or more impurities (this solution may or may not be a direct or indirect product of the homogenization, clarification and/or first anion exchange chromatography step as discussed above) to an anion exchange resin for the purpose of carrying out the first anion exchange step, and thereby obtaining a first anion exchange product that contains the AnxA5 protein; as well as

subjecting the first anion exchange product, directly or indirectly, to an affinity chromatography step, and thereby obtaining a first affinity chromatography product that contains the released AnxA5 protein.

Preferably, according to the method of the second aspect of the present invention, the affinity chromatography step may include binding of the AnxA5 protein to the immobilized heparin, wherein the binding is optionally promoted by the addition of calcium ions, and furthermore, the AnxA5 protein is optionally eluted from the immobilized heparin with an elution buffer containing a chelator. calcium ions such as EDTA.

In addition, as described in Example 3, Applicant has found that Tween 80 is particularly beneficial in the heparin affinity chromatography step (compared to other non-ionic detergents, including other Tweens such as Tween 20). Incorporating Tween 80, eg, about 0.1% (w/v), in the buffers used in the heparin affinity chromatography step can help elute the AnxA5 protein in a single peak, reduce pressure, and prevent precipitation.

Accordingly, in a third aspect of the present invention, there is provided a method for isolating and/or purifying a protein comprising an annexin A5 (AnxA5) sequence from a solution containing an AnxA5 protein and one or more contaminants, the method comprising subjecting a solution comprising an AnxA5 protein and one or more more impurities (which may or may not be a direct or indirect product of the homogenization, clarification and/or first anion exchange chromatography step as discussed above), the heparin affinity chromatography step in the presence of Tween 80 (preferably in the presence of 0.1% Tween 80 ), resulting in a first affinity chromatography product that contains the released AnxA5 protein.

Thus, both the second and third aspects of the invention, which may be used independently or in combination (i.e., the heparin affinity chromatography step in the second aspect of the invention, may include Tween 80 (e.g., about 0.1% (w/v)) in the buffers used in the heparin affinity chromatography step,

More generally, however, the first aspect of the invention may include the step of subjecting the released AnxA5 protein to an affinity chromatography step, and thereby obtaining a first affinity chromatography product that contains the released AnxA5 protein. It may be particularly preferred that the AnxA5 protein in the first anion exchange product, eg obtained by the method described above, be directly or indirectly subjected to an affinity chromatography step (eg, after sterilizing filtration and/or addition of additional components).

Accordingly, in one embodiment of the first aspect of the present invention (which may be combined with one or both features of the second and third aspects of the present invention), the method includes the steps of:

(a) a biomass homogenate containing the released AnxA5 protein as described above is clarified using the clarification method as described above, and thus a clarified product containing the released AnxA5 protein is obtained, and

(b) the AnxA5 protein in the clarified product is exposed to an anion exchange resin to carry out the first anion exchange step as described above, and thus a first anion exchange product is obtained which contains the AnxA5 protein; as well as

(c) The AnxA5 protein in the first anion exchange product is subjected (directly or indirectly) to an affinity chromatography step.

Preferably, the affinity chromatography step includes binding of the AnxA5 protein to immobilized heparin, and optionally, binding is stimulated by the addition of calcium ions.

Accordingly, the AnxA5 product can be conditioned to the heparin affinity step by adding any one or more calcium ions (e.g. CaCl ₂ ), a non-ionic detergent (preferably Tween 80), and optionally buffering (e.g. Tris buffer at pH 7.4) . Without limitation, Applicant has demonstrated a beneficial effect when the filtered anion exchange product is diluted about 8-fold with a dilution buffer containing 20 mM Tris, pH 7.4, 0.1% Tween 80, and 2 mM CaCl ₂ .

Therefore, it may be preferred that the AnxA5 product be conditioned with polysorbate 80, and more preferably when polysorbate 80 has a final concentration of from about 0.01% 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%, 0.9 %, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. The term "about" in this context refers to ±50%, 40%, 30%, 20%, 10% or 5% of the specified value.

Dilution with calcium promotes binding of annexin A5 to heparin during heparin-immobilized chromatography. This interaction, in comparison with the ionic interaction, is slow. The contact time is important, so chromatography is preferably carried out with ≤ 100 cm/h.

The conditions used when loading an affinity chromatography column (eg, a heparin affinity chromatography column) allow binding of the AnxA5 protein to heparin. That is, affinity chromatography is usually performed in a positive mode with respect to the AnxA5 protein.

An affinity chromatography column (eg, a heparin affinity chromatography column) can be loaded with the desired level of AnxA5 product. For example, loading can be carried out in a volume of about or more than 5 g per liter of column resin volume, for example, 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 specified value. In practice, Applicant has found that loading about 20 to 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 loadings, Applicant has found that the presence of Polysorbate 80 in the feed mixture is particularly useful in preventing AnxA5 protein precipitation or insolubility. If polysorbate 80 is not used, precipitation and back pressure increase occur and the affinity chromatography step becomes less efficient. See examples 3 and 4.

After loading 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, without limitation, that a suitable wash protocol includes a two step wash. For example, the first wash step can be performed using a first wash buffer containing calcium (eg, 20 mM Tris, pH 7.4, 0.1% Tween 80, 2 mM CaCl ₂ ). The volume for the first rinsing step may vary depending on the desired result and the basic properties of the rinsing solution used. Without limitation, the Applicant has found, for example, that the typical wash buffer above can be successfully used in a 15 OK wash. In the second wash step, the wash buffer may contain less calcium than the first wash, or preferably no calcium (eg, 20 mM Tris, pH 7.4; 0.1% Tween 80). The volume for the second washing step may vary depending on the desired result and the basic properties of the washing solution used in the second washing step. Without limitation, Applicant has found, for example, that the typical second-stage wash buffer above can be successfully used with 2 OCs for a wash.

Thereafter, the AnxA5 protein is eluted from the affinity chromatography column using an elution buffer, and thus a first affinity chromatography product is obtained which contains the released AnxA5 protein. In the case of a heparin affinity chromatography column, it may be preferable to use an elution buffer including a calcium metal ion chelator such as EDTA or EGTA.

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

During elution, in order to increase the concentration of the product, it may be preferable to reduce the flow rate to less than 100 cm/h. For example, without limitation, in the examples, Applicant allowed concentrated elution by reducing the flow rate to ≤ 60 cm/h during elution. A full elution peak, for example, can be obtained by starting with an increase in the UV signal at 0.05 EOP to 0.05 EOP in a descending peak representing about 7 OC. Preferably, the elution profile shows a single peak, which is most ideally a sharp peak.

Accordingly, the first affinity chromatography product contains the released AnxA5 protein and a calcium ion chelator such as EDTA or EGTA, optionally the calcium ion chelator is present in the range from about 0.1 mM to 500 mM, for example, from about 1 mM to about 100 mM , more typically in the range of about 2 mM to about 50 mM, more preferably in the range of about 5 mM to about 15 mM, and most preferably about 10 mM. The term "about" in this context may include a value of ± 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the specified concentration(s).

The affinity chromatography step may represent the most powerful purification step in a process design. The 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 protein forms of AnxA5 that are able to combine with calcium can bind to heparin. Thus, an affinity chromatography step may be useful in order to help distinguish between a product with a correct and an irregular structure. In addition, high lean ratios are achieved in the intermediate step because a highly specific interaction is combined with a specific elution regimen via calcium chelation with EDTA. Therefore, there is a strong decrease in the level of endotoxin and HCP, combined with a moderate decrease in the content of DNA. Typically, the level of endotoxin, which is already reduced (preferably by about 97%), is further reduced during the preceding anion exchange step (preferably by about 99%), thereby preferably adjusting endotoxin levels to 0.03% of its levels in the clarified product before the first anion exchange capture step.

G. Anion exchange purification

In a further embodiment of the first, second, and/or third aspects of the present invention, the AnxA5 product (eg, obtained in the affinity chromatography step) can be further purified, directly or indirectly, in the anion exchange purification step.

In the case where the AnxA5 product obtained from the affinity chromatography step is indirectly further purified by the anion exchange purification step, the affinity chromatography steps and the anion exchange purification step can be separated by adding one or more conditioning agents to the product obtained from the affinity chromatography step.

Suitable additives may include, for example, a diluent (eg water) to further dilute the AnxA5 protein in the product from the affinity chromatography step; buffer (eg Tris, eg 35 mM, pH 8); a non-ionic detergent (eg, polysorbate, more preferably Tween 80, for example, at a concentration of about 0.1% w/v); and/or one or more additional additives based on the fourth aspect of the present invention, as discussed below.

It is understood that the fourth aspect of this invention is based on the Applicant's recognition that calcium metal ion chelators (eg EDTA) can adversely affect the efficiency of the anion exchange steps. Free EDTA (or other chelator) can directly bind to functional anion exchange groups and thereby reduce the capacity as well as the separation achieved in the anion exchange step. There are certain limitations when performing an anion exchange on a product from a heparin affinity chromatography step in which the AnxA5 protein is bound to heparin in the presence of calcium ions, followed by the use of a calcium metal ion chelator (e.g., EDTA) to elute the bound AnxA5 protein. As a consequence, the eluted AnxA5 product from the heparin affinity chromatography step contains high levels of calcium ion chelator. It is generally desirable to be able to further purify the AnxA5 product with an additional anion exchange step, but the use of a calcium ion chelator can be difficult during this additional anion exchange step. On the other hand, attempts to remove the calcium metal ion chelator prior to the anion exchange step are time consuming and hence costly. Therefore, prior art methods, including, for example, dialysis steps for buffer exchange, are slow and inefficient, and therefore increase production costs. In addition, the incorporation of a calcium metal ion chelator into the AnxA5 product at the anion exchange step may be an important component to prevent calcium-dependent binding of the AnxA5 protein to impurities, including endotoxin. Therefore, it would be convenient and effective to introduce an additive that blocks or reduces the binding of the calcium ion chelator to the anion exchange resin, which would allow the anion exchange step to be carried out without the trouble and expense associated with dialysis and without preventing the positive effect of the calcium metal ion chelator in the anion exchange step.

Applicant has realized that this can be achieved by including in the AnxA5 protein product prior to anion exchange one or more types of additional selected metal ions, wherein the additional selected metal ions are selected such that the calcium metal ion chelator has a binding affinity for the selected metal ions that is is higher than the binding affinity for the anion exchange resin, but lower than its binding affinity for calcium ions. The choice of appropriate additional metal ions will depend on the nature of the calcium ion chelator and the nature of the anion exchange resin. For example, in the case of using EDTA as a calcium ion chelator, Mg ^{2+ ions} are generally useful for the purpose of this invention and can be added to the AnxA5 protein product prior to the anion exchange step.

Accordingly, in a fourth aspect of the present invention, there is provided a method for isolating and/or purifying a protein containing an annexin A5 (AnxA5) sequence from a composition that contains the AnxA5 protein and a calcium metal ion chelator, characterized in that the method comprises exposing the composition to an anion exchange resin to carry out an anion exchange step and thus isolating and/or purifying the AnxA5 protein from the composition and

additionally characterized by the fact that the anion exchange step is carried out in the presence of additional selected metal ions,

wherein the additional selected metal ions are selected such that the calcium metal ion chelator has a binding affinity for the selected metal ions that is higher than the binding affinity for the anion exchange resin but lower than its binding affinity for calcium ions.

Preferably, additional selected metal ions are mixed with the composition, which contains the AnxA5 protein and the calcium metal ion chelator, before the composition is exposed to the anion exchange resin. Solutions used in subsequent anion exchange steps (eg, wash solutions and/or elution buffers) may or may not include additional selected metal ions. Therefore, in one embodiment of this aspect of the invention, the step of conducting the anion exchange step in the presence of additional selected metal ions involves adding additional selected metal ions to the composition that contains the AnxA5 protein and a calcium metal ion chelator, prior to exposing said composition to the anion exchange resin.

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

The calcium metal ion chelator may be present in the composition in excess and/or at a concentration ranging from about 0.1 mM to 500 mM, such as from about 1 mM to about 100 mM, more typically in the range from about 2 mM to about 50 mM, for example, at 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 a value of ± 0.5, 0.4, 0.3, 0.2, or 0.1 mm from the specified value. In the above context, the term "excess" may mean that sufficient calcium metal ion chelator is present to remove any divalent ion, which may cause the AnxA5 protein to bind to the immobilized heparin on the column in the preceding affinity chromatography step, whereby the AnxA5 protein may be released from columns into solution, and then applied directly or indirectly to the anion-exchange purification step.

In one exemplary embodiment of the fourth aspect of this invention, the selected metal ions are divalent cations such as Mg ^{2+ ions}

Preferably, the selected metal ions are present in the anion exchange step in an amount effective to reduce (e.g., about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more) or prevent interaction between the calcium ion chelator and the anion exchange resin during the process of bringing the composition into contact with the anion exchange resin. For example, selected metal ions may be present in the anion exchange step in an amount effective to reduce or prevent interaction between the calcium ion chelator and the anion exchange resin during loading of the composition onto the anion exchange resin and/or in one or more wash steps in which the AnxA5 protein binds to anion exchange resin, and impurities are removed by washing.

It may be preferred that selected metal ions are present in the anion exchange step in an amount effective to increase the binding of the AnxA5 protein to the anion exchange resin in the presence of a calcium ion chelator and thereby decrease (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more) loss of AnxA5 protein in the anion exchange filtrate compared with the level of losses observed when no selected metal ions are used in the anion exchange step.

It may be preferred that the selected metal ions are present in 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.

In addition, it may be preferred that the calcium metal ion chelator be EDTA and the metal ions chosen be Mg ^{2+ ions} , and more preferably the molar ratio of Mg ^{2+ ions} to EDTA be in the range of 0.5:1 to 2: 1, most preferably at least 1:1 or >1:1.

In the context of the fourth aspect of the present invention, in a further embodiment of the present invention, the composition which contains the AnxA5 protein and a calcium metal ion chelator and which is exposed to the anion exchange resin may be a direct or indirect product of a prior process which includes the step of subjecting the AnxA5 protein to an affinity step. chromatography and eluting the AnxA5 protein with a calcium ion chelator, resulting in an affinity chromatography product that is a composition containing the AnxA5 protein and a calcium metal ion chelator. For example, the preceding affinity chromatography step may include the binding of the AnxA5 protein to the immobilized heparin, wherein the binding is optionally stimulated by the addition of calcium ions and furthermore, the AnxA5 protein is optionally eluted from the immobilized heparin with an elution buffer containing a calcium ion chelator such as EDTA. .

In addition, it may be preferable not to perform a dialysis step between the preceding affinity chromatography step and the anion exchange step and/or not to remove the calcium ion chelator from the product of the preceding affinity chromatography step prior to using the direct or indirect product in the anion exchange step.

In the context of a fourth aspect of this invention, in a further embodiment of this invention, selected metal ions are added to the composition before or during the anion exchange step.

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

Optionally, prior to the (second) anion exchange purification step, one or more environmental parameters of the released AnxA5 protein are adjusted, selected from the group consisting of AnxA5 protein concentration, pH, conductivity, calcium ion chelator level, and non-ionic detergent level, or selected additional metal ion level ( according to the fourth aspect of the present invention).

Typically, the anion exchange resin is equilibrated prior to contacting the (second) anion exchange resin in the purification step and the AnxA5 product. To this end, you can use any suitable method of balancing. For example, the anion exchange resin can be equilibrated with a buffer (eg, 20 mM Tris, pH 7.4), a non-ionic detergent (eg, 0.1% polysorbate, preferably Tween 80), and salt (eg, 25 mM NaCl). Any suitable equilibrium volume may be used; without limitation, Applicant has found 3 column volumes (CV) as a usable volume in the exemplary embodiment of the present invention.

Preferably, the (second) anion exchange step is performed in positive mode with respect to the AnxA5 protein, and therefore the AnxA5 protein is transiently bound to the anion exchanger during the anion exchange step, typically a wash solution is passed through a column to remove contaminants from the bound AnxA5 protein, and then, the second anion exchange product, which contains the released AnxA5 protein, is obtained by applying the elution buffer to the anion exchange resin to release the bound AnxA5 protein.

The use of strong anion exchange resins is preferred. Strong anion exchange resins are well known in the art and examples include quaternary ammonium functional resins such as type I resin containing trialkylammonium chloride or hydroxide, or type II resins containing dialkyl 2-hydroxyethylammonium chloride or hydroxide. Without limitation, one example of a suitable anion exchange resin for the second purification step is Source 15Q. Source 15Q anion exchange resin can be defined as a polymeric strong anion exchanger and can additionally be characterized by the presence of a quaternary ammonium ligand, with an average particle size distribution of the total volume of about 15 microns (d50v), a polystyrene / divinylbenzene matrix and / or pressure / flow parameters of about 400 cm / h, 1000 kPa, when evaluating it as a FineLine 100 column with a layer height of 10 cm.

Without limitation, an additional example of a suitable anion exchange resin for the second purification step is Capto Q ImpRes. Capto Q ImpRes anion exchange resin can be defined as a strong anion exchanger and can be further characterized by the presence of a quaternary amine ligand, high flux agarose matrix, with an average particle size of about 36-44 µm (d50v), an ionic capacity of about 0.15-0.18 mmol Cl ^- /ml medium, binding capacity/ml chromatographic medium > 55 mg BSA and > 48 mg β-lactoglobulin and/or pressure/flow parameters of about 300 kPa at min. 220 cm / h, when assessing it as a column with a diameter of 1 m with a layer height of 20 cm.

Without wishing to be bound by theory, Applicant has found that the use of Capto Q ImpRes anion exchange resin for the (second) anion exchange purification step can be particularly advantageous when the AnxA5 protein to be purified comes from a recombinant source (such as E. coli BL21 (DE3), which additionally produces an overexpressing PGL as described in Aon et al (Appl. Env. Microbiol., 2008, 74(4): 950-958; the content of which is incorporated herein by reference) that does not or causes a low level of protein gluconoylation AnxA5 expressed by it The term "low" in this context may include meaning that the level of gluconoylation is less (e.g., less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% , 10% 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) than the level of gluconoylation of the AnxA5 protein that is expressed in E. coli strain BL21 (DE3) (e.g. , as is widely commercially available, and as described in Marder et al., 2 014, BMC Biotechnology, 14:33). For example, it is possible that the level of gluconoylated AnxA5 protein in the product that is applied to the Capto Q ImpRes anion exchange resin for the (second) anion exchange purification step is in the range of 0.5 to 30%, or 0.5 to 20%, or 0 .5 to 15%, or 0.5 to 10% of the total AnxA5 protein content in the product used. Gluconoylated Anx5 variants can, for example, be measured and quantified by ultra high performance liquid chromatography (UHPLC) or high performance liquid chromatography (HPLC) using appropriate anion exchange or reverse phase columns. Various peaks can be further identified and characterized using mass spectroscopy (MS).

Applicant has found that the use of Capto Q ImpRes anion exchange resin for the (second) step of anion exchange purification, when the AnxA5 protein to be purified has no or low levels of gluconoylation, results in an even more efficient process (compared to, say, using an anion exchange resin Source 15Q for the (second) anion exchange step) because Capto Q ImpRes anion exchange resin has high binding capacity, is suitable for high flow rates without back pressure, can be packed in a higher layer and has a lower price. The quality and purity of the final product is maintained regardless of whether Source 15Q anion exchange resin or Capto Q ImpRes is used. However, switching from Source 15Q anion exchange resin (particle size 15 µm) to Capto Q ImpRes anion exchange resin (particle size 40 µm) is believed to provide a performance increase that can be more than about five times (5x) by reducing the time required for the entire process to function (particularly since the second anion exchange step used for purification is one of the most labor-intensive steps) and can reduce the overall manufacturing cost by more than 50%. Eliminating the use of resin with a very small bead size (<30 µm) provides high flow rates and allows the chromatography column to be packed on a higher resin bed without unacceptable back pressure. This increases throughput because for a given size (column diameter) more resin can be packed into the column and therefore more protein can bind, while at the same time allowing the system to run faster due to the higher flow rate.

After loading onto an anion exchange resin in the (second) purification step, in positive mode, the AnxA5 protein temporarily binds to the resin and can be washed to reduce/remove impurities. Any suitable washing conditions may be used. For example, the wash solution may contain, consist essentially of, or consist of an aqueous solution of buffer (eg, 20 mM Bis-Tris, pH 7) and salt (eg, 25 mM NaCl) and optionally a non-ionic buffer (eg, polysorbate, preferably Tween 80 , for example, at a concentration of about 0.1 wt./vol.). Any suitable rinsing volume may be used; without limitation, Applicant has found 3 column volumes (CV) as suitable volumes in the exemplary embodiment of the present invention.

The bound AnxA5 protein is then released from the anion exchange resin using an elution buffer. For this purpose, any suitable elution buffer may be used. For example, the elution buffer may contain, consist essentially of, or consist of an aqueous buffer solution (e.g., 20 mM Bis-Tris, pH 7), salt at a concentration higher than the wash solution (e.g., 180 mM NaCl, optionally ± 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 or 5 mM) and optionally a non-ionic buffer (eg polysorbate, preferably Tween 80, for example at a concentration of about 0.1 w/v). Any suitable elution volume may be used; without limitation, Applicant found 33 column volumes (CVs) by increasing the concentration of elution buffer from 0 to 100% in a linear gradient to obtain a usable elution volume in the exemplary embodiment of the present invention.

Accordingly, the AnxA5 protein is released into the elution buffer, resulting in a purified product of the (second) anion exchange. As discussed in Example 1, the 0.1% Tween 80 process increased product yield after the intermediate step by about 30%.

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

The purification step is mainly necessary to reduce the level of impurities associated with the product, for example, the separation of various annexin A5 isoforms. In addition, the purification step achieves the highest depletion ratio for residual DNA and greatly reduces the level of HCP. Endotoxin levels, already reduced after the intermediate step, are further reduced by about 99% (when used in combination with prior cell homogenization, nuclease treatment, clarification, anion exchange capture, filtration (e.g. sterile filtration step) and heparin affinity step), in whereby the levels of endotoxin are adjusted to 0.0003% of its levels in the clarified product prior to the first AX step.

Accordingly, in a further embodiment of the first, second, third and/or fourth aspects of the present invention, there is provided a method for recovering and/or purifying a recombinantly expressed intracellular protein comprising an annexin A5 (AnxA5) sequence from a host cell with a cell wall or culture according to the first aspect of this of the invention and preferably, said method comprising isolating and/or purifying a recombinantly expressed intracellular AnxA5 protein from a culture of host cells, wherein the culture has a volume of at least about 100 L, about 200 L, about 300 L, about 400 L , approx. 500 l, approx. 600 l, approx. 700 l, approx. 800 l, approx. 900 l, approx. 1000 l, approx. 2000 l, approx. 3000 l, approx. 8000 l, approx. 9000 l, approx. 10,000 l, approx. 20,000 l, approx. 30,000 l, approx. 40,000 l, approx. l, about 100,000 liters or more, while:

(a) the method includes releasing an intracellular protein from a host cell in the presence of a homogenization buffer containing a non-ionic detergent according to the first aspect of the present invention;

(b) optionally, wherein the release step corresponds to any one or more embodiments of the first aspect of the present invention as described above in subsection C;

(c) optionally further, the method comprising the step of purifying the biomass homogenate according to any one or more embodiments of the present invention as described above in subsection D; as well as

(d) wherein the method further comprises the step of directly or indirectly exposing the released AnxA5 protein to an anion exchange resin to perform a first anion exchange step and thereby obtaining a first anion exchange product that contains the released AnxA5 protein according to any one or more embodiments of this inventions as described above in subsection E; as well as

(e) wherein the method further comprises the step of subjecting the released AnxA5 protein, directly or indirectly, to an affinity chromatography step according to any of the first, second and/or third aspects of the present invention as described above in section F, and

(f) wherein the product of the affinity chromatography step is a composition that contains the AnxA5 protein and a calcium metal ion chelator; as well as

(g) wherein the direct or indirect product of the affinity chromatography step, which contains the AnxA5 protein and a calcium metal ion chelator, is subjected to an anion exchange step according to any of the embodiments of the present invention described above in this section (i.e., in section G).

H. Composition of the product

The method according to any one of the first, second, third or fourth aspects of the present invention may further comprise, preferably at the end of the method, one or more additional steps selected from the group consisting of concentration, buffer exchange, conditioning and filtration (e.g. sterile filtration step ) and optionally a final storage step for the AnxA5 protein containing protein in a sterile container.

For example, one of the additional steps used to prepare the product may be ultrafiltration/diafiltration (UV/DF), optionally, wherein the product of the UV/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 higher.

In another example, one of the additional steps used to prepare the product may be the addition of a non-ionic surfactant, preferably Polysorbate, and more preferably Tween 80. concentrations of about 0.05% (w/w) (for example, ± 0.05, 0.04, 0.03, 0.02 or 0.01% w/v).

In another example, one of the additional steps used to prepare the product may be a filtration step (such as a sterilizing filtration step), for example using a 0.45-0.2 µm or 0.22 µm filter.

As indicated above, the method according to the first, second, third or fourth aspects of the present invention may end with the step of sterilizing filtration and placing a sterile filtered product containing AnxA5 protein in a sterile container.

The final concentration of AnxA5 protein in the filled container can be adjusted as needed. Without limitation, Applicant has exemplified a final concentration of 10 mg/ml. A suitable concentration may be, for example, 1-125 mg/ml, 2-100 mg/ml, 5-50 mg/ml, 7-30 mg/ml, or about 10-20 mg/ml.

Optionally, the methods of this invention can provide the final sterile AnxA5 protein product in a non-phosphate buffer (such as Bis-Tris or Tris buffer) at a pH of about 7.4 (e.g. ±0.5, 0.4, 0.3, 0, 2 or 0.1 pH units) containing about 150 mM NaCl (for example, 50, 40, 30, 20 or 10 mM), about 1 mM CaCl ₂ (for example, ± 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 administration), as it matches the pH of human blood and provides proper solubility for the stable AnxA5 protein. At pH values below 7, and in particular up to about pH 6, the AnxA5 protein loses solubility and may begin to precipitate.

NaCl may be a suitable medium to maintain the AnxA5 product in monomeric form during storage. Accordingly, the final sterile AnxA5 protein product can be obtained using the methods of this invention, wherein the concentration of NaCl present maintains the AnxA5 protein in a form that is predominantly (i.e., greater than 50% such as 60%, 70%, 80%, 85% , 90%, 95%, 96%, 97%, 98%, 99% or essentially 100%) monomeric. Percentage levels of monomers can be readily determined using techniques well known in the art such as gel permeation chromatography (GPC).

In one embodiment of the invention, the methods of the invention produce a final sterile, therapeutically acceptable AnxA5 protein product with a total yield of greater than 1 g of AnxA5 protein per liter of host cell culture, more preferably at least about 1.5 g/L, 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 indicated value.

In another embodiment of this invention, using the methods of this invention, a final sterile, therapeutically acceptable AnxA5 protein product is obtained with a total recovery of AnxA5 protein of at least about 24 wt.% (for example, ± 10, 9, 8, 7, 6, 5, 4, 3, 2, 1%) or more of the AnxA5 protein present in the host cell culture. This can be determined, for example, by measuring the soluble AnxA5 protein in the original homogenate (which can be fixed and measured by centrifuging an aliquot of the homogenate and checking the AnxA5 level in the supernatant) and in the final purified product.

In addition, at any time during the process (if necessary, prior to filling the sterile container) and generally after the final purification step as described above, the AnxA5 protein can be chemically modified. For example, the AnxA5 protein can be PEGylated. PEGylated annexin A5 is described in WO 02/067857. PEGylation is a method well known to those skilled in the art in which a polypeptide or peptidomimetic compound (for purposes of this invention, the AnxA5 protein) is modified such that one or more polyethylene glycol (PEG) molecules are covalently attached to the side chain of one or more the number of amino acids or their derivatives. This is one of the most important molecular modification techniques in structural chemistry (MASC). Other MASC methods can also be used; such methods can improve the pharmacodynamic characteristics of the molecule, for example, extend its in vivo half-life. The PEG protein conjugate is formed by first activating the PEG moiety, whereby it will interact with and bind to the protein or peptidomimetic compound of the invention. PEG fragments vary considerably in molecular weight and conformation, with early fragments (monofunctional PEGs, mPEGs) being linear with molecular weights of 12 kDa or less, and later fragments having increased molecular weights. PEG2, one of the latest innovations in PEG technology, involves the coupling of a 30 kDa (or less) mPEG to the amino acid lysine (although PEGylation may extend to adding PEG to other amino acids), which subsequently reacts to form a branched structure that functions as a linear mPEG with a 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 the references cited therein. Benefits of PEGylation include decreased renal clearance, which for some products allows for more consistent adsorption after administration, and limited distribution, which may result in more consistent and stable plasma concentrations and therefore improved clinical efficacy (Harris et al. (2001) Clin Pharmacokinet 40, 539-551). Other benefits 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). In the event that the AnxA5 protein is chemically modified, it may be desirable to perform one or more additional purification steps such as reducing or removing unreacted components and/or selecting a homogeneous population of the chemically modified AnxA5 protein for inclusion in the final product. A person skilled in the art knows a suitable method for purifying chemically modified proteins from a reaction process.

The final product may or may subsequently be formulated to form a pharmaceutical or veterinary composition.

The final product may be presented in the form of a dosage form. For example, a dosage form may contain about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg of AnxA5 protein (with "about" referring to ±0.5, 0.4, 0, 3, 0.2 or 0.1 mg) or more, for example, in the range of 0.1 to 1000 mg or 1 to 100 mg.

The pharmaceutical or veterinary composition may contain the AnxA5 protein in admixture with a pharmaceutically or veterinarily acceptable adjuvant, diluent, or carrier, which is generally chosen based on the intended route of administration and standard pharmaceutical practice. The composition may be in the form of immediate, sustained or controlled release. Preferably, the composition is a dosage containing a daily dose or unit, a daily portion of the dose, or an appropriate proportion of its active ingredient.

The phrase "pharmaceutical or veterinary acceptable" means compositions that do not result in an undesirable, allergic or other adverse reaction when administered to an animal or human, if such administration is necessary. The preparation of such pharmaceutical or veterinary compositions is known to those skilled in the art in light of this disclosure, as exemplified in Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference. In addition, when administered to animals or humans, it should be understood that formulations must meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Biological Standards Office.

As used herein, the term "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, medicinal agents, drug stabilizers, excipients, disintegrating agents such as materials and combinations thereof, which are known to the person skilled in the art. Any conventional carrier may be used in therapeutic or pharmaceutical compositions unless it is incompatible with the active ingredient.

Pharmaceutical or veterinary compositions according to the invention may or may not be intended and thus formulated in a manner suitable for parenteral, intravenous, intraarterial, intraperitoneal, intramuscular, intraocular, intracranial, intracerebral, intraosseous, intraventricular, intrathecal, or subcutaneous administration. , administration from a drug-eluting stent, administration by infusion methods, or topical administration (eg, in a form suitable for dermal application, such as a cream or ointment, inhalation, administration in ophthalmic/eye drops, otic/ear drops, or through mucous membranes of the body). Sterile injectable solutions can be obtained by incorporating the active compound in the required amount of a suitable solvent with various other ingredients listed above, followed by sterilization, if necessary. The pharmaceutical compositions are most conveniently administered as a sterile aqueous solution which may contain other substances, such as sufficient salts or glucose, to provide a solution that is isotonic with blood. Aqueous solutions should be suitably buffered (preferably to pH 3 to 9) if necessary. The preparation of suitable pharmaceutical formulations under sterile conditions is readily accomplished using standard pharmaceutical techniques well known to those skilled in the art.

Pharmaceutical or veterinary compositions of this invention may furthermore be formulated as a powder, such as a sterile powder, which may be a lyophilized powder.

A therapeutically effective amount of AnxA5 protein to be administered to a patient, e.g., a patient, based on a daily dosage level, may be from 0.01 to 1000 mg of AnxA5 protein per adult (e.g., about 0.001 to 20 mg per kg of patient body weight, e.g., 0 01-10 mg/kg, for example, more than 0.1 mg/kg and up to or less than 20, 10, 5, 4, 3, or 2 mg/kg, for example, about 1 mg/kg), administered as single or divided doses.

The physician will in any case determine the actual dose that will be most suitable for any individual patient, and this dose will vary according to the age, weight and response of the particular patient. The above doses are typical for the average case. Of course, there may be occasional instances where higher or lower dosage ranges may be used, and such ranges are included within the scope of this invention.

For veterinary use, the compound of this invention is administered as a suitable acceptable composition according to standard veterinary practice, and the veterinarian will determine the schedule and route of administration of doses that will be most suitable for a particular animal.

I.Product Features

Using the methods of this invention receive the product AnxA5, as defined above. Thus, the product obtained using the claimed method is also an additional, fifth aspect of the present invention.

In a further embodiment of the present invention, using the method of any of the aspects of the present invention, a product containing the AnxA5 protein can be obtained in a purity suitable for an injectable pharmaceutical agent for human use. Typically, the process described herein removes process-related contaminants to acceptable levels, such as host cell protein below 20 ng per mg AnxA5 protein, DNA below 10 μg per mg AnxA5 protein, and endotoxins below 1 EU per mg protein. AnxA5.

In a further embodiment of the present invention, using the method of any of the aspects of the present invention, a product can be obtained containing a protein without AnxA5, in particular a host cell protein (excluding the recombinantly expressed AnxA5 protein), at a level of less than 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5 ng or less per mg of AnxA5 protein. The FDA and EMA require that the host cell protein level be less than 100 ng/mg, and Applicant has demonstrated a host cell protein level of less than 20 ng/mg. The protein content of the host cells can, for example, be measured using antibodies against the host cell protein using sandwich ELISA or other EMA and FDA approved methods.

In a further embodiment of this invention, using the method according to any of the aspects of this invention, a product containing endotoxin in an amount of less than 100, 90, 80, 70, 60, 50 45, 40, 35, 30, 35, 20, 15 and preferably less than 10, 5 or 1 EU per mg of AnxA5 protein, and/or preferably, the method produces a product in the form of a dosage form, and the product contains less than 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 35, 20, 15 and preferably less than 10, 5 or 1 EU per dosage. FDA and EMA require endotoxin levels to be less than 100 EU/dose (maximum allowed is 350 EU/dose); the expected level is less than 10 EU/mg at a dose of 10 mg. Within these parameters, the dosage form may contain about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg of AnxA5 protein or more (for example, in the range of 0.1 to 1000 mg or 1 to 100 mg). The amount of endotoxins can, for example, be measured using methods based on the LAL test or other methods approved by the EMA and FDA.

In a further embodiment of the present invention, using the method of any one of the aspects of the present invention, a product can be produced containing nucleic acid (e.g., DNA) levels, such as nucleic acid (e.g., DNA) levels of a host cell, of less than 1000 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, less than 20 pg per mg of AnxA5 protein, less than 15 pg per mg of AnxA5 protein, less than 10 μg 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. The amount of DNA, for example, can be measured using quantitative polymerase chain reaction (qPCR) methods or other EMA and FDA approved methods.

In a particularly preferred embodiment of the present invention, a product having any one or more characteristics selected from the list consisting of:

- AnxA5 protein concentration, which is usually about 8-12 g/l;

host cell protein levels equal to or less than 100 ng/mg and more preferably 20 ng/mg (as determined by ELISA);

- host cell DNA levels equal to or below 100 pg/mg; and more preferably 10 pg/mg

- endotoxin levels equal to or below 35 EU/mg and more preferably 1 EU/mg,

- purity > 95% as determined by size exclusion chromatography;

- bioburden < 1 cfu/ml (as defined in European Pharmacopoeia 2.6.12);

- transparent, colorless appearance, free of visible particles; as well as

- in this case, the main band detected by Western blot analysis corresponds to the reference annexin A5.

In yet another particularly preferred embodiment of the present invention, the AnxA5 protein in the product may have a low level of gluconoylation. The term "low" in this context may include meaning that the level of gluconoylation is less (e.g., less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% 9% , 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) than the level of gluconoylation of the AnxA5 protein that is expressed in the E. coli strain BL21 (DE3) (for example, as it is widely commercially available, and as described in Marder et al., 2014, BMC Biotechnology, 14:33) For example, it is possible that the level of gluconoylated AnxA5 protein in a product ranges from 0.5 to 30% or from 0.5 to 20 % or 0.5 to 15% or 0.5 to 10% of the total AnxA5 protein content of the product In other words, it may be that the level of gluconoylated AnxA5 protein in the product is below 40%, for example below 30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, and preferably substantially up to 0% Gluconoylated Anx5 variants can, for example, be measured and quantified assessed using HPLC or HPLC chromatography instruments using appropriate anion exchange or reverse phase columns.

Accordingly, in a fifth aspect of the present invention, there is provided a composition comprising an AnxA5 protein, wherein the composition is a direct or indirect product (resulting from a direct or indirect) process according to any one of the first, second, third or fourth aspects of the present invention. Optionally, said composition is a pharmaceutically acceptable and/or veterinarily acceptable composition.

J. Medical and veterinary applications

The sixth aspect of the present invention also provides a composition according to the fifth aspect of the present invention for use in medicine. In other words, in a sixth aspect of the present invention, there is provided a method comprising administering to a human or animal in need thereof a therapeutically effective amount of the composition of the fifth aspect of the present invention.

In some embodiments of the sixth aspect of the present invention, the composition of the fifth aspect of the present invention may be used:

(a) to prevent or reduce the risk of thrombosis (eg, atherothrombosis) and/or plaque rupture, or for administration to patients at risk, including, but not limited to, patients with systemic lupus erythematosus (SLE) and/or patients, who have or have had (or are at risk of) upper respiratory tract infections or other infections (including pneumococcal infection) that may cause elevated levels of antiphospholipid-related antibodies, or to treat (actively or prophylactically) or reduce the risk of thromboembolism, hemorrhagic or vasculitic stroke , myocardial infarction, angina or intermittent claudication, unstable angina, other forms of severe angina or transient ischemic attacks (TIA), for example, as further described in WO 2005/099744 (the contents of which are incorporated herein by reference);

(b) to treat, prevent or reduce the risk of vascular dysfunction, angina pectoris, coronary heart disease, peripheral arterial disease, systolic hypertension, migraine, type 2 diabetes and erectile dysfunction, reduce the intensity of ischemic pain and/or treat diseases associated with vascular rupture, for example, as described in WO 2009/077764 (the contents of which are incorporated into this description by reference);

(c) for the prevention or treatment of restenosis (in particular, the formation or thickening of the neointima) or vascular inflammation, for example, as described in WO 2009/103977 (the contents of which are incorporated herein by reference);

(d) for use in inhibiting the activity of oxidized cardiolipin (oxCL), as well as for treating, preventing and/or reducing the risk of developing a cardiovascular disease, an autoimmune disease or an inflammatory pathological condition, for example, as described in WO 2010/069605 (contents which is incorporated herein by reference), including but not limited to the following diseases: cardiovascular disease (CVD), diabetes II, Alzheimer's disease, dementia in general, rheumatic diseases, atherosclerosis, high blood pressure, acute and/or chronic inflammatory conditions, myocardial infarction, acute coronary syndrome, stroke, transient ischemic attack (TIA), lameness, angina, type I diabetes, rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, Reiter's syndrome, systemic lupus erythematosus, dermatomyositis, Sjögren's syndrome, erythematosus lupus, multiple sclerosis, myasthenia gravis, asthma, encephalitis, inflammatory bowel disease, chronic obstructive pulmonary disease (COPD), arthritis, including osteoarthritis, idiopathic inflammatory myopathies (IIM), dermatomyositis (DM), polymyositis (PM), myositis with included bodies, allergic conditions and/or osteoarthritis in mammals; as well as

(e) to prevent and/or reduce peri- or postoperative complications after surgery, such as complications after vascular surgery, especially peripheral, for example, as described in WO 2012/136819 (the contents of which are incorporated herein by reference).

In a further embodiment of the sixth aspect of the present invention, the composition of the fifth aspect of the present invention may be used in a prophylactic or therapeutic method for treating, preventing or reducing the risk of hematological disorders, including but not limited to sickle cell anemia.

In a further embodiment of the sixth aspect of the present invention, the composition of the fifth aspect of the present invention may be used in a prophylactic or therapeutic method for treating, preventing or reducing the risk of acute and chronic vascular inflammation, primary or secondary vasculitis, including, but not limited to, vasculitis with autoimmune components and /or drug-induced vasculitis. Accordingly, the present invention also provides a prophylactic or therapeutic method for treating, preventing, or reducing the risk of vasculitis, including Behçet's disease, cutaneous vasculitis, eosinophilic granulomatosis with polyangitis (EGR), giant cell arteritis, granulomatosis with polyangitis (GPA), immunoglobulin A-associated vasculitis ( IgAB), microscopic polyangiitis (MPA), polyarteritis nodosa (PAN), polymyalgia rheumatica, and takaisu arteritis. Of particular interest may be polymyalgia rheumatica.

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

In a further embodiment of the sixth aspect of the present invention, the composition of the fifth aspect of the present invention may be used in a prophylactic or therapeutic method for (i) preventing or reducing the rate of transmission of a viral infection; (ii) prevent or protect against viral infection; or (iii) treating a viral infection in a subject, wherein the viral infection is caused by a virus selected from the group consisting of:

(a) a virus capable of causing hemorrhagic fever (HHF), and

(b) a virus that contains phosphatidylserine (PS) and mediates cell infection and/or internalization by binding to PS.

Accordingly, in yet another embodiment, the present invention provides a composition according to the fifth aspect of the present invention for use in a prophylactic or therapeutic method for preventing or reducing the rate of transmission of a viral infection in a subject, wherein the viral infection is due to a virus capable of causing hemorrhagic fever (HHF).

Thus, the present invention provides a prophylactic or therapeutic method for preventing or reducing the rate of transmission of a viral infection in a subject, wherein the viral infection is caused by a virus capable of causing hemorrhagic fever (HHF), comprising administering to the subject a therapeutically effective amount of a composition according to the fifth aspect of the present invention.

In other words, this embodiment of the present invention provides a composition according to the fifth aspect of the present invention for use in the manufacture of a medicament for prophylaxis or treatment by preventing or reducing the rate of transmission of a viral infection in a subject, wherein the viral infection is caused by a virus capable of causing hemorrhagic fever (HHF). ).

In another embodiment, the present invention provides a composition according to the fifth aspect of the present invention for use in a prophylactic or therapeutic method for preventing or reducing the rate of transmission of a viral infection in a subject, wherein the viral infection is caused by a virus that contains phosphatidylserine (PS) and mediates infection of a cell and/or internalization through FS binding.

Thus, the present invention provides a prophylactic or therapeutic method for preventing or reducing the rate of transmission of a viral infection in a subject, wherein the viral infection is caused by a virus that contains phosphatidylserine (PS) and mediates cell infection and/or internalization by binding to the PS, wherein the method comprises administering to a subject a therapeutically effective amount of a composition according to the fifth aspect of this invention.

In other words, this embodiment of the present invention provides a composition according to the fifth aspect of the present invention for use in the manufacture of a medicament for prophylaxis or treatment by preventing or reducing the rate of transmission of a viral infection in a subject, wherein the viral infection is due to a virus that contains phosphatidylserine (PS) and mediates cell infection and/or internalization via PS binding.

In another embodiment, the present invention provides a composition according to the fifth aspect of the present invention for use in a prophylactic or therapeutic method for preventing or protecting against a viral infection in a subject, wherein the viral infection is due to a virus capable of causing hemorrhagic fever (HHF).

Thus, the present invention provides a prophylactic or therapeutic method for preventing or protecting against a viral infection in a subject, wherein the viral infection is caused by a virus capable of causing hemorrhagic fever (HHF), comprising administering to the subject a therapeutically effective amount of a composition according to the fifth aspect of the present invention.

In other words, this embodiment of the present invention provides a composition according to the fifth aspect of the present invention for use in the manufacture of a medicament for prophylaxis or treatment by preventing or protecting against a viral infection in a subject, wherein the viral infection is caused by a virus capable of causing hemorrhagic fever (HHF) .

In another embodiment, the present invention provides a composition according to the fifth aspect of the present invention for use in a prophylactic or therapeutic method of preventing or protecting against a viral infection in a subject, wherein the viral infection is caused by a virus that contains phosphatidylserine (PS) and mediates cell infection and/or internalization through association with FS.

Thus, the present invention provides a prophylactic or therapeutic method for preventing or protecting against a viral infection in a subject, wherein the viral infection is caused by a virus that contains phosphatidylserine (PS) and mediates cell infection and/or internalization by binding to the PS, the method comprising administering to the subject a therapeutically effective amount of a composition according to the fifth aspect of the present invention.

In other words, this embodiment of the present invention provides a composition according to the fifth aspect of the present invention for use in the manufacture of a medicament for prophylaxis or treatment by preventing or protecting against a viral infection in a subject, wherein the viral infection is caused by a virus that contains phosphatidylserine (PS) and mediates cell infection and/or internalization via PS binding.

In another embodiment, the present invention provides a composition according to the fifth aspect of the present invention for use in a prophylactic or therapeutic method of treating a viral infection in a subject, wherein the viral infection is due to a virus capable of causing hemorrhagic fever (HHF).

Thus, the present invention provides a prophylactic or therapeutic method for treating a viral infection in a subject, wherein the viral infection is caused by a virus capable of causing hemorrhagic fever (HHF), comprising administering to the subject a therapeutically effective amount of a composition according to the fifth aspect of the present invention.

In other words, this embodiment of the present invention provides a composition according to the fifth aspect of the present invention for use in the manufacture of a medicament for the prophylactic or therapeutic effect by treating a viral infection in a subject, wherein the viral infection is due to a virus capable of causing hemorrhagic fever (HHF).

In another embodiment, the present invention provides a composition according to the fifth aspect of the present invention for use in a prophylactic or therapeutic method of treating a viral infection in a subject, wherein the viral infection is caused by a virus that contains phosphatidylserine (PS) and mediates cell infection and/or internalization by binding to FS.

Thus, the present invention provides a prophylactic or therapeutic method for treating a viral infection in a subject, wherein the viral infection is caused by a virus that contains phosphatidylserine (PS) and mediates cell infection and/or internalization by binding to a PS, the method comprising administering to the subject therapeutically an effective amount of the composition according to the fifth aspect of the present invention.

In other words, this embodiment of the present invention provides a composition according to the fifth aspect of the present invention for use in the manufacture of a medicament for the prophylaxis or therapeutic effect by treating a viral infection in a subject, wherein the viral infection is due to a virus that contains phosphatidylserine (PS) and mediates the infection cells and/or internalization via PS binding.

According to another embodiment of the present invention, there is provided a composition according to the fifth aspect of the present 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 bacteria capable of causing hemorrhagic fever. fever (BGL).

In other words, this embodiment of the present invention provides 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 (HHF) or bacteria capable of causing hemorrhagic fever (BHF), while the method includes administering to a subject a therapeutically effective amount of a composition according to the fifth aspect of the present invention.

In other words, this embodiment of the present invention provides a composition according to the fifth aspect of the present 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 (HHF) or bacteria that can cause hemorrhagic fever (BHF).

According to another embodiment of the present invention, there is provided a composition according to the fifth aspect of the present 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 bacteria capable of causing hemorrhagic fever (HHF).

In other words, this embodiment of the present invention provides 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 bacteria capable of causing hemorrhagic fever (BHF), the method comprising administering to the subject a therapeutically effective amount of a composition according to the fifth aspect of the present invention.

In other words, this embodiment of the present invention provides a composition according to the fifth aspect of the present 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 (HHF) or bacteria capable of causing hemorrhagic fever (BHF).

According to another embodiment of the present invention, a composition according to the fifth aspect of the present invention is provided for use in a method of treating a subject who has been in contact with a biological material present in the body of another subject or excreted by another subject who is 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 bacteria capable of causing hemorrhagic fever (BHF).

In other words, this embodiment of the present invention provides a method of treating a subject who has been in contact with biological material present in another subject or secreted by another subject that is infected or suspected of being infected with a pathogen capable of causing hemorrhagic fever, such as a virus, capable of causing hemorrhagic fever (HHF) or bacteria capable of causing hemorrhagic fever (BHF), the method comprising administering to a subject a therapeutically effective amount of a composition according to the fifth aspect of the present invention.

In other words, this embodiment of the present invention provides a composition according to the fifth aspect of the present invention for use in the manufacture of a medicament for the treatment of a subject who has been in contact with a biological material present in the body of another subject or secreted by another subject who is infected or suspected of having infection with a pathogen capable of causing hemorrhagic fever, such as a virus capable of causing hemorrhagic fever (HHF) or bacteria capable of causing hemorrhagic fever (BHF).

According to these above embodiments of the present invention, the pathogen capable of causing hemorrhagic fever may be VHF.

Viral hemorrhagic (or hemorrhagic) fevers (VHFs) are a group of various 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 bleeding, and in many cases they can all progress to high-level hyperthermia, shock, and death.

A subject suspected of being infected with a pathogen capable of causing hemorrhagic fever, such as a virus capable of causing hemorrhagic fever (HHF) or bacteria capable of causing hemorrhagic fever (BHF), may be a subject with a history of exposure to the disease (e.g., while performing professional duties as a healthcare worker or when a family member is infected) and/or may be an individual who has one or more signs or symptoms of infection prior to confirmation of the diagnosis.

The signs and symptoms of VHF are characterized by fever and an increased tendency to bleed (hemorrhagic diathesis). Symptoms of VHF often also include flushing of the face and chest, small red or purple spots (petechiae), open bleeding, swelling due to edema, low blood pressure (hypotension), and shock. Often there is a feeling of general malaise, muscle pain (myalgia), headache, vomiting and diarrhea. The severity of symptoms varies depending on the type of virus, the "VHF syndrome" (increased capillary permeability, hemorrhagic diathesis, and circulatory failure leading to shock) occurring in most patients with filovirus hemorrhagic fevers (eg, Ebola and Marburg), Crimean-Congo hemorrhagic fever (CCHF), and South American hemorrhagic fevers, and in a small proportion of patients with dengue fever, Rift Valley fever (RVF), and Lassa fever.

According to a sixth aspect of the present invention, the VHF may be Ebola, wherein the subject may present with one or more symptoms of Ebola, such as symptoms selected from primary clinical symptoms such as excessive or profuse sweating, fever, myalgia, general malaise, and /or chills; and/or flu-like symptoms, optionally accompanied by gastrointestinal symptoms; maculopapular rash, petechiae, conjunctival hemorrhage, epistaxis, melena, hematemesis, shock and/or encephalopathy; leukopenia (for example, associated with increased apoptosis of lymphoid cells), thrombocytopenia, elevated levels of aminotransferase, thrombin and/or partial thromboplastin, fibrin cleavage products found in the blood, and/or disseminated intravascular coagulation (DIC).

As a rule, confirmation of the diagnosis is carried out in a reference laboratory with increased safety requirements. Laboratory results vary somewhat between viruses, but in general there is a decrease in total white blood cell (especially lymphocyte) count, a decrease in platelet count, an increase in serum liver enzymes, and a decrease in blood clotting ability, as measured by an increase in prothrombin (PT) and activated partial thromboplastin time (aPTT). Hematocrit may be elevated. Serum urea and creatine levels may increase, but this will depend on the patient's hydration status. Bleeding time tends to increase.

For example, being a BSL-4 agent, confirmatory clinical laboratory diagnosis of viremia during the acute phase of an Ebola virus infection is possible using suitable laboratory equipment. The tests that can be used depend on the stage of the disease.

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

Later, in the course of the disease, studies can be applied that include: a) IgM and IgG ELISA using authentic viral antigens, and in case of death, autopsy tissues can be examined for antigen detection using immunostaining methods, b) immunohistochemical detection of Ebola virus antigen (Zaki et al, J Infect Dis, 1999;179(Suppl. 1): S36e47., the contents of which are incorporated herein by reference in its entirety) and c) in-situ hybridization for viral RNA detection.

The details of each of these methods have been summarized in Saijo et al., Clin Vaccine Immunol 2006, 13: 444e51, the contents of which are incorporated herein by reference in their entirety.

An ELISA-based assay has been standardized by the CDC for the detection of specific antibodies to Ebolavirus. The assay was highly sensitive and has been shown to be able to detect antibodies in the sera of people exposed to Ebola 10 years ago. A cell-based plaque assay and end point titration (TCID50) assay for the detection and quantification of filoviruses has also been developed 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).

For example, VHF may be a virus from a family selected from Filoviridae, Arenaviridae, Bunyaviridae, Flaviviridae, or Rhabdoviridae.

The Arenaviridae family includes viruses that cause Lassa fever, Argentinean, Bolivian, Brazilian and Venezuelan hemorrhagic fevers, and the Lujo virus.

The Bunyaviridae family includes members of the genus Hantavirus that cause hemorrhagic fever with renal syndrome (HFRS), Crimean-Congo hemorrhagic fever virus (CCHF) of the genus Nairovirus, Garissa virus (Garissa) and Ilesha virus (Ilesha) of the genus Orthobunyavirus and fever virus Rift Valley (RVL) from the genus Phlebovirus.

The family Filoviridae includes the Ebola virus and the Marburg virus.

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

2 fatal and 2 non-fatal cases of hemorrhagic fever have also been reported in the Bas-Congo region of the Democratic Republic of the Cong due to an isolated member of the Rhabdoviridae family. Non-fatal cases have been reported in healthcare workers treating two other patients, suggesting the possibility of human-to-human transmission of the virus.

Accordingly, for example, in one embodiment of the present invention of particular interest, the present invention may be applied to viruses of the Filoviridae family such as Ebola virus and Marburg virus. In another embodiment of the invention of particular interest, the invention may be applied to viruses of the Flaviviridae family, such as dengue virus.

Accordingly, the present invention provides a composition according to the fifth aspect of the present invention for use in a prophylactic or therapeutic method as described above for (i) preventing or reducing the rate of transmission of an Ebola infection; (ii) preventing or protecting against Ebola infection; or (iii) treatment of an Ebola infection in a subject who is infected or suspected of being infected with Ebola virus, or if the subject has been or is expected to be in contact with another subject who is or is suspected of being infected with Ebola virus, or if the subject has been or is suspected of being infected with Ebola that will come into contact with biological material present in the body of another subject or excreted by another subject who is infected or suspected of being infected with the Ebola virus.

Accordingly, the present invention provides a composition according to the fifth aspect of the present invention for use in a prophylactic or therapeutic method as described above for (i) preventing or reducing the rate of transmission of Marburg infection; (ii) preventing or protecting against Marburg infection; or (iii) treating a Marburg infection where the subject is or is suspected of being infected with Marburg virus, or the subject has been or is expected to be in contact with another subject who is or is suspected to be infected with Marburg virus, or the subject has been or is suspected of being infected with Marburg virus that will be in contact with biological material present in the body of another subject or secreted by another subject who is infected or suspected of being infected with Marburg virus.

Accordingly, the present invention provides a composition according to the fifth aspect of the present invention for use in a prophylactic or therapeutic method as described above for (i) preventing or reducing the rate of transmission of dengue fever virus; (ii) preventing or protecting against dengue virus; or (iii) treatment of dengue virus, where the subject is infected or suspected of being infected with dengue fever virus, or the subject has been or is expected to be in contact with another subject who is infected or suspected of being infected with dengue fever virus, or the subject has been or is expected to be in contact with biological material present in the body of another subject or excreted by another subject who is infected or suspected of being infected with dengue fever virus.

The present invention also provides a composition according to the fifth aspect of the present invention for use in the method described above for the purpose of treating, preventing and/or slowing the progression of an infection in a subject caused by VHF or BGL.

The present invention also provides a composition according to the fifth aspect of the present invention for use in the method described above, in order to prevent, reduce the severity, prevent the development and/or slow the progression of direct and/or mediated bacterial or viral damage to the immune and/or vascular system in a subject, caused by BGL or VHF.

For example, this invention can be used to prevent, reduce the severity, interfere with the development and/or slow the progression of direct and/or mediated bacterial or viral damage to the immune system in a subject, for example, in the case of Ebola infection. For example, bacterial or viral damage can be selected from damage to the innate immune response, damage to the acquired humoral response, damage to dendritic cells, damage to the regulation of the production of inflammatory factors, for example, production of interferon (including IL1 production), damage to macrophages, and/or damage to monocytes.

This invention can be used to prevent, reduce the severity, prevent the development and/or slow the progression of bleeding (hemorrhages), hypotension, lowering blood pressure, shock or death in a subject.

This invention can be used to prevent, reduce the severity, hinder the development and/or slow the progression of virus-induced nitric oxide damage to the vascular endothelium of a subject.

The present invention provides a composition according to the fifth aspect of the present invention for use in a method of preventing, reducing the severity, preventing the development and/or slowing the progression of an activation, death and/or disruption of the lesion of the vascular endothelium or its endothelial cells, in a subject infected or suspected of infection with a pathogen capable of causing hemorrhagic fever, such as VHF or BHF. The integrity of the vascular endothelium or its endothelial cells can be determined, for example, by the degree of cellular or vascular epithelial leakage and/or by detecting one or more hemorrhagic elements or edema formation and/or dehydration of the subject.

The present invention provides a composition according to the fifth aspect of the present invention for use in a method for preventing, reducing the severity, preventing the development and/or slowing down the progression of a lesion, activation, death and/or disruption of the integrity of the vascular endothelium or its endothelial cells in a subject who has been or is expected to that will be in contact with another subject who is infected or suspected of being infected with a pathogen capable of causing hemorrhagic fever, such as VHF or BHF.

The present invention provides a composition according to the fifth aspect of the present invention for use in a method for preventing, reducing the severity, preventing the development and/or slowing the progression of a lesion, activation, death and/or disruption of the integrity of the vascular endothelium or its endothelial cells in a subject who has been or is expected to that will be in contact with biological material present in the body of another subject or secreted by another subject who is infected or suspected of being infected with a pathogen capable of causing hemorrhagic fever, such as VHF or BHF.

In a further embodiment of the present invention, a composition according to the fifth aspect of the present invention is provided for use as described above with reference to various embodiments of the present invention in a prophylactic or therapeutic method, wherein the viral infection is caused by a virus that contains phosphatidylserine (PS) and mediates the infection cells and/or internalization via PS binding. Alternatively, the viral infection may be caused by a virus that contains one or more other types of phospholipids that bind with annexin A5 and/or other fragments that bind with annexin A5.

Viruses that contain phosphatidylserine (PS) and mediate cell infection and/or internalization by binding to PS may particularly include viruses with an envelope that contains phosphatidylserine (PS), especially in the outer layer. The content of PS in the virus can be determined using methods known in the art, for example, using an ELISA assay to measure the binding of annexin A5 to the virus. A suitable method may, for example, include measuring the binding of hemagglutinin (HA)-labelled annexin A5 to anti-HA antiserum by ELISA as described in Moller-Tank, et al, 2013, J. Virol., 87(15 ), 8327-8341 (the contents of which are incorporated herein by reference).

A group of viruses of particular interest to the present invention includes those that mediate cell infection and/or internalization by binding to a phosphatidylserine-mediated entry-enhancing receptor (PVEER). PVEERs are discussed in Moller Tank, et al, 2013, J. Virol., 87(15), 8327-8341, and one example is T-cell immunoglobulin mucin 1 (TIM-1) receptor. Other examples may include TIM-4, Gas6 or protein S/Axl, Mer and Tyro3, and MFG-E8/integrin αvβ3 or αvβ5

Ebola is an example of one virus of particular interest that contains phosphatidylserine (PS) and mediates cell infection and/or internalization through PS binding to TIM-1. Moller Tank, et al., 2013, J. Virol., 87(15), 8327-8341.

The present invention has determined that annexin A5 and the composition of the fifth aspect of the present invention can be used to inhibit or interrupt FS-mediated infection and/or internalization of cells by viruses such as Ebola virus via PVEERs such as TIM-1 and therefore can be suitable for use in a prophylactic or therapeutic method of (i) preventing or reducing the rate of transmission of a viral infection in a subject, (ii) preventing or protecting against a viral infection; or (iii) treating a viral infection in a subject, wherein the viral infection is due to a virus that contains phosphatidylserine (PS) and mediates cell infection and/or internalization by binding to the PS.

Phosphatidylserine (PS)-containing viruses may, for example, be selected from the group consisting of a virus of the Filoviridae family (eg, Ebola and Marburg virus); families Flaviviridae; hepatitis A virus; alpha viruses; baculoviruses; and arenaviruses. Viruses can infect people, or only people. The viruses can infect animals, or only non-human animals, such as any one or more animals selected from the group consisting of dogs, cats, cattle, sheep, pigs, goats, rodents, camels, domestic animals and wild animals.

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

TIM-1, also known as HAVCR1 and KIM-1, has been identified as a human asthma susceptibility gene (Mclntire et al, 2003, Nature 425:576). One published amino acid sequence for the human TIM-1 protein is shown as:

MHPQVVILSLILHLADSVAGSVKVGGEAGPSVTLPCHYSGAVTSMCWRGSC SLFTCQNGIVWTNGTHVTYRKDTRYKLLGDLSRRDVSLTIENTAVSDSGVYC CRVEHRGWFNDMKITVSLEIVPPKVTTTPIVTTVPTVTTVRTSTTVPTTTTVPM TTVPTTTVPTTMSIPTTTTVLTTMTVSTTTSVPTTTSIPTTTSVPVTTTVSTFVPP MPLPRQNHEPVATSPSSPQPAETHPTTLQGAIRREPTSSPLYSYTTDGNDTVTE SSDGLWNNQTQLFLEHSLLTANTTKGIYAGVCISVLVLLALLGVIIAKKYFF KKEVQQLSVSFSSLQIKALQNAVEKEVQAEDNIYIENSLYATD (SEQ ID NO: 2).

TIM-1 is a type I membrane protein with an extracellular region containing an IgV domain, a mucin-rich domain, and a membrane-proximal stalk containing N-linked glycosylation sites (Ichimura et al, 1998, J, Biol, Chem. 273(7): 4135-42). The ΤIΜ-1 domain of IgV has a disulfide-dependent conformation in which the CC' loop is folded on the GFC β strands, resulting in a characteristic "cleft" formed by the CC' and FG loops (Santiago et al, 2007, Immunity 26(3):299-310 ). The cleft formed by the CC' and FG loops is the binding site for phosphatidylserine (Kobayashi et al, 2007, Immunity 27(6):927-40). Antibodies directed to 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 with allergic asthma (Sonar et al, 2010, J. Clin. Invest 120:2767-81).

Another 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 PVEERs to bind to the PS presented therein. The AnxA5 protein in the composition of the fifth aspect of the present invention preferably also has the ability to bind PS and according to this embodiment of the present invention is able to compete with PVEER for PS binding.

Accordingly, in another embodiment of the present invention, the composition of the fifth aspect of the present invention can be used in a method that inhibits the binding of phosphatidylserine to TIM-1 (or other PVEER).

For example, it may be prophylactically or therapeutically useful in inhibiting, reducing or preventing infection of cells by viruses that contain phosphatidylserine (PS) and mediate cell infection and/or internalization by PS.

Alternatively, it may be prophylactically or therapeutically useful for other medical conditions characterized by PS binding to TIM-1 (or other PVEERs). TIM-1 associated disorders are discussed below.

Therefore, in another embodiment, the present invention provides a method for inhibiting or reducing the binding of TIM-1 or another PVEER to phosphatidylserine, the method comprising contacting a first cell that expresses TIM-1 or another PVEER with an amount of the composition of the fifth aspect of this of the invention, which is effective for inhibiting or reducing the binding of a first cell to a second cell that contains phosphatidylserine on its surface, or to a virus that contains phosphatidylserine (PS) on its surface. This method may be an in vivo or in vitro method. In the case of an in vivo method, it may be to treat or prevent a pathological condition characterized by PS binding to TIM-1 or another PVEER.

In other words, this embodiment of the present invention also provides a composition according to the fifth aspect of the present invention for use in a prophylactic or therapeutic method to inhibit or reduce the binding of TIM-1 or other PVEER to phosphatidylserine in an indicated patient.

In another embodiment, the present invention provides a method for inhibiting or reducing PS binding to TIM-1 or another PVEER on a dendritic cell, the method comprising contacting a dendritic cell that expresses TIM-1 or another PVEER with a fifth amount of the composition. aspect of the present invention that is effective to inhibit or reduce PS binding to a dendritic cell. This method may be an in vivo or in vitro method. When used in vivo, the method may be to treat or prevent a pathological condition characterized by PS binding to TIM-1 or another PVEER on a dendritic cell.

In other words, this embodiment of the present invention also provides a composition according to the fifth aspect of the present invention for use in a prophylactic or therapeutic method to inhibit or reduce the binding of PS to TIM-1 or other PVEER on a dendritic cell in a patient having this indication.

A method for treating or preventing an inflammatory or autoimmune condition is also described, the method comprising administering to a mammal having an inflammatory or autoimmune condition a pharmaceutical composition comprising a therapeutically effective amount of the composition of the fifth aspect of the present invention.

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

A method for treating or preventing asthma is also described, the method comprising administering to a mammal having asthma a pharmaceutical composition comprising a composition according to the fifth aspect of the present invention.

In other words, this embodiment of the present invention also provides a composition according to the fifth aspect of the present invention for use in a prophylactic or therapeutic method for preventing, treating or reducing the severity of asthma.

A method for treating or preventing an atopic disorder is also described, 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 present invention. The atopic disorder may be, for example, atopic dermatitis, contact dermatitis, urticaria, allergic rhinitis, angioedema, latex allergy, or allergic lung disease (eg, asthma, allergic bronchopulmonary aspergillosis, or hypersensitivity pneumonitis).

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

The composition of the fifth aspect of the present invention is used as described herein for the treatment or prevention of various TIM-1 associated disorders and other PVEER associated disorders, including immunological disorders such as inflammatory and autoimmune disorders.

The term "treatment" means the administration of a substance or composition described herein in an amount, in a method and/or schedule effective to improve a condition, symptom or parameter associated with a disorder, or to prevent the progression or exacerbation of a disorder (including secondary damage caused by the disorder) either to a statistically significant extent or to an extent determined by a person skilled in the art.

A subject at risk diagnosed with or having one of these disorders may be administered the composition of the fifth aspect of the present invention in an amount and over time to provide an overall therapeutic effect. The composition according to the fifth aspect of the present invention can be administered alone (monotherapy) or in combination with other agents (combination therapy), either in admixture or by separate, simultaneous or sequential administration. In the case of combination therapy, the amount and time of administration may be such as to provide, for example, an additional or synergistic therapeutic effect. In addition, the administration of a composition according to the fifth aspect of the present invention (with or without a second agent) can be used as a primary, for example, first line therapy, or as a secondary therapy, for example, for subjects who demonstrate an insufficient response to previously administered therapy (t i.e. non-AnxA5 protein therapy).

Diseases or pathological conditions that can be treated using the composition of the fifth aspect of the present invention described herein include, for example, ischemia-reperfusion injury (for example, ischemia-reperfusion injury of organs, such as ischemia-reperfusion injury of the liver or kidneys ), allergies, asthma, inflammatory bowel disease (IBD), Crohn's disease, transplant rejection, pancreatitis, and delayed-type hypersensitivity (DTH).

Additional diseases or conditions that can be treated using the composition of the fifth aspect of the present invention described herein include, for example, autoimmune disorders.

Systemic 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.

Examples of other organ-specific or systemic autoimmune diseases that can be treated using the composition of the fifth aspect of this invention described herein are myasthenia gravis, autoimmune hemolytic anemia, Chagas disease, Graves' disease, idiopathic thrombocytopenia purpura (IPT), 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) can be treated with annexin A5 as described herein.

Additional TIM-1 related diseases or conditions that can be treated using the composition of the fifth aspect of the present invention described herein include, for example, graft versus host disease (GVHD). GVHD is a T cell mediated disease condition that can be treated using Annexin A5 described herein. GVHD is initiated when host antigens are recognized as foreign by donor T cells. Often fatal in human patients with bone marrow transplantation (BMT), GVHD can be acute or chronic. Acute and chronic forms of GVHD are characterized by the development of Th1 and Th2 antigen-specific 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, intestines, liver, and other organs. Chronic GVHD appears later (100 days after BMT) and is characterized by overproduction of immunoglobulin (Ig), including autoantibodies, and damage to the skin, kidneys, and other organs caused by Ig precipitation. In almost 90% of patients, acute GVHD progresses to chronic GVHD. Chronic GVHD appears to be a Th2-mediated T cell (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 cytotoxicity is a characteristic feature of acute GVHD. The consequences of donor cytotoxicity directed against the host organism can be different. First, host lymphocytes are rapidly degraded, resulting in acute GVHD mice with profound immunosuppression. Second, donor lymphocytes become grafted and proliferate in the host's spleen, and their cytotoxic activity can be directly measured in vitro with cell lines expressing host antigens that can be recognized (as foreign) by donor cells. Third, the disease becomes fatal as additional tissues and cell populations are destroyed.

Additional TIM-1 associated diseases or conditions that can be treated using the composition of the fifth aspect of the present invention described herein include, for example, atopic disorders. Atopic disorders are characterized by the expression by cells of the immune system, including activated T cells and APCs, of cytokines, chemokines, and other molecules that are characteristic of Th2 responses, such as the cytokines IL-4, IL-5, and IL-13, along with several others. Thus, such atopic disorders will be treated using the composition of the fifth aspect of the present invention as described herein. Atopic disorders include airway hypersensitivity and distress syndrome, atopic dermatitis, contact dermatitis, urticaria, allergic rhinitis, angioedema, latex allergy, and allergic lung pathology (eg, asthma, allergic bronchopulmonary aspergillosis, and hypersensitivity pneumonitis).

Additional TIM-1 associated diseases or conditions that can be treated using the composition of the fifth aspect of the present invention described herein include, for example, numerous immune or inflammatory disorders. Immune or inflammatory diseases include, but are not limited to, allergic rhinitis, autoimmune hemolytic anemia; blackening acanthosis; Addison's disease; alopecia areata; universal alopecia; amyloidosis; anaphylactoid purpura; anaphylactoid reaction; aplastic anemia; ankylosing spondylitis; cranial arteritis; Takayasu's giant cell arteritis; temporal arteritis, ataxia-telangiectasia; autoimmune oophoritis; autoimmune orchitis; autoimmune polyendocrine insufficiency; Behcet's disease; Berger's disease; Buerger's disease; bronchitis; bullous pemphigus; chronic mucocutaneous candidiasis; Kaplan syndrome; syndrome after myocardial infarction; postpericardiotomy syndrome; carditis; celiac sprue; Chagas disease; Chediak-Higashi syndrome; Churg-Strauss disease; Cogan's syndrome; cold agglutinin disease; CREST syndrome; Crohn's disease; cryoglobulinemia; cryptogenic fibrous alveolitis; herpetiform dermatitis; dermatomyositis; diabetes; Diamond-Blackfan syndrome; diGeorge syndrome; discoid lupus erythematosus; eosinophilic fasciitis; episcleritis; persistent elevated erythema rheumatoid erythema; erythema multiforme; erythema nodosum; familial Mediterranean fever; Felty's syndrome; pulmonary fibrosis; glomerulonephritis, anaphylactoid glomerulonephritis; autoimmune glomerulonephritis; poststreptococcal glomerulonephritis; post-transplant glomerulonephritis; membranous glomerulopathy; Goodpasture's syndrome; immune-mediated granulocytopenia; annular granuloma; allergic granulomatosis; granulomatous myositis; Graves' disease; Hashimoto's thyroiditis; hemolytic disease of the newborn; idiopathic hemochromatosis; Schonlein-Genoch purpura; chronic active and chronic progressive hepatitis; histiocytosis X; hypereosinophilic syndrome; idiopathic thrombocytopenic purpura; Job's syndrome; juvenile dermatomyositis; juvenile rheumatoid arthritis (juvenile chronic arthritis); Kawasaki disease; keratitis; dry keratoconjunctivitis; Landry-Guillain-Barré-Strohl syndrome; lepromatous leprosy; Loeffler's syndrome; lupus; Lyell's syndrome; Lyme disease; lymphomatoid granulomatosis; systemic mastocytosis; mixed connective tissue disease; multiple mononeuritis; McCle-Wells syndrome; syndrome of mucocutaneous lymph nodes; syndrome of mucocutaneous lymph nodes; multicenter reticulohistiocytosis; multiple sclerosis; myasthenia gravis; fungoid mycosis; systemic necrotizing vasculitis; nephrotic syndrome; cross syndrome; panniculitis; paroxysmal cold hemoglobinuria; paroxysmal nocturnal hemoglobinuria; pemphigoid; pemphigus; erythematous pemphigus; leaf-shaped pemphigus; ordinary pemphigus; allergy of pigeon breeders; nodular polyarteritis; rheumatic polymyalgia; polymyositis; idiopathic polyneuritis; Portuguese familial polyneuropathies; preeclampsia/eclampsia; primary biliary cirrhosis; progressive systemic sclerosis (scleroderma); psoriasis; psoriatic arthritis; pulmonary alveolar proteinosis; pulmonary fibrosis, Raynaud's phenomenon/syndrome; Reidel's thyroiditis; Reiter's syndrome, relapsing polychondritis; rheumatic fever; rheumatoid arthritis; sarcoidosis; scleritis; sclerosing cholangitis; serum sickness; Cesari's syndrome; Sjögren's syndrome; Stevens-Johnson syndrome; Still's disease; subacute sclerosing panencephalitis; sympathetic ophthalmia; systemic lupus erythematosus; transplant rejection; ulcerative colitis; undifferentiated connective tissue disease; chronic urticaria; cold urticaria; uveitis; vitiligo; Weber-Christian disease; Wegener's granulomatosis or Wiskott-Aldrich syndrome.

Hereinafter, the present invention will be described with reference to one or more non-limiting examples.

Examples

The following examples are included to demonstrate specific embodiments of the present invention. It will be appreciated by those skilled in the art that the methods described in the following examples are methods discovered by the inventors of the present invention to function properly in the practice of the present invention, and thus may be considered the preferred methods for its implementation. However, those skilled in the art, in light of this disclosure, will appreciate that many changes can be made to the specific embodiments described without departing from the spirit and scope of the invention to obtain similar or similar results.

Comparative Example 1

The method of Marder et al., 2014, BMC Biotechnology, 14:33 involves processing 1 liter of culture and consists of two centrifugation steps 38,900 g lasting 30 minutes, while the first centrifugation step precipitates annexin A5 associated with cell debris, and the second step centrifugation precipitated cell debris while keeping annexin A5 in solution.

The following analysis is provided to calculate the consequences associated with scaling up the product in the Marder et al. process from 1 L to a commercially significant culture volume of 1000 L.

Considering the best centrifuges currently available with the best rotors for maximum throughput, these centrifuges can hold 6x250 ml = 1.5 liters and can reach 30,200-38,400 g. Examples are the expensive carbon fiber rotor (Fiberlite F14- 6 x 250y fixed angle) for use in Thermo Scientific™ Sorvall™ LYNX super high speed centrifuges or JLA-16.250 fixed angle rotor with aluminum and biosafety cap, 6 x 250 ml, 38400 x g for use in Beckmancoulters Avanti JXN-26 . With these modern high performance high efficiency centrifuges, in order to load the centrifuge, start the centrifugation and accelerate to the required speed, spend 30 minutes at maximum dynamic overload to ensure a thorough sedimentation and then stop so as not to disturb the sediment, and then empty the rotor, it will be necessary about 45 minutes.

That is, the best centrifuges currently available can process 1.5 liters in 45 minutes.

Marder et al. indicated (in the section entitled "Purification") that 3 g wet cell weight was suspended in 30 ml buffer prior to sonication and centrifugation. Accordingly, the wet cell weight (WCW) concentration used by Marder et al. during centrifugation (3 g in 30 ml buffer) = 9.1% WCW.

Marder et al. also indicated (under the heading "Bioreactor Culture" on the second page) an average value of 27.48 g (DCW) L-1 (SD = 1.96) for the biomass concentration. Therefore, the concentration by dry cell weight (DCW) in the Marder fermenter was 27.48 g/l = 2.748%. It is known that 1 gram of DCW = about 4 grams of wet cell weight (WCW). Therefore, in the cell concentration fermenter, the concentration of WCW was 2.748 × 4 = 11.0%. If this volume is adjusted to 1000 liters of culture, and with caution assuming a 5% cell loss during production with 1000 liters, then the WCW in a 1000 liter tank using Marder's method would be = 1000 x 11% x 0.95 = 104 .5 kg WCW.

With the Marder centrifugation method, a WCW concentration of 9.1% was used during the centrifugation step. Therefore, 104.5 kg of WCW from a 1000 L culture must be diluted to a concentration of 9.1% WCW, requiring centrifugation of a full volume of 1148 L.

If one optimistically assumes that two high-performance centrifuges are in the bioproduction facility, then the first centrifuge can be used to precipitate annexin with cellular debris (first centrifugation), while the second centrifuge can run a second centrifuge in parallel when annexin is in solution , followed by sedimentation of cellular debris.

The total centrifugation time for a 1148 L solution is calculated assuming that the centrifuges can process 1.5 liters in 45 minutes = 1148/1.5 = 766 centrifugations of 45 minutes each = 34,470 minutes = 574.5 hours.

Assuming that the bio-production facilities operate 12 hours a day, the treatment of WCW from a 1000 L tank according to the method of Marder et al. will take 48 working days or (assuming five working days per week) 10 weeks of centrifugation alone.

In total, it will take about two weeks for fermentation, post-processing and other operations. At the manufacturing facility, it would take 12 weeks to prepare and process cells from a 1000 L culture, assuming that biomanufacturing capacity is fully committed to this one process, and therefore no other production can be made at the same facilities. This is despite the optimistic assumption of having two centrifuges. If only one centrifuge is used, the production time is 22 weeks for one batch of 1000 liters.

In contrast, as discussed below, the methods of this invention can process a 1000 L culture in as little as two weeks, i.e. about 6 times faster (which also provides a higher quality product with a much higher yield than the Marder et al. ).

The cost of manufacturing is directly proportional to the manufacturing time, since the manufacturing plant will be busy, and there can be no other production with the same capacities.

It is calculated that the amount of Annexin A5 produced in the process of this invention will be 2-3 times higher per batch compared to the amount of product obtained by the method of Marder et al. As a result, the cost of manufacturing one gram of annexin A5 protein according to the Marder method is 12-18 times higher (6 x 2-3).

Moreover, the purity of the protein obtained by the method of Marder is not suitable for use in the human body. Despite elaborate centrifugations, only one anion exchange chromatography step is used, which essentially does not meet the requirement to achieve sufficient purity in terms of both manufacturing-related impurities (especially endotoxin) and product-related variants. Marder does not provide any data on levels of endotoxin or other impurities, indicating a lack of suitability for pharmaceutical use.

In addition, during very slow centrifugation operations according to the Marder method, it is necessary that the annexin A5 protein be in an unstable environment for a long period of time. This is likely to result in product degradation or product modification and is another disadvantage of long manufacturing times, with negative consequences for product quality.

Example 1

Abbreviations:

AP Aqua Purificata (purified water) NF national formulary BV Bovenau NL normal liter KIEF capillary isoelectric focusing NMWCO nominal monoisotopic molecular weight limit CX cation exchange chromatography n/prov. not carried out CFU colony forming unit well not specified CR room with column OP optical density CRG Room class with column PAAG polyacrylamide gel electrophoresis OK column volume PBS phosphate buffered saline solution DF diafiltration PC Polycarbonate LP medicinal product PETG polyethylene terephthalate copolyester modified with glycol NRP downstream process PES Polyethersulfone LS drug substance Ph.Eur. European pharmacopoeia EE unit of endotoxin PP Polypropylene EVA ethylene-vinyl acetate copolymer BCP Polypropylene glycol EVOH Ethylene-vinylethanol copolymer PVDF Polyvinylidene fluoride FF direct flow test/diffusion method QA Quality control department FIO for informational purposes only QC quality management department G gram RHB Richter-Helm BioLogics hour hours HOF reverse phase chromatography H Hanover rpm rpm BCH host cell protein CT room temperature (20-25°C) HH Hamburg SDS Sodium dodecyl sulfate ID inner diameter EH size exclusion chromatography IOC ion exchange chromatography DFT tangential flow filtration IPC control during the process TMD transmembrane pressure L liter UV Ultrafiltration LPV laminar air flow GRP upstream process PEnp low density polyethylene USP U.S.P MCB main cell bank v/v volume fraction min minutes W.B. Western blot ECO main reference standard WCB working cell bank n/a not applicable WFI Water for injections

Introduction:

Recombinant protein annexin A5 ~36 kDa, containing 320 amino acids, is expressed in the cytoplasm of E. coli BL21/pHIP.ANXA5. The recombinant annexin A5 protein is mainly produced in its soluble form. For this purpose, a heat-inducible pHIP expression plasmid carrying the coding sequence for annexin A5 is used. The selectable marker is a kanamycin resistance gene. The MCB of the respective clone has been identified and extensively characterized.

The production process is expanding from a laboratory scale equivalent to a 3L fermentation volume to a large scale equivalent to a 100L fermentation volume.

The developed process includes efficient anion exchange capture from the initial lysate followed by an affinity step on immobilized heparin in the presence of calcium. This intermediate affinity step is highly specific for annexin A5. High resolution anion exchange chromatography is used as the final purification step. The purification step allows you to separate the impurities associated with the product. The formulation was ultradiafiltered using a 10 kDa NMWCO cassette.

This example describes and evaluates planned adaptations/changes to the manufacturing process, defines the operating parameters and necessary measures to ensure a successful migration, and provides acceptance criteria to determine its success. Successful transfer is demonstrated by the results of the downstream process involving adaptation to a lab scale procedure for scaling up production resulting in drugs with comparable yield and product quality.

The overall goal of the project is to develop a method for manufacturing cGMP for annexin A5.

Procedure - comparison and evaluation of methods

This section evaluates process parameters, raw materials, consumables, buffers, and equipment.

In FIG. 2 is a schematic overview of the complete manufacturing process for Annexin A5.

Table 1 compares and evaluates the raw materials used in the 3L and 100L processes.

Table 1. Comparison of raw materials used in the 3 l and 100 l methods

Method for 3 l Method for 100 l Raw material Provider Quality Provider Quality Tris(hydroxymethyl)aminomethane (TRIS) Merck Ph.Eur. Merck Ph.Eur. Magnesium chloride heptahydrate Merck Ph.Eur. Merck Ph.Eur. Sodium chloride Merck Ph.Eur. Merck Ph.Eur. Twin-80 (polysorbate) Merck Ph.Eur. Merck Ph.Eur. Sodium hydroxide Merck Ph.Eur. Merck Ph.Eur. Benzonase nuclease Merck/Novagen Purity > 90% Merck Purity > 90% CaCl ₂ x 2H ₂ O Merck Ph.Eur. Merck Ph.Eur. Titriplex III Merck Ph.Eur. Merck Ph.Eur. Bis Tris
1,3-bis[tris(hydroxymethyl)methylamino]propane Merck Ultrol grade Sigma BioUltra Water RHB HH Bidest/Ampuva (Fresenius) Fresenius
Biochrom Ph.Eur. ethanol Merck Emprove Merck EMSURE® ACS,ISO Hydrochloric acid Merck Emprove exp.Ph.Helv Merck p.a. orthophosphoric acid Merck Emprove exp.Ph.Eur Merck Ph.Eur. Sodium hydroxide 33% Reher & Ramsden n/a --- ---

Table 2 compares and evaluates the consumables used in the 3L and 100L methods. Consumables (sampling devices and connecting tubes) should not affect the quality and yield of the product during the NRP. All materials used meet the required specification.

Table 2. Consumables list

Method for 3 l Method for 100 l Consumable Provider Quality Provider Quality Depth filter
Cuno SP 60 3M, CUNO USP class VI 3M, CUNO USP class VI Q Sepharose XL
(AX resin) GE Healthcare Pharmacopoeia article of the manufacturer GE Healthcare Pharmacopoeia article of the manufacturer Heparin HyperD M
(AF resin) Pall Pharmacopoeia article of the manufacturer Pall Pharmacopoeia article of the manufacturer Source15Q
(AX resin) GE Healthcare Pharmacopoeia article of the manufacturer GE Healthcare Pharmacopoeia article of the manufacturer Filter 0.2 µm
Sartopore 2 0.45-0.2 µm Sartorius USP class VI Sartorius USP class VI Filter 0.2 µm
EKV, supor Pall USP class VI Pall USP class VI UV/DF cassette, Hydrosart, 10 kDa membrane Sartorius USP class VI Sartorius USP class VI

General Notes:

• All consumables used are disposable or product oriented.

• Bags used during the process to store the buffers and as a container for the intermediate product with a PE/EVOH layer (CX5-14 film) were supplied by Sartorius. The pouches have been validated by the manufacturer for, for example, sterility, low levels of endotoxin, and leachables and extractables.

• All other consumables, including those in contact with the product, such as tubing, connectors, sampling systems or sample containers are suitable for use in the appropriate process step. This typically includes USP Class VI certification, sterility, and/or low endotoxin if applicable. Platinum vulcanized silicone tubing is used throughout the NRP, with the exception of C-Flex tubing, which is pre-pouched. All consumables used are animal-free or TSE-certified.

Table 3 compares the equipment used in the 3L and 100L processes in the manufacture of Annexin A5.

Table 3. List of used equipment

Method for 3 l Method for 100 l Equipment Type of Manufacturer Type of Manufacturer Thermometer n/a Hanna ≥ 0-100°C Amarell Pipette n/a eppendorf various eppendorf Pump n/a Watson Marlow 604 U/R Watson Marlow Pump n/a Watson Marlow 505DU Watson Marlow Pump n/a n/a 1000S QuattroFlow HPLC system 1 Akta 100 GE Healthcare BioProcess GE Healthcare Capture column XK50 GE Healthcare BPG300 GE Healthcare intermediate column XK50 GE Healthcare BPG300 GE Healthcare Cleansing column Fine Line 70 GE Healthcare Fineline200 GE Healthcare Ultrafiltration system n/a n/a UFDF-H1 PALL Photometer Ultrospec 3100 GE Healthcare Ultrospec x300 GE Healthcare pH/conductivity meter +
Printer MPC227 Mettler Toledo CG
HI
730p Schott
Hanna
W.T.W. camera with
laminar air flow (LAF) n/a Herasafe HS Herasafe Magnetic stirrer n/a IKA RET/REO IKA Vacuum pump n/a KNF - - Device for
filter integrity checks n/a n/a AquaWIT
Exacta PALL
Millipore

Media, buffers and solutions as indicated in Table 4. Buffer specifications are adapted only with respect to conductivity and based on buffer preparations analyzed. Buffers were prepared prior to running, checked for specification, microfiltered (0.2 µm filter) and stored (shelf life ≤ 3 months at room temperature). Buffer sampling (as reference; for analysis: endotoxin, bioburden) was performed at the time of application.

Table 4. List of media and solutions

Method for 3 l Method for 100 l Buffer Composition Application Composition buffer for
homogenization 1 50 mM Tris; pH 7.4, 1 mM MgCl ₂ ; 1% Twin 80 Homogenization 50 mM Tris; pH 7.4, 1 mM MgCl ₂ ; 1% Twin 80 Conditioning buffer for post homogenization 1 1% Twin 80 (w/v) capture 1% (v/v) Tween 80, 4 mM EDTA, pH 8.0 Conditioning buffer for post homogenization 2 0.5 M EDTA capture AX
buffer A 20 mM Tris, 25 mM NaCl, 0.1% Tween 80, pH 7.4 capture 20 mM Tris, 25 mM NaCl, 0.1% Tween 80, pH 7.4 AX
buffer B 20 mM Tris, 300 mM NaCl, 0.1% Tween 80, pH 7.4 capture 20 mM Tris, 300 mM NaCl, 0.1% Tween 80, pH 7.4 AX
CIP1 2M NaCl capture 2M NaCl AX
C.I.P. 2 1M NaOH capture 1M NaOH AF
buffer A 20 mM Tris, 25 mM NaCl, 2 mM CaCl ₂ 0.1% Tween 80, pH 7.4 Intermediate 20 mM Tris, 25 mM NaCl, 2 mM CaCl ₂ 0.1% Tween 80, pH 7.4 AF
wash buffer 20 mM Tris, 25 mM NaCl, 0.1% Tween 80, pH 7.4 Intermediate 20 mM Tris, 25 mM NaCl, 0.1% Tween 80, pH 7.4 AF
buffer B 20 mM Tris, 10 mM EDTA, 25 mM NaCl, 0.1% Tween 80, pH 7.4 Intermediate 20 mM Tris, 10 mM EDTA, 25 mM NaCl, 0.1% Tween 80, pH 7.4 AF
C.I.P. 1 50 mM Tris, 2 M NaCl, pH 7.4 Intermediate 50 mM Tris, 2 M NaCl, pH 7.4 AF
C.I.P. 2 0.1 M NaOH Intermediate 0.1 M NaOH AF
Storage 25% EtOH, 1 M NaCl Intermediate 25% EtOH, 1 M NaCl AX 2 air conditioning 35 mM Tris, 0.1% Tween 80, 12.5 mM MgCl ₂ , pH 8.0 cleaning 35 mM Tris, 0.1% Tween 80, 12.5 mM MgCl ₂ , pH 8.0 AX
buffer A 20 mM Bis-Tris, 25 mM NaCl, pH 7.4 cleaning 20 mM Bis-Tris, 25 mM NaCl, pH 7.4 AX
buffer B 20 mM Bis-Tris, 180 mM NaCl, pH 7.4 cleaning 20 mM Bis-Tris, 180 mM NaCl, pH 7.4 UV/DF
buffer 20 mM Bis-Tris, 150 mM NaCl, 1 mM CaCl ₂ , pH 7.0 Compound 20 mM Bis-Tris, 150 mM NaCl, 1 mM CaCl ₂ , pH 7.0 Conditioning after UV/DF 10% Tween 80, 20 mM Bis-Tris, 150 mM NaCl, 1 mM CaCl ₂ , pH 7.0 Compound 10% Tween 80, 20 mM Bis-Tris, 150 mM NaCl, 1 mM CaCl ₂ , pH 7.0

In an industrial plant, a fed-batch fermentation process with the possibility of scaling up production has been developed and expanded. The subsequent purification process includes three chromatography steps. After benzonase treatment, the filtered and diluted feed stream was applied to an AX chromatography system (Q Sepharose XL, GE Healthcare) as the first capture step. The eluate was conditioned by dilution with intermediate purification using affinity chromatography (Heparin Hyper DM, Pall). The AF pool was diluted and used in the final AX chromatography step (Source15 Q, GE Healthcare). Finally, the change in concentration and buffer was carried out using UV/DF.

A successful test run was carried out during the NRP, with a fermentation volume equivalent to 3 liters, to demonstrate proper performance for all process steps.

Target ranges were determined at a small scale and used to evaluate the result obtained at a scale up.

Method comparison

In the following sections, the laboratory scale downstream process (LRP) is described in detail based on a trial run performed on a process scale equivalent to a 3 L fermentation volume. Typically, in addition to loading in the first capture step, the chromatographic steps were performed on a laboratory scale using an Aekta Explorer system. On a large scale, all chromatographic steps were performed using the Bioprocess system. The scaling factor for the RRP was 33 (from 3 L of _GRP to 100 L of _GRP ).

1.1.1. Biomass resuspension, benzonase treatment and cell disruption

After fermentation, the biomass was collected by centrifugation and stored at -20°C. Post-treatment was started by thawing the biomass and resuspending in homogenization buffer 1. Pre-homogenized benzonase, previously diluted in homogenization buffer (3.300 U/l _WRP or 1.850 U/l _{resuspended biomass} ) was added to the resuspended cells. The resuspension ratio was set to 1 g biomass/10 ml. Homogenization was carried out in three cycles with 600 bar to achieve a high degree of homogeneity, which is preferable for the next capture step. Homogenization does not require active cooling, given that it is desirable to increase the temperature to 40°C in order to ensure optimal cleavage of nucleic acids by benzonase. When manufactured on a small scale, temperatures were in the range of 36-40°C.

In FIG. 8 is a flow chart of the biomass resuspension, benzonase treatment and cell disruption process.

1.1.2. Clarification, conditioning and capture chromatography

After homogenization, the lysate was clarified by filtration using a Cuno 60 SP depth filter (0.6-0.2 µm). This step was carried out in order to further reduce the level of nucleic acids and obtain a solution with a reduced number of particles, which can be used for capture chromatography. The depth filter was pre-washed with water according to the manufacturer's instructions.

After filtration, the lysate was diluted twice with 1% Tween 80. EDTA was added to give a final concentration of 2 mM.

This conditioned pool was used offline with a peristaltic pump for AX capture chromatography. The AX capture column was equilibrated with two column volumes (CV) (20 mM Tris pH 7.4, 0.1% Tween 80, 25 mM NaCl) at a linear velocity of 200 cm/hr.

After loading, the column was washed off-line with equilibration buffer with 5 OK, and then transferred to the chromatographic system for an additional wash of 5 OK. Elution of annexin A5 was performed with a 9 OC elution step using a higher salt concentration (20 mM Tris, pH 7.4; 0.1% Tween 80, 300 mM NaCl). Fractionation was determined by increasing the UV _{280 nm} signal by 0.1 optical density units (EOD) to 0.2 OOD in the descending peak. The entire elution peak was further processed.

A two-step CIP procedure was performed to regenerate and clean the column (step 1: 2 M NaCl for 3 CC 100 cm/h; upflow/step 2: 1 M NaOH, 3 SC, incubation > 15 h, 40 cm/h upflow ). The column was then stored in 20 mM NaOH.

In FIG. 3 shows a flowchart of an exemplary AX capture chromatography process.

Generally, the capture step can be thought of as a conditioning step to improve the performance of the intermediate step. At this stage, the product is concentrated and the load matrix changes significantly. In addition, there is a strong decrease in the level of endotoxin (about 97%) and a moderate decrease in DNA and HCC.

1.1.3. Intermediate step - affinity chromatography

The resulting AX elution pool (250 ml/L _WSP ) was filtered (Sartopore2 0.45-0.2 µm) before intermediate chromatography.

The filtered AX pool was then diluted 8 times (dilution buffer: 20 mM Tris, pH 7.4, 0.1% Tween 80, 2 mM CaCl ₂ ). Dilution with calcium promotes binding of annexin A5 to heparin during heparin-immobilized chromatography. This interaction, in comparison with the ionic interaction, is slow. The contact time is extremely important, so chromatography was carried out with ≤ 100 cm/h.

Carried out two stages of washing. Wash step 1 was performed with 15 TC (20 mM Tris, pH 7.4, 0.1% Tween 80, 2 CaCl ₂ ) followed by a second wash step with 2 TC with calcium-free buffer (20 mM Tris, pH 7.4, 0.1% Tween 80).

Elution was 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), which chelates calcium ions. The chelation reaction specifically elutes annexin A5, which can only bind to heparin in the presence of calcium. To ensure concentrated elution during elution, the flow rate was reduced to ≤ 60 cm/h. A complete elution peak was obtained starting with an increase in UV signal at 0.05 EOP to 0.05 EOP in a descending peak representing about 7 OC. The elution profile showed one sharp peak.

A two-step CIP procedure was performed to regenerate and clean the column (step 1: 2 M NaCl for 3 CC 100 cm/h; upflow/step 2: 0.1 M NaOH, 3 CC, incubation for > 15 h, 40 cm/h upstream). The column was then stored in 1 M NaCl in 25% EtOH.

In FIG. 4 shows a block diagram of an intermediate affinity chromatography process.

The intermediate step is the most powerful cleaning step in the process diagram. Annexin A5 binds to calcium ions. In this calcium-bound state, the product can form a highly specific bond with heparin. Only properly folded forms of AnxA5 annexin that are able to combine with calcium can bind to heparin. Thus, with the help of chromatography, it is possible to distinguish between a product with a correctly and incorrectly folded structure. In addition, high lean ratios are achieved in the intermediate step because a highly specific interaction is combined with a specific elution regimen via calcium chelation with EDTA. Therefore, there is a strong decrease in endotoxin levels (a further decrease of about 99%) and HCC, combined with a moderate decrease in DNA content.

The combined endotoxin reduction effect of the first AX capture step (about 97%) and the intermediate affinity chromatography step (about 99%) results in an annexin A5 product in which endotoxin levels are reduced to about 0.03% of the clarified product levels measured before the first stage AX.

1.1.4. Purification - Chromatography AX

The resulting AF elution pool (300 ml/L _WSP ) was diluted twice (35 mM Tris, pH 8, 0.1% Tween 80, 12.5 mM MgCl ₂ ) and filtered (Sartopore2 0.45-0.2 μm) before step purification chromatography. As a result of dilution, the conductivity of the AX load decreases, but complexes of free EDTA molecules with Mg ions are also formed. Otherwise, free EDTA binds to the column, thus reducing mainly the capacity at this stage, but also the separation.

The cleaning resin is Source15 Q with an average resin diameter of 15 µm. It is a high resolution cleaning resin that has the disadvantage of high back pressure. Therefore, chromatography was carried out at a speed of 100 cm/h. Loading should be <16 g/l of resin in order to ensure proper clearance.

Washing after loading was performed using buffer A (20 mm Bis-Tris pH 7, 25 mm NaCl) for 3 OK. Elution was performed using a linear gradient to 100% B (20 mM Bis-Tris pH 7; 180 mM NaCl) in 33 OC. This chromatographic step is mainly designed to remove impurities associated with the product. Various forms of annexin A5 were eluted from 40-100% B, starting with a major peak, a second reduced peak, and several smaller peaks that followed.

The first major peak was taken from 0.05 EOP to the trough between peak 1 and peak 2 representing about 7 OC.

A two-step CIP procedure was performed to regenerate and clean the column (step 1: 2 M NaCl for 3 CC 100 cm/h; upflow/step 2: 1 M NaOH, 3 SC, incubation > 15 h, 40 cm/h upflow ). The column was then stored in 25 mM NaCl.

Recent results from small trials have demonstrated the beneficial effects of Tween 80. The 0.1% Tween 80 process increased yield after the intermediate step by about 30%. Loading at the stage of cleaning was limited to the amount of resin 16 g/l to ensure proper resolution. Increasing yield also influences the action plan for scaling up production. The calculated column size in the purification step was planned with two cycles. Increasing the yield of the product necessitates 4-5 cycles if the total amount is processed on a scale of 100 liters of _GRP .

In FIG. 2 shows a block diagram of the final chromatographic purification step of AX.

The purification step is mainly necessary to reduce the level of impurities associated with the product, for example, the separation of various annexin A5 isoforms. In addition, the purification step achieves the highest depletion ratio for residual DNA and greatly reduces the level of HCC. The level of endotoxin, already reduced after the intermediate step, is further reduced by about 99%, resulting in endotoxin levels adjusted to 0.0003% levels in the clarified product before the first AX step.

1.1.5. Ultra/diafiltration and annexin A5 formulation

The AX pool was directly exposed to UV/DF to increase the product concentration and change the buffer. After buffer exchange, Tween 80 was added to give a final concentration of 0.05%, and the drug substance was subjected to sterilizing filtration.

At the first stage of the process, the AX pool was concentrated 6-8 times. This was followed by buffer exchange with 8-10 volumes of diafiltration in preparation buffer containing no Tween (20 mM Bis-Tris, 150 mM NaCl, 1 mM CaCl ₂ pH 7 or pH 7.4). After changing the buffer, a second concentration was carried out to achieve a final concentration of 12 g/l. This allowed a first cassette wash to be added and Tween 80 to be added to a final concentration of 0.05% to reach a final concentration of 10 g/l after sterilizing filtration. The UV/DF step was run with a low TMP of 0.9-1.1 bar to minimize overcoating.

In FIG. 6 shows a flowchart of the ultra/diafiltration process and annexin A5 formulation.

2. Target values and acceptance criteria

The following target values and acceptance criteria are defined to evaluate the performance and scale of the process compared to a small scale NRP. Target values were determined based on IPC/bulk analysis in a trial run. The key process parameters were characterized to indicate the key steps of the process and improve the reliability of its performance.

2.1. The main parameters of the method

Table 5 shows the main parameters of the method. Achievement of the target ranges in the implementation of the method indicates a successful increase in its scale.

Table 5 Method Step Parameters and Corresponding Target Values

Stage Parameter Target Range Homogenization Temperature after homogenization 34-40°C pressure 600 bar cycles 3 Resuspension factor 1 g/10 ml Capture stage
AX chromatography
(Q Sepharose XL) loading volume [g/l _resin ]
[10 L _GRP / l _resin ]] 30–50
10-12 Elution criteria
Elution criteria [UV 280, 2 mm] Main Peak
Pool (0.1 EOP-0.2 EOP) Elution volume [OK] 5-10 intermediate stage
AF chromatography
(Heparin HyperD) loading volume [g/l resin] 20 - 30 Elution criteria [UV 280, 2 mm] full peak
Pool (0.1 EOP - 0.1 EOP) Elution volume [OK] 2-3 Cleaning stage
AX chromatography
(Source15Q) loading volume [g/l resin] 10 – 23 Elution criteria [UV 280, 2 mm] main peak from 0.05 EOP to the trough between peak 1 and peak 2 Elution volume [OK] 5–10 UV/DF - Hydrosart 10 kDa Inlet pressure [bar] 0.8 - 1.2

2.2. The main parameters of the method

A high degree of suspension homogeneity prior to performing capture chromatography is essential. To achieve this goal, three cycles of homogenization can be applied. Moreover, by increasing the temperature during homogenization, it is possible to obtain temperature values of the lysate in the range of 37°C. This is important for benzonase activity, which has a direct impact on the filtration step and capture efficiency.

Combining with the purification step is also important as this step is used to separate the impurities associated with the product. The UV/DF step was performed under medium TMP conditions in order to minimize the formation of a coating layer.

Table 6. Important process parameters

Stage Parameter Target Range Homogenization Temperature after homogenization 34-40°C Homogenization Cycle time 3 cleaning Loading 10-16 g/l resin Consolidation after cleaning UV signal (2 mm) main peak from 0.05 EOP to the trough between peak 1 and peak 2 UV/DF TMD 0.9-1.1 bar

2.3. Control during the implementation of the method

Table 7 shows the elements of control during the implementation of the method. Achievement of the target ranges during the implementation of the method indicates the successful scaling up. The target ranges were set only based on observations in previous small scale runs that were performed with the changes made.

Stage
classification:
method for 3 l method for 100 l Analysis Laboratory Target range Resuspension for homogenization
IPC-D01 Resuspension for homogenization
IPC-03.1 Content:
SDS-PAGE electrophoresis [g/l] Bioanalytics HH > 5 g/l GRP SDS-PAGE (soluble and insoluble fraction on the recovered. gel) [ratio sol./insoluble. in %] Bioanalytics HH < 40% insoluble cell destruction
(after cycle 3)
IPC-D05 cell destruction
(after cycle 3)
IPC-03 Content:
SDS-PAGE electrophoresis [g/l] Bioanalytics HH > 5 g/l GRP SDS-PAGE (soluble and insoluble fraction on the recovered. gel) [ratio sol./insoluble. in %] Bioanalytics HH < 40% insoluble Lightening
IPC-D07 Lightening
IPC-04 Content:
SDS-PAGE electrophoresis [g/l] Bioanalytics HH n/a Conditioning for capture
IPC-D07a Dilution in real time
to capture
IPC-05.1 Content:
SDS-PAGE electrophoresis [g/l] Bioanalytics HH TBD Identity:
SDS-PAGE-Coomassie electrophoresis Bioanalytics HH TBD Purity:
BCH (WB) Bioanalytics HH related sample Purity:
BCH (ELISA) [ng/mg] Bioanalytics HH TBD Purity:
DNA (qPCR) [pg/mg] Bioanalytics HH TBD Purity:
Endotoxin
[EU/mg]; [EU/ml] L+S TBD AX capture
FT
IPC-D08 AX capture
FT
IPC-05.2 Identity:
SDS-PAGE-Coomassie electrophoresis Bioanalytics HH related sample AX capture
elution pool
IPC-D09 AX capture
elution pool
IPC-05 Content:
AX-HPLC [g/l] PANATecs n/a Content:
SDS-PAGE electrophoresis [g/l] Bioanalytics HH TBD Identity:
SDS-PAGE-Coomassie electrophoresis Bioanalytics HH related sample Identity:
IEF Bioanalytics HH related sample Purity:
BCH (WB) Bioanalytics HH related sample Purity:
BCH (ELISA) [ng/mg] Bioanalytics HH TBD Purity:
DNA (qPCR) [pg/mg] Bioanalytics HH Downgraded from IPC-D07a Purity:
Endotoxin
[EU/mg]; [EU/ml] L+S Downgraded from IPC-D07a Purity:
AX-HPLC [%] PANATecs TBD AX capture
Salt CIP
IPC-D10 AX capture
Salt CIP
IPC-05.3 Optional data analysis Bioanalytics HH n/a filtering 0.2 µm capture pool
IPC-D11 filtering 0.2 µm capture pool
IPC-06.1 n/a Bioanalytics HH n/a Conditioning for intermediate AF
IPC-D12 Conditioning for intermediate AF
IPC-06.2 Content:
AX-HPLC [g/l] PANATecs n/a Intermediate AF
FT + flush
IPC-D13 Intermediate AF
FT
IPC-06.3 Content:
AX-HPLC [g/l] PANATecs n/a Identity:
SDS-PAGE-Coomassie electrophoresis Bioanalytics HH related sample Intermediate AF
Elution pool
IPC-D14 Intermediate AF
Elution pool
IPC-06 Content:
AX-HPLC [g/l] PANATecs n/a Content:
UV 280 [g/l] QC H TBD Identity:
SDS-PAGE-Coomassie electrophoresis Bioanalytics HH related sample Identity:
IEF Bioanalytics HH related sample Purity:
BCH (WB) Bioanalytics HH related sample Purity:
BCH (ELISA) [ng/mg] Bioanalytics HH Downgraded from IPC-D09 Purity:
DNA (qPCR) [pg/mg] Bioanalytics HH Downgraded from IPC-D09 Purity:
Endotoxin
[EU/mg]; [EU/ml] L+S Downgraded from IPC-D09 Purity:
AX-HPLC [%] PANATecs TBD Purity:
EH [%] QC BV TBD filtering 0.2 µm intermediate pool
IPC-D17 filtering 0.2 µm intermediate pool
IPC-07.1 Optional data analysis Bioanalytics HH n/a Cleaning AX
FT + flush
IPC-D18 Cleaning AX
FT
IPC-07.2 Identity:
SDS-PAGE-Coomassie electrophoresis
FQKA-HB005 Bioanalytics HH related sample Cleaning AX
Salt CIP
IPC-D20 Cleaning AX
Salt CIP
IPC-07.3 Optional data analysis Bioanalytics HH n/a - Purification of the AX fraction
IPC-07.4 Identity:
IEF Bioanalytics HH related sample Purity:
AX-HPLC [%] PANATecs TBD - Purification of the AX fraction
IPC-07.5 Identity:
IEF Bioanalytics HH related sample Purity:
AX-HPLC [%] PANATecs TBD - Purification of the AX fraction
IPC-07.6 Identity:
IEF Bioanalytics HH related sample Purity:
AX-HPLC [%] PANATecs TBD - Purification of the AX fraction
IPC-07.7 Identity:
IEF Bioanalytics HH related sample Purity:
AX-HPLC [%] PANATecs TBD Cleaning AX
Elution pool
IPC-D19 Cleaning AX
Elution pool
IPC-07 Content:
AX-HPLC [g/l] PANATecs TBD Content:
UV 280 [g/l] QC H TBD Identity:
SDS-PAGE-Coomassie electrophoresis Bioanalytics HH related sample Identity:
IEF Bioanalytics HH related sample Purity:
BCH (WB) Bioanalytics HH related sample Purity:
BCH (ELISA) [ng/mg] Bioanalytics HH Downgraded from IPC-D14 Purity:
DNA (qPCR) [pg/mg] Bioanalytics HH Downgraded from IPC-D14 Purity:
Endotoxin
[EU/mg]; [EU/ml] L+S Downgraded from IPC-D14 Purity:
AX-HPLC [%] PANATecs TBD Purity:
EH [%] QC BV TBD Post concentration
IPC-D21 Post concentration
IPC-08.1 Optional data analysis Bioanalytics HH n/a Post-diafiltration/conc.
IPC-D22 Post-diafiltration/conc.
IPC-08.2 Content:
UV 280 [g/l] QC H TBD Post adding cassette wash and adding Twin 80
IPC-D23 Post adding cassette flush
IPC-08.3 Content:
UV 280 [g/l] QC H TBD Bulk substance, including Tween 80
IPC-D24 Bulk substance, including Tween 80
IPC-08 Content:
UV 280 [g/l] QC H > 10 g/l Bulk substance
sterilizing filtered
IPC-D25 Bulk substance
sterilizing filtered
IPC-09 Identity:
SDS-PAGE-electrophoresis restored. Bioanalytics HH Complies with the standard Identity:
IEF Bioanalytics HH The main band corresponds to the standard Content:
UV 280 [g/l] QC H 8-12 g/l Purity:
BCH (WB) Bioanalytics HH related sample Purity:
BCH (ELISA) [ng/mg] Bioanalytics HH 100 ng/mg Purity:
DNA (qPCR) [pg/mg] Bioanalytics HH 100 pg/mg Purity:
Endotoxin L+S 35 EU/mg Purity:
AX-HPLC [%] PANATecs TBD Purity:
EH [%] QC BV > 95 Bioburden
Ph. Eur. 2.6.12 L+S < 1 cfu/ml Activity Bioassay HH TBD pH QC-Hannover 6.8 - 7.2 Appearance QC-Hannover Transparent, colorless, free of visible particles Purity:
SDS-PAGE-electrophoresis unrecovered. Silver QC BV > 90% Content:
AX-HPLC [g/l] PANATecs TBD Identity:
Western blot Bioanalytics HH The main band corresponds to the standard Osmolality TECHPharm TBD

3. Conclusions

The above manufacturing method is well adapted for large scale manufacturing without any limitation to scale up to 10,000 liters if needed.

After a trial run using an upscaled scale up to 200 L in a GMP pilot plant, the method resulted in a product containing 1.8 pg of host cell DNA per mg of AnxA5 protein; 16.6 ng host cell protein per mg AnxA5 protein; and 0.1 EU per mg of AnxA5 protein.

During the entire manufacturing process, the annexin A5 protein remained in solution in an active form, while it did not temporarily bind to the chromatographic resins.

When using a 1000 L culture, the total time required by the manufacturer to carry out the process will be one week for fermentation, product harvesting, cell disruption, and pre-chromatography processing, and another week for post-chromatography processing. Thus, the time to implement all the steps of the method at the GMP facility will be 2 weeks. The time taken is scale-independent and ideally suited to the industry standard, and the method can be used by any contract manufacturing organization (CMO) or pharmaceutical manufacturer.

As noted above, for comparison, for the method of Marder et al. it takes about 12 weeks to process a 1000 L culture.

Moreover, the yield of the product in the implementation of the present method is 2-3 times higher than that of the method of Marder et al. As a result, the cost of manufacturing one gram of Annexin A5 drug substance according to the Marder method is 12-24 times higher (6-8 x 2-3).

Moreover, in addition to providing a purification process that is faster and has a higher product yield compared to the process described in the method of Marder et al., the method of the present invention also results in a product with a higher degree of purity. As discussed above in Comparative Example 1, the degree of purity of the protein obtained by the Marder method is not suitable for use in the human body. Despite elaborate centrifugations, the Marder process employs only one anion exchange chromatography step, which essentially fails to meet the requirement to achieve sufficient purity in terms of both manufacturing-related impurities (especially endotoxin) and product-related variants. Marder does not provide any data on levels of endotoxin or other impurities, indicating a lack of suitability for pharmaceutical use.

In contrast, the method of this application results in a high purity Annexin A5 protein product having the characteristics listed below:

- concentration, as a rule, is about 8-12 g/l;

host cell protein levels equal to or below 100 ng/m, more typically below 20 ng/mg (as determined by ELISA);

- host cell DNA levels equal to or below 100 pg/mg; more typically below 10 pg/mg;

- endotoxin levels equal to or below 35 EU/mg; more typically below 1 EU/mg,

- purity > 95% as determined by size exclusion chromatography;

- bioburden < 1 cfu/ml (as defined in European Pharmacopoeia 2.6.12);

- transparent, colorless appearance, free of visible particles; as well as

- in this case, the main band detected by Western blot analysis corresponds to the reference annexin A5.

The process was repeated using Capto Q ImpRes for the second purification step instead of Source 15Q. This has provided an even more efficient process, as Capto Q ImpRes anion exchange resin has high binding capacity, is suitable for high flow rates with low back pressure, can be packed in a higher layer and is cheaper. Maintained the quality and purity of the final product.

Compared to Source 15Q, Capto Q ImpRes resin features:

• More than double the capacity per gram/liter of resin

• Allows more than 2 times the flow rate at the same back pressure

• Can be packed with a higher layer, typically 35-60% higher, resulting in higher capacity per column; as well as

• The cost is two times lower when buying per liter of resin.

Example 2

The example illustrates a comparison of anion exchange (AX) capture and affinity capture using heparin chromatography.

Capture AX and capture affinity chromatography with heparin were compared in terms of recovery and purity of annexin A5 in the capture eluate. Both strategies were compared in parallel experiments under optimized conditions.

Analysis parameters:

Chromatography AX:

Serial mode: 500 µl resin (75% suspension)

Buffer AX A: 20 mM Na phosphate; pH 7; 5 mM EDTA; 250 mM NaCl

Buffer AX B: 20 mM Na phosphate; pH 6.5; 5 mM EDTA

Chromatography AX: load: 10 ml pre-filtered lysate

CIP: 1 M NaOH

Affinity chromatography with heparin:

Serial mode: 500 µl resin (75% suspension)

Loading: 10 ml pre-filtered lysate; + 10 mM CaCl ₂

Buffer AF A: 50 mM Tris, pH 7.4, 5 mM CaCl ₂

Buffer AF B: 50 mM Tris; pH 7.4; 40 mM EGTA; 50 mM NaCl

CIP: 3 M NaCl

Table 8. Comparison of AX Chromatography and Capture Affinity Chromatography with Heparin

Resin Recovery (%) Purity (determined based on SDS-PAGE-R&D) (%) AX Q Sepharose XL 70-90 10-20 affinity Heparin Hyper D 30-40 85-95 Affinity (after AX) Heparin Hyper D
(Q Sepharose XL) 70-80 85-95

These results demonstrate that performing AX capture prior to affinity chromatography does not significantly increase purity, but has a significant effect on the productivity of the heparin step. In addition, an expensive affinity resin may be useful for a long time if used as an intermediate step. The amount of product obtained is important.

Capture with AX can be considered as a conditioning step that allows the high specific affinity step to be effectively applied using heparin chromatography.

Example 3

Partially purified annexin A5 product was obtained using methods similar to Example 1, up to the first step of anion exchange capture chromatography.

Briefly, recombinant E. coli expressing annexin A5 was resuspended in homogenization buffer (50 mM Tris, 1 mM MgCl, 1% Tween 20, pH 7.5) with 3200 U benzonase and homogenized with three cycles at 600 bar pressure. . The temperature after homogenization was measured at 36°C. A clarification step was then carried out with Cuno 60 SP 0.6-0.2 μm, and the clarified solution was diluted 1:2 in 1% Tween 20 with the addition of EDTA. The capture post-conditioning 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 using buffer B (20 mM Tris, 300 mM NaCl, 0.1% Tween 20, pH 7.4). The resulting product was then subjected to sterilizing filtration using a 0.2 μm filter.

The resulting sterile filtered anion exchange product was purified by heparin affinity chromatography and analyzed under various conditions.

The heparin affinity chromatography conditions used were as follows:

Heparin HyperD M 50 ml OK Diluted Load Volume (ml) 1173 Volume after loading (ml) 5 Volume flow through the system (ml) 1145 The volume of the simulated elution pool (A5-B6)
50 to 50 mEOP downstream) (mL)
-UV 2mm- 150 Volume of IPC samples (ml) - Flow rate (cm/h) 100 (elution 60)

Sterilizing filtration of the capture elution pool was performed with Sartopore 2 0.45-0.2 µm. This pool was diluted 8 times with buffer A (1050 ml) from heparin chromatography. The resulting pool had a pH of 7.4 and a conductivity of 6.8 mS. Elution was carried out with a reduced flow rate of 60 cm/h.

Assays 1 and 2 were performed to determine the effect of Tween 80 using wash buffer A and elution buffer B as follows:

Analysis 1:

Buffer A 20 mM Tris, 25 mM NaCl, 2 mM CaCl ₂ , 0.1% Tween 20, pH 7.4

Buffer B 20 mM Tris, 10 mM EDTA, 25 mM NaCl, 0.1% Tween 20, pH 7.4

Analysis 2:

Buffer A 20 mM Tris, 25 mM NaCl, 2 mM CaCl ₂ , 0.1% Tween 20, 0.1% Tween 80 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 of analysis 1 are shown in FIG. 7A and the results of analysis 2 are shown in FIG. 7B.

The results obtained demonstrate that after the addition of Tween 80, a shift of the eluate to a single peak was observed, in contrast to the separated peaks of the eluate under standard conditions (analysis 1). Apparently, Twin 80 stabilizes annexin A5. A possible explanation for the change in elution profile is that

theoretical precipitation on the column is prevented. Two main positive effects can be described when using Tween 80 in the heparin affinity chromatography step:

- Reduced pressure: The pressure on the column, which increased from 0.5 to 2-3 bar when loaded, clearly decreased to 0.5 bar. This is especially useful when making a product on a large scale. The reason for the increase in pressure may be a small amount of sediment observed after a long incubation.

- Prevention of precipitation: The second positive effect observed during elution. When fractionating the eluate, the highly concentrated fractions of the main peak tend to precipitate. After combining the fractions, precipitation is no longer observed; suggest that this effect is associated with a very high concentration of annexin A5 in the main peak; moreover, the formation of the precipitate appears to be a reversible process. It was not possible to prevent precipitation in the main peak by increasing the salt concentration in the eluate. In contrast, the addition of Tween 80 also prevented precipitation in the main peak fractions.

Based on these results, it seems appropriate to additionally add Tween 80 to all intermediate chromatographic buffers, since it has a stabilizing effect on annexin A5.

Example 4

Partially purified annexin A5 product was obtained using methods similar to Example 1, up to the first step of anion exchange capture chromatography.

1.25 ml of the anion exchange step product was then mixed with 8.75 ml of various forms of assay dilution buffer. The mixtures were then incubated at ambient temperature and visually assessed after 30 minutes, 18 hours and 4 days.

The results are shown in the table below:

An approach dilution buffer Point in time (30 min) Point in time (18h) Point in time (4 days) A 20 mM Tris, 25 mM NaCl, 2 mM CaCl ₂ , 0.1% Tween20 pH 7.4 _, Transparent, barely visible opalescence Opalescence, the first signs of sediment formation Opalescence and partial precipitation B 20 mM Tris, 50 mM NaCl, 2 mM CaCl ₂ , 0.1% Tween 20 pH 7.4 Transparent, barely visible opalescence Opalescence, the first signs of sediment formation Opalescence and partial precipitation C 200 mM glycine, 20 mM Tris, 25 mM NaCl, 2 mM CaCl ₂ , 0.1% Tween 20 pH 7.4 Transparent, barely visible opalescence Opalescence, the first signs of sediment formation Opalescence and partial precipitation D 200 mM arginine, 20 mM Tris, 25 mM NaCl, 2 mM CaCl ₂ , 0.1% Tween 20 pH 7.4 Transparent, barely visible opalescence Opalescence, the first signs of sediment formation Opalescence and partial precipitation E 20 mM Tris, 25 mM NaCl, 2 mM CaCl ₂ , 0.1% Tween 20, 0.1% Tween 80, pH 7.4 Transparent, barely visible opalescence Opalescent, no sign of sedimentation Opalescent, no sign of sedimentation F 20 mM Tris, 25 mM NaCl, 2 mM CaCl ₂ , 0.1% Tween 20 pH 8.0 Transparent, barely visible opalescence Opalescence, the first signs of sediment formation Opalescence and partial precipitation

These results demonstrated the benefit of adding Tween 80 (ie Polysorbate 80) to prevent precipitation of the product in the sample. This is important in relation to conditioning the AnxA5 product prior to application to a chromatographic column, such as an affinity chromatography column, in order to reduce precipitation and prevent backpressure buildup during column operation.

Claims (153)

1. A method for isolating and/or purifying a recombinantly expressed intracellular protein containing the annexin A5 (AnxA5) sequence from an endotoxin-producing host cell with a cell wall, wherein said method comprises releasing the intracellular protein from the host cell, characterized in that said step of releasing the intracellular AnxA5 protein is carried out in the presence of a homogenization buffer containing a non-ionic detergent selected from Tween 20 and Tween 80, and wherein the method does not include any centrifugation steps to isolate and/or purify the AnxA5 protein after it has been released from the host cell, and/or in the method, the AnxA5 protein remains in solution at all steps of the method, except when it is temporarily bound with any chromatographic resins, and where the concentration of free calcium ions in the homogenization buffer during the step of releasing the intracellular AnxA5 protein from the host cell or after the release of the AnxA5 protein from the host cell, but before any subsequent chromatographic purification occurs, is less than 10 mM. 2. The method of claim 1 wherein the non-ionic detergent is Tween 80. 3. The method according to claim 1 or 2, wherein the step of releasing the intracellular AnxA5 protein is carried out in the presence of a homogenization buffer containing an amount of a non-ionic detergent that is effective to reduce or prevent the binding of annexin A5 to endotoxin. 4. The method according to any one of the preceding claims, characterized in that the step of releasing the intracellular AnxA5 protein is carried out in the presence of a homogenization buffer containing from 0.01 to 10% (w/w) non-ionic detergent, for example from 0.02 to 5 % (wt./wt.), from 0.05 to 2% (wt./wt.) or about 1% (wt./wt.) non-ionic detergent. 5. A method according to any one of the preceding claims for isolating and/or purifying a recombinantly expressed intracellular protein containing the annexin A5 (AnxA5) sequence from a host cell with a cell wall, said method comprising releasing the AnxA5 intracellular protein from the host cell, wherein the concentration of free calcium ions in the homogenization buffer during the release of the intracellular AnxA5 protein from the host cell or after the release of the intracellular AnxA5 protein from the host cell, but before any subsequent chromatographic purification occurs, is less than 5 mM, 1 mM, more preferably less than 500 µM or essentially zero and/or wherein the homogenization buffer contains a calcium metal ion chelator or is modified to include a calcium metal ion chelator after the release of the intracellular AnxA5 protein. 6. The method of claim 5, wherein the calcium metal ion chelator is selected from EDTA or a salt thereof, EGTA or a salt thereof, but most preferably is EDTA. 7. The method of claim 5 or 6 wherein the level of free calcium ions and/or the amount of calcium metal ion chelator is effective to reduce or prevent binding of annexin A5 to host cell wall components. 8. The method according to any one of paragraphs. 5-7, characterized in that the homogenization buffer contains or is brought before or after the release of the AnxA5 protein to such a composition that it contains from 0.01 to 500 mm, for example, from 0.05 to 100 mm, from 0.5 to 20 mM, 1 to 15 mM, 2 to 10 mM, or about 4 mM, a calcium metal ion chelator, and preferably wherein the calcium metal ion chelator is EDTA. 9. The method according to any of the preceding claims, characterized in that said method comprises isolating and/or purifying the recombinantly expressed intracellular AnxA5 protein from a culture of host cells, and this culture has a volume of at least 100 l, 500 l, 1000 l , 5000 l or 10 000 l. 10. A method according to any one of the preceding claims, comprising the step of mixing biomass from the host cell culture in homogenization buffer at a concentration of about 10 g of biomass per ml of homogenization buffer. 11. The method according to any one of the preceding claims, wherein said step of releasing the intracellular AnxA5 protein from the host cell in homogenization buffer comprises lysing, disrupting, homogenizing, ultrasonicizing, or applying pressure to the host cell, resulting in a cell wall and the cell membrane barrier of the host cell is destroyed and, as a result, the intracellular AnxA5 protein is released, and optionally this step does not include exposure to osmotic shock and/or a freeze/thaw step. 12. The method of claim 11, wherein the step of releasing the intracellular AnxA5 protein from the host cell comprises high pressure homogenization, e.g. one or more high pressure homogenization cycles in the range of about 400 bar to about 2500 bar, preferably three homogenization cycle at approx. 600 bar or two homogenization cycles at approx. 800 bar. 13. A method according to any one of the preceding claims, wherein the step of releasing the intracellular AnxA5 protein can result in a biomass homogenate containing the released AnxA5 protein. 14. The method according to claim 13, characterized in that the biomass homogenate additionally contains one or more, as a rule, all of the impurities selected from the group consisting of proteins of the host cell, components of the cell wall of the host cell, the membrane of the host cell , host cell nucleic acid, and endotoxin. 15. The method according to claim 13 or 14, further comprising the step of clarifying the biomass homogenate, resulting in a clarified product containing the released AnxA5 protein. 16. The method of claim 15, wherein the step of clarifying the biomass homogenate comprises treating the homogenate with a nuclease, such as nuclease A, preferably nuclease A from Serratia marescens, and optionally including the nuclease in a homogenization buffer until intracellular AnxA5 protein is released. 17. The method according to claim 15 or 16, characterized in that the step of clarifying the biomass homogenate preferably includes, after treatment with the nuclease according to claim 15 or 16, the step of passing the biomass homogenate containing the released AnxA5 protein through a filter such as a cellulose or polypropylene filter, preferably wherein the filter is a depth filter and/or preferably the filter has a cutoff pore size of less than 4 μm; and the filtrate is a clarified product containing the released AnxA5 protein. 18. The method of any one of the preceding claims, further comprising the step of exposing the released AnxA5 protein to an anion exchange resin to perform the first anion exchange step and thereby producing a first anion exchange product that contains the released AnxA5 protein. 19. The method according to claim 18, characterized in that the clarified product containing the released AnxA5 protein obtained by the method according to claim 17 is subjected to a first anion exchange step, resulting in a first anion exchange product that contains the released AnxA5 protein. 20. The method according to claim 18 or 19, characterized in that, before the first anion exchange step, one or more environmental parameters of the released AnxA5 protein selected from the group consisting of pH, conductivity, calcium ion chelator level and non-ionic detergent level are adjusted. 21. The method according to any one of paragraphs. 18-20, characterized in that the released AnxA5 protein, which is subjected to an anion exchange step, is prepared 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 is diluted with a non-ionic detergent, for example, in order to obtain a final concentration of non-ionic detergent from 0.01 to 1% (w/v), more preferably about 0.1% (w/v). 22. The method according to any one of paragraphs. 18-21, characterized in that the AnxA5 protein binds during the anion exchange step, and the first anion exchange product, which contains the released AnxA5 protein, is obtained by applying a wash solution and/or elution buffer to the anion exchange resin to release the bound AnxA5 protein, optionally the elution buffer contains NaCl, eg about 300 mM NaCl. 23. The method according to any one of paragraphs. 1-22, further comprising performing an affinity chromatography step on the released AnxA5 protein, resulting in a first affinity chromatography product that contains the released AnxA5 protein. 24. The method according to p. 23, characterized in that the AnxA5 protein in the product of the first anion exchange obtained according to any one of paragraphs. 18-22 are subjected to an affinity chromatography step. 25. The method according to p. 24, including the steps in which: (a) a biomass homogenate containing the released AnxA5 protein according to claim 13 or 14 is clarified according to any one of paragraphs. 15, 16 or 17, resulting in a clarified product containing the released AnxA5 protein, as well as (b) the AnxA5 protein in the clarified product is exposed to an anion exchange resin in order to carry out the first anion exchange step according to any one of paragraphs. 18-22, and thus obtain the product of the first anion exchange, which contains the protein AnxA5; and (c) wherein the AnxA5 protein in the first anion exchange product is subjected to the affinity chromatography step according to paragraph 23 or 24. 26. The method according to any one of paragraphs. 23-25, characterized in that the step of affinity chromatography includes the binding of the AnxA5 protein to immobilized heparin, and optionally the binding is stimulated by the presence of calcium ions. 27. The method of claim 26 wherein the AnxA5 protein is eluted from the immobilized heparin with an elution buffer containing a calcium ion chelator such as EDTA. 28. The method according to any one of paragraphs. 23-27, characterized in that the first affinity chromatography product contains the released AnxA5 protein and a calcium ion chelator such as EDTA or EGTA, optionally in the range of 0.1 to 500 mM, more preferably about 10 mM. 29. A method for isolating and/or purifying a protein containing the annexin A5 (AnxA5) sequence from a composition that contains the AnxA5 protein and a calcium metal ion chelator, characterized in that the method includes: exposing the composition to an anion exchange resin to carry out the anion exchange step and thereby isolating and/or purifying the AnxA5 protein from the composition, and where the anion exchange step is carried out in the presence of additional selected metal ions, and wherein the additional selected metal ions are selected such that the calcium metal ion chelator has a binding affinity for the selected metal ions that is higher than its binding affinity for the anion exchange resin, but lower than its binding affinity for calcium ions, while the AnxA5 protein remains in solution at all stages of anion exchange chromatography, except when it temporarily binds to the specified chromatographic resin. 30. The method of claim 29 wherein the calcium metal ion chelator is selected from EDTA or a salt thereof, EGTA or a salt thereof, but most preferably is EDTA. 31. The method according to p. 29 or 30, characterized in that the calcium metal ion chelator is present in the composition in excess and / or at a concentration of about 0.1 mm, 0.5 mm, 1 mm, 5 mm, 10 mm, 15 mm , 20 mM or more. 32. The method according to any one of paragraphs. 29-31, characterized in that the selected metal ions are divalent cations such as Mg ^{2+ ions} . 33. The method according to any one of paragraphs. 29-32, characterized in that the selected metal ions are present in the anion exchange step in an amount effective to reduce or prevent interaction between the calcium ion chelator and the anion exchange resin during the process of exposing said composition to the anion exchange resin. 34. The method according to any one of paragraphs. 29-33, characterized in that the selected metal ions are present at the anion exchange stage in an amount effective to increase the binding of the AnxA5 protein to the anion exchange resin in the presence of a calcium ion chelator and thereby reduce the loss of the AnxA5 protein during the passage of the flow through the anion exchange stage compared to the level of losses , observed in those cases when the selected metal ions are not present at the anion-exchange stage. 35. The method according to any one of paragraphs. 29-34, characterized in that the selected metal ions are present at the anion exchange stage, for example, as a result of adding to the composition containing the AnxA5 protein and the metal calcium ion chelator, before exposing this composition to the anion exchange resin, at a concentration of from about 1 to about 100 mm , for example, about 2 to about 50 mM, about 5 to about 25 mM, about 10 to about 15 mM, or about 12.5 mM. 36. The method according to any one of paragraphs. 29-35, characterized in that the calcium metal ion chelator is EDTA, and the selected metal ions are Mg ^{2+ ions} , and preferably the molar ratio of Mg ^{2+ ions} to EDTA is in the range from 0.5:1 to 2:1, most preferably at least 1:1 or higher. 37. The method according to any one of paragraphs. 29-36, characterized in that a composition that contains an AnxA5 protein and a calcium metal ion chelator and is exposed to an anion exchange resin is a direct or indirect product of a preceding process that includes the step of performing an affinity chromatography step for the AnxA5 protein and eluting the AnxA5 protein with a calcium ion chelator, resulting in an affinity chromatography product that is a composition containing the AnxA5 protein and a calcium metal ion chelator. 38. The method according to claim 37, characterized in that the preceding affinity chromatography step includes binding of the AnxA5 protein to immobilized heparin, and optionally the binding is stimulated by the addition of calcium ions. 39. The method of claim 38, wherein the AnxA5 protein is eluted from the immobilized heparin using an elution buffer containing a calcium ion chelator such as EDTA and EGTA. 40. The method according to any one of paragraphs. 37-39, characterized in that between the previous affinity chromatography step and the anion exchange step, the dialysis step is not carried out and / or the removal of the calcium ion chelator from the product of the previous affinity chromatography step is not carried out before direct or indirect use of the product at the anion exchange step. 41. The method according to any one of paragraphs. 29-40, characterized in that the selected metal ions are added to the composition before or during the anion exchange step. 42. The method according to any one of paragraphs. 1-28, characterized in that said method includes an anion exchange step according to any one of paragraphs. 29-41. 43. A method for isolating and/or purifying a recombinantly expressed intracellular protein containing the sequence of annexin A5 (AnxA5) from an endotoxin-producing host cell with a cell wall or from a culture of this host cell, as defined in paragraph 9, this method includes : (a) releasing an intracellular protein from the host cell in the presence of a homogenization buffer containing a non-ionic detergent according to any one of paragraphs. 1-4; (b) performing a method on the released AnxA5 protein, directly or indirectly, which comprises i. the step of exposing this released AnxA5 protein directly or indirectly to an anion exchange resin to carry out the first anion exchange step and thereby obtain a first anion exchange step product that contains this released AnxA5 protein, and ii. performing an affinity chromatography step on this released AnxA5 protein of the product of the first anion exchange chromatography directly or indirectly, wherein optionally, said released AnxA5 protein is exposed to an anion exchange resin in the presence of a calcium ion chelator, and the product of the affinity chromatography step is a composition that contains the AnxA5 protein and a calcium metal ion chelator; and (c) wherein the direct or indirect product of the affinity chromatography step, which contains the AnxA5 protein and the calcium metal ion chelator, is subjected to an anion exchange step according to any one of paragraphs. 29-42; and the AnxA5 protein remains soluble except for transient binding to the anion exchange and affinity chromatography phases during steps (a)-(c) of this method. 44. The method of claim 43, wherein the release step comprises the step of mixing biomass from the host cell culture in homogenization buffer at a concentration of about 10 g of biomass per ml of homogenization buffer, and/or wherein the release step includes lysis, disruption, homogenization, processing sonicating or squeezing the host cell so that the cell wall and cell membrane barrier of the host cell is destroyed, and thus the intracellular AnxA5 protein is released, and optionally, where this step does not include the use of osmotic shock and/or a freeze-thaw step; optionally, where the homogenization comprises high pressure homogenization, such as one or more high pressure homogenization cycles from about 400 bar to about 2500 bar, preferably three homogenization cycles at about 600 bar or two homogenization cycles at about 800 bar. 45. The method according to p. 43 or 44, in which the method includes the stage of clarification of the biomass homogenate, thus obtaining a clarified product containing the released protein AnxA5; optionally wherein the step of clarifying the biomass homogenate comprises treating the homogenate with a nuclease such as nuclease A, preferably Serratia marescens nuclease A, and optionally the nuclease is added to the homogenization buffer prior to the release of the intracellular AnxA5 protein, and/or the step of clarifying the biomass homogenate preferably includes after the nuclease treatment a pass step a biomass homogenate containing the released AnxA5 protein through a filter; such as a cellulose or polypropylene filter, preferably in which the filter is a depth filter and/or preferably in which the filter has a cut-off limit of less than 4 microns; and the filtered stream is a clarified product containing the released AnxA5 protein. 46. The method according to any one of paragraphs. 43-45, wherein the affinity chromatography step (ii) comprises binding of the AnxA5 protein to immobilized heparin, and wherein said binding is stimulated by providing the presence of calcium ions. 47. The method of claim 46 wherein the AnxA5 protein is eluted from the immobilized heparin with an elution buffer containing a calcium ion chelator such as EDTA. 48. The method according to p. 47, in which the affinity chromatography of step (ii) includes carrying out the stage of affinity chromatography with immobilized heparin in the presence of Tween 80, preferably in the presence of 0.1% Tween 80, in relation to a solution containing AnxA5 protein and one or more impurities, thereby obtaining the first affinity chromatography product that contains the released AnxA5 protein. 49. The method of claim 48, wherein the AnxA5 protein remains in solution throughout the entire process, including any upstream or downstream steps, except when it is temporarily associated with any chromatographic resins. 50. The method according to any of the preceding claims, characterized in that said method comprises, preferably at the end of the method, as defined by any of the preceding claims, one or more additional steps selected from the group consisting of concentration, buffer exchange, conditioning and filtration, for example sterilizing filtration, and optionally the final step of storing the product containing the AnxA5 protein in a sterile container. 51. The method according to p. 50, characterized in that one of the additional steps is diafiltration, while optionally the product of the diafiltration step contains the AnxA5 protein at a concentration of at least about 1 mg/ml, 5 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 50 mg/ml, 100 mg/ml or higher. 52. The method according to claim 50 or 51, characterized in that a 0.45-0.2 µm filter or a 0.22 µm filter is used for filtration, and the filtration is preferably a sterilizing filtration step. 53. The method according to any one of paragraphs. 50-52, characterized in that sterilizing filtration is the final purification step before storing the product containing the AnxA5 protein in a sterile container. 54. The method according to any of the preceding claims, characterized in that said method includes the steps necessary to obtain the final sterile protein product AnxA5 in a phosphate-free buffer, such as Bis-Tris or Tris buffer, at a pH of about 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 AnxA5 protein concentration in the final sterile AnxA5 protein product is about 10 mg /ml 55. The method according to any one of the preceding claims, characterized in that said method produces the final sterile AnxA5 protein product, wherein NaCl is present at a concentration that maintains the AnxA5 protein in a form that is predominantly monomeric. 56. The method according to any of the preceding claims, characterized in that said method provides a total product yield of more than 1 g of AnxA5 protein per 1 liter of host cell culture, more preferably at least about 1.5 g/L, even more preferably in the range from about 2 to about 4 g/l. 57. The method according to any one of the preceding claims, characterized in that said method provides a total amount of AnxA5 protein that is about 24 wt.% of the AnxA5 protein present in the culture of the host cell. 58. The method according to any one of the preceding claims, characterized in that said method produces a product containing a host cell protein that differs from the recombinantly expressed AnxA5 protein at a level of less than 100, 90, 80, 70, 60, 50, 40, 30, 20 ng or less per mg of AnxA5 protein. 59. The method according to any of the preceding claims, characterized in that said method provides a product containing an endotoxin level of less than 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 35, 20, 15 and preferably less than 10, 5 or 1 EU per mg of AnxA5 protein, and/or preferably, this method provides a product in the form of a dosage form, while the product contains less than 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 35, 20, 15 and preferably less than 10, 5 or 1 EU per dosing unit. 60. A method according to any one of the preceding claims, wherein said method provides a product containing host cell nucleic acid levels of less than 1000 pg per mg of AnxA5 protein, preferably less than 100 pg per mg of AnxA5 protein, more preferably less than 10 pg per mg AnxA5 protein. 61. The method of any one of the preceding claims, comprising the step of chemically modifying the AnxA5 protein, and optionally also containing one or more additional purification steps, such as steps to reduce or remove unreacted components and/or as steps to select a homogeneous population of chemically modified AnxA5 protein. 62. An AnxA5 protein-containing composition for use in annexin A5 protein therapy, characterized in that said composition is obtained directly or indirectly using a method according to any one of paragraphs. 46-49 and paragraphs dependent on them, and where (a) the composition has been subjected to a sterile filtration step and is a sterile composition; (b) the composition is stored in a sterile container; (c) endotoxin is contained in the composition in an amount of less than 100, 50, 20, 10, 5, or 1 EU per mg of AnxA5 protein; (d) a nucleic acid, such as a host cell nucleic acid, is present in the composition at a level of less than 1000 pg, 100 pg, or 10 pg per mg of AnxA5 protein; and (e) the AnxA5 protein does not contain a His-tag. 63. The composition according to p. 62, characterized in that the specified composition is a direct or indirect product of the method according to any one of paragraphs. 46-49, wherein in said method the affinity chromatography step comprises binding the AnxA5 protein to the immobilized heparin, where this binding is promoted by the presence of calcium ions, and wherein the AnxA5 protein is eluted from the immobilized heparin using an elution buffer containing a calcium ion chelator such as EDTA. 64. The composition according to claim 62 or 63, which contains the AnxA5 protein at a concentration of at least about 1 mg/ml, 5 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 50 mg/ml, 100 mg/ml or higher. 65. The composition according to any one of paragraphs. 62-64, characterized in that the specified composition contains a sterile protein product AnxA5 in a non-phosphate buffer, such as Bis-Tris or Tris buffer at a pH of about 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 AnxA5 protein concentration in the final sterile AnxA5 protein product is about 10 mg/mL. 66. The composition according to any one of paragraphs. 62-65, characterized in that NaCl is present at a concentration that maintains the AnxA5 protein in a form that is predominantly monomeric. 67. The composition according to any one of paragraphs. 62-66 which contains a non-AnxA5 protein, such as a host cell protein, at a level of less than 20 ng per mg of AnxA5 protein, wherein the host cell is optionally a prokaryotic cell, such as a Gram-positive or Gram-negative cell, and, in in particular, it may be a Gram-negative endotoxin-producing bacterial cell, and optionally additional host cell protein is present in the composition at a detectable level, albeit less than 20 ng per mg of AnxA5 protein. 68. The composition according to any one of paragraphs. 62-67, which contains endotoxin at a detectable level, in an amount of less than 100, 50, 20, 10, 5, or 1 EU per mg of AnxA5 protein, in the composition. 69. Composition according to any one of paragraphs. 62-68, which contains endotoxin at a detectable level, in an amount of less than 100, 50, 20, 10, 5, or 1 EU per mg of dosing, in the composition. 70. The composition according to any one of paragraphs. 62-69, which contains a nucleic acid, such as a host cell nucleic acid, at a level of less than 1000 pg, 100 pg, or 10 pg per mg of AnxA5 protein, optionally, the host cell is a prokaryotic cell, such as a gram-positive or gram-negative cell, and in particular may be a gram-negative endotoxin-producing bacterial cell, and further optionally, the host cell nucleic acid is present in the composition at a detectable level, albeit less than 1000 pg, 100 pg, or 10 pg per mg of AnxA5 protein. 71. Composition according to any one of paragraphs. 62-70, characterized in that the level of gluconoylated AnxA5 protein in the composition is in the range from 0.5 to 30%, or from 0.5 to 20%, or from 0.5 to 15%, or from 0.5 to 10 % of the total AnxA5 protein in the product. 72. The composition according to any one of paragraphs. 62-71, characterized in that the level of gluconoylated AnxA5 protein in the composition is below 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2% or 1%, and preferably is essentially 0%. 73. Composition according to any one of paragraphs. 62-72, characterized in that the AnxA5 protein does not contain one or more RGD motifs. 74. The composition according to any one of paragraphs. 62-73, where the AnxA5 protein is chemically modified and/or where the AnxA5 protein is a fusion protein containing the AnxA5 protein and a fusion partner. 75. Composition according to any one of paragraphs. 62-74, characterized in that said composition is a pharmaceutically acceptable and/or veterinarily acceptable composition. 76. The composition of claim 75 in the form of a pharmaceutical or veterinary composition that is suitable for parenteral, intravenous, intraarterial, intraperitoneal, intramuscular, intraocular, intracranial, intracerebral, intraosseous, intracerebroventricular, intrathecal, subcutaneous, or topical administration. 77. The composition according to claim 76, which is suitable for topical application and is a cream, ointment or is in the form of eye drops. 78. The composition of claim 62, wherein the AnxA5 protein in the composition is 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. 79. The composition according to claim 78, where the composition is in the form of application with immediate, delayed or controlled release. 80. The composition of claim. 62 in the form of a pharmaceutical or veterinary composition, which is a drug-eluting stent. 81. The composition according to any one of paragraphs. 75-80, where the composition is for the treatment, prevention and/or reduction of the risk of developing a cardiovascular disease, an autoimmune disease, or an inflammatory condition. 82. Composition according to any one of paragraphs. 75-80, where the composition is intended to prevent or reduce the risk of thrombosis. 83. Composition according to any one of paragraphs. 75-80, wherein the composition is for treating, preventing, or reducing the risk of retinal vein occlusion. 84. A composition according to any one of claims 75-80, wherein the composition is for the treatment, prevention, or risk reduction of hematological disorders, including but not limited to sickle cell anemia. 85. The composition according to any one of paragraphs. 75-80, where the composition is for the treatment, prevention or reduction of the risk of pulmonary fibrosis. 86. Composition according to any one of paragraphs. 75-80, where the composition is intended for the treatment, prevention and/or reduction of the risk of atherosclerosis, acute coronary syndrome, stroke, lameness, angina pectoris, coronary heart disease, peripheral arterial disease, systolic hypertension, thromboembolism, hemorrhagic or vasculitic stroke, myocardial infarction, angina or intermittent claudication, unstable angina, other forms of severe angina or transient ischemic attacks (TIA). 87. Composition according to any one of paragraphs. 75-80, where the composition is for the treatment, prevention or reduction of the risk of vascular dysfunction. 88. Composition according to any one of paragraphs. 75-80, where the composition is for reducing ischemic pain. 89. Composition according to any one of paragraphs. 75-80, where the composition is for the prevention or treatment of restenosis, such as the formation or thickening of the neointima. 90. The composition according to any one of paragraphs. 75-80, where the composition is for the treatment, prevention or reduction of the risk of ischemia-reperfusion injury, ischemia-reperfusion injury of an organ, such as ischemia-reperfusion injury of the liver or kidney, graft rejection, graft-versus-host disease (GVHD), including acute and / or chronic GVHD, or for the treatment of a subject after transplantation. 91. The composition according to any one of paragraphs. 75-80, wherein the composition is for preventing and/or reducing peri- or postoperative complications after surgery, such as complications after vascular surgery, eg peripheral vascular surgery. 92. The composition according to any one of paragraphs. 75-80, where the composition is for the prevention or treatment of vascular inflammation, for the treatment, prevention or reduction of the risk of acute and chronic vascular inflammation or carditis. 93. The composition according to any one of paragraphs. 75-80, where the composition is for the treatment, prevention and/or reduction of the risk of developing acute and/or chronic inflammatory conditions and/or inflammatory bowel disease. 94. The composition according to any one of paragraphs. 75-80, where the composition is for the treatment, prevention and/or reduction of the risk of developing rheumatic diseases and/or arthritis, including osteoarthritis, rheumatoid arthritis, psoriatic arthritis. 95. Composition according to any one of paragraphs. 75-80, where the composition is for the treatment, prevention and/or reduction of the risk of developing Alzheimer's disease, dementia in general, multiple sclerosis or myasthenia gravis. 96. The use of a composition according to any one of paragraphs. 62-80 in the way: treatment, prevention and / or reduction of the risk of developing cardiovascular diseases, autoimmune diseases or inflammatory conditions, prevent or reduce the risk of thrombosis, treating, preventing or reducing the risk of retinal vein occlusion, treating, preventing or reducing the risk of hematological disorders, including but not limited to sickle cell anemia, treating, preventing or reducing the risk of pulmonary fibrosis, treatment, prevention and / or reduction of the risk of atherosclerosis, acute coronary syndrome, stroke, lameness, angina pectoris, coronary heart disease, peripheral arterial disease, systolic hypertension, thromboembolism, hemorrhagic or vasculitic stroke, myocardial infarction, angina pectoris or intermittent claudication, unstable angina pectoris, other forms of severe angina or transient ischemic attacks (TIA), treatment, prevention or reduction of the risk of vascular dysfunction, reduction of ischemic pain, prevention or treatment of restenosis, such as formation or thickening of the neointima, treating, preventing or reducing the risk of ischemia-reperfusion injury, ischemia-reperfusion injury of an organ such as ischemia-reperfusion injury of the liver or kidneys, transplant rejection, graft-versus-host disease (GVHD), including acute and/or chronic GVHD, or for the treatment of a subject after transplant, preventing and/or reducing peri- or postoperative complications after surgery, such as complications after vascular surgery, such as peripheral vascular surgery, prevention or treatment of vascular inflammation, treatment, prevention or reduction of the risk of acute and chronic vascular inflammation or carditis, treatment, prevention and/or reduction of the risk of developing acute and/or chronic inflammatory conditions and/or inflammatory bowel disease, treating, preventing and/or reducing the risk of developing rheumatic diseases and/or arthritis, including osteoarthritis, rheumatoid arthritis, psoriatic arthritis, or treating, preventing and/or reducing the risk of developing Alzheimer's disease, dementia in general, multiple sclerosis or myasthenia gravis. 97. A method of treating a human or animal in need of therapy with an annexin A5 protein composition according to any one of paragraphs. 62-80, characterized in that it includes the introduction of a therapeutically effective amount of the AnxA5 protein according to any one of paragraphs. 62-80 subject. 98. The method of claim 97, wherein the method is for one of the following: treating, preventing and/or reducing the risk of developing cardiovascular diseases, autoimmune diseases or inflammatory conditions, preventing or reducing the risk of thrombosis, treating, preventing or reducing the risk of retinal vein occlusion, treating, preventing or reducing the risk of hematological disorders, including but not limited to sickle cell anemia, treating, preventing or reducing the risk of pulmonary fibrosis, treatment, prevention and / or reduction of the risk of atherosclerosis, acute coronary syndrome, stroke, lameness, angina pectoris, coronary heart disease, peripheral arterial disease, systolic hypertension, thromboembolism, hemorrhagic or vasculitic stroke, myocardial infarction, angina pectoris or intermittent claudication, unstable angina pectoris, other forms of severe angina or transient ischemic attacks (TIA), treatment, prevention or reduction of the risk of vascular dysfunction, reduction of ischemic pain, prevention or treatment of restenosis, such as formation or thickening of the neointima, treating, preventing or reducing the risk of ischemia-reperfusion injury, ischemia-reperfusion injury of an organ such as ischemia-reperfusion injury of the liver or kidneys, transplant rejection, graft-versus-host disease (GVHD), including acute and/or chronic GVHD, or to treat a subject after transplantation, preventing and/or reducing peri- or postoperative complications after surgery, such as complications after vascular surgery, such as peripheral vascular surgery, prevention or treatment of vascular inflammation, treatment, prevention or reduction of the risk of acute and chronic vascular inflammation or carditis, treatment, prevention and/or reduction of the risk of developing acute and/or chronic inflammatory conditions and/or inflammatory bowel disease, treating, preventing and/or reducing the risk of developing rheumatic diseases and/or arthritis, including osteoarthritis, rheumatoid arthritis, psoriatic arthritis, and treating, preventing and/or reducing the risk of developing Alzheimer's disease, dementia in general, multiple sclerosis or myasthenia gravis.

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