JP2021161033A - Highly pure histidine-rich glycoprotein, and production method and use thereof - Google Patents

Abstract

To provide a highly pure and stable human histidine-rich glycoprotein (HRG)-containing composition reduced in the amount of plasmin and plasminogen, which is a plasmin precursor, from ingredients originating from blood plasma or recombinant animal cells inevitably contaminated with the plasmin and plasminogen, and thus provide an HRG formulation effective as a pharmaceutical.SOLUTION: A production method of a human HRG-containing composition reduced in the amount of plasmin and/or plasminogen comprises treating, with an anion exchange resin under a condition causing a mass overload of HRG, a human HRG-containing solution originating from animals, cells or tissues thereof producing plasmin and/or plasminogen.SELECTED DRAWING: None

Description

The present invention relates to a composition containing a high-purity and stable histidine-rich glycoprotein (hereinafter, also referred to as “HRG”), a method for producing the same, and an application thereof. More specifically, the present invention comprises treating an HRG-containing material contaminated with plasmin and / or plasminogen with an anion exchange resin under mass overload (hereinafter also referred to as "Mass OverLoad") conditions. The present invention relates to a method for producing a stable HRG-containing composition containing reduced plasmin and / or plasminogen, an HRG-containing composition obtained by the method, and various pharmaceutical uses thereof.

Histidine-rich glycoprotein (HRG) is a plasma protein normally present in human blood and is a multidomain protein isolated by Heimburger et al. In 1972. HRG is a factor that plays an extremely important role in maintaining the calm of circulating neutrophils and vascular endothelial cells, and the cascade of septic pathologies progresses from a decrease in blood HRG. It has been reported that supplementation with HRG has a dramatic survival-maintaining effect on clearly reduced mice (Non-Patent Document 1).

Various purification methods such as cation exchange chromatography, anion exchange chromatography, metal immobilization affinity chromatography, size exclusion chromatography, and heparin affinity chromatography are used to purify HRG. Since it has a histidine-rich region in the center of its amino acid sequence that specifically binds to metal ions such as copper, cobalt, and nickel, the first step of purification is followed by immobilized metal affinity chromatography (IMAC). By using anion exchange chromatography in the second step, it is possible to prepare HRG with higher purity (Non-Patent Document 2).

However, it is known that HRG is degraded by serine proteases plasmin and kallikrein (Non-Patent Documents 3 and 4), and it is an issue to suppress the degradation of HRG in the industrial production of HRG preparations. It has become. The human HRG gene is known, and recombinant production using host cells that do not produce plasmin such as Escherichia coli, yeast, and plants is possible, but since HRG is a glycoprotein, its physiological activity and safety are taken into consideration. In this case, it is necessary to use higher eukaryotic cells such as mammals capable of sugar chain modification similar to humans as a host, and contamination with plasmin or the like is unavoidable.

Purification on ice and addition of a protease inhibitor have been adopted as countermeasures against HRG degradation that occurs during the manufacturing process (Non-Patent Document 2), but the degradation has not been sufficiently suppressed. In addition, regarding the addition of a protease inhibitor, not only is the high addition cost a problem when considering industrial production as a pharmaceutical product, but also the protease inhibitor itself has pharmacological activity, so that it remains in the final HRG preparation. , May induce unintended side effects. Therefore, when the protease inhibitor is used for industrial production, the residual amount in the final preparation is strictly controlled, that is, the protease inhibitor is substantially not contained in the final preparation containing HRG as the main component. HRG stability is required. Even when protease inhibitors are not used in industrial production, HRG stability is naturally required.

In Non-Patent Document 2, as a result of examining the amount of plasminogen in purified HRG after immobilized metal affinity chromatography using cobalt and anion exchange chromatography using Western blotting, the contamination of plasminogen was found. It is stated that it was about 13% or less of the amount of HRG protein, but it was unclear to what extent the amount of plasmin mixed in the HRG solution should be reduced to improve the stability of HRG.

Mass OverLoad is a phenomenon described as a non-linear region in Langmuir's adsorption isotherm, that is, a change in retention time and a change in peak shape that occurs at a protein concentration exceeding the number of adsorption sites. Non-Patent Document 5 describes Mass OverLoad of proteins in anion exchange chromatography, and Non-Patent Document 6 describes Mass OverLoad of peptides in reverse-phase chromatography. However, there are many contaminating proteins and competitive adsorption. No suggestion has been made regarding the industrial application as a protease separation and removal technique in a complex chromatography step in which a protein can occur.

EBioMedicine DOI: 10.1016 / j.ebiom.2016.06.003 IUBMB Life. 2013 Jun; 65 (6): 550-63. Doi: 10.1002 / iub.1168. Epub 2013 Apr 10. Thromb Res. 1985 Dec 1; 40 (5): 653-61. Biochem J. 2009 Oct 23; 424 (1): 27-37. Doi: 10.1042 / BJ20090794. Journal of Chromatography A, 760 (1997) 89-103 Journal of Chromatography A, In press, corrected proof, Available online 19 December 2019, Article 460789

An object of the present invention is to reduce the amount of plasmin / plasminogen from plasma or recombinant animal cell-derived raw materials inevitably contaminated with plasmin or its precursor, plasminogen, and to contain high-purity and stable HRG. To provide a composition, and thus to provide an HRG formulation that is effective as a pharmaceutical.

As a result of diligent studies to achieve the above object, the present inventors have obtained a load density of a human plasma-derived HRG-containing solution contaminated with plasmin / plasminogen, which causes Mass OverLoad during the process of HRG elution of the column. After contacting with an anion exchange resin so as to adsorb HRG, most of the HRG loaded on the ion exchanger is plasmin / plasminose by shortening the column retention time by Mass OverLoad of HRG in the column elution process. By efficiently eluting with most of the gen retained in the column and collecting the eluate until just before the elution of plasmin / plasminogen begins, the amount of plasmin / plasminogen mixed can be reduced to the total protein. We succeeded in obtaining an HRG solution with a high recovery rate, which was dramatically reduced to the order of 10 ppm to 100 ppm with respect to the amount. Similar results were obtained even when this purification step was carried out at room temperature. Furthermore, by subjecting the obtained eluate to a further chromatography step, it was finally successful in obtaining a high-purity HRG-containing composition in which the amount of plasmin / plasminogen mixed was sufficiently reduced. Various known concentrations of plasmin were added to the obtained high-purity HRG-containing composition, and the mixture was incubated at 4 ° C or 25 ° C for 24 hours to examine the decomposition rate of HRG over time. Since HRG degradation is significantly promoted when plasmin of 650 ppm or more is contained, reducing the amount of plasmin / plasminogen mixed so as to be less than 650 ppm with respect to the total protein amount stabilizes HRG. It became clear that it is important for enhancing sex. Therefore, it was confirmed that the anion exchange resin treatment using Mass OverLoad is an extremely effective means for improving the stability of HRG.
As a result of further research based on these findings, the present inventors have completed the present invention.

That is, the present invention is as follows.
[1] Anion exchange of a human histidine-rich glycoprotein (HRG) -containing solution derived from an animal producing plasmin and / or plasminogen or its cells or tissues under the condition that HRG causes Mass OverLoad. A method for producing a human HRG-containing composition having a reduced amount of plasmin and / or plasminogen, which comprises treating with a resin.
[2] The process is the following step:
(1) A step of contacting and adsorbing an HRG-containing solution with an anion exchange resin so that HRG has a load density that causes Mass OverLoad in the process of column elution, and (2) HRG adsorbed on the anion exchange resin. The method according to [1], which comprises a step of eluting with an eluent under the condition that the plasmin / plasminogen is not completely eluted.
[3] Plasmin and / or plasminogen are reduced so that the increase in HRG degradation rate after incubating the HRG-containing composition at 25 ° C. for 24 hours is 9.2% or less, [1] or The method according to [2].
[4] The method according to [1] or [2], wherein the amount of plasmin and / or plasminogen mixed is less than 650 ppm with respect to the total amount of protein.
[5] The method according to [1] or [2], wherein the amount of plasmin and / or plasminogen mixed is less than 100 ppm with respect to the total amount of protein.
[6] The method according to any one of [1] to [5], wherein the HRG-containing solution is derived from human plasma or a fraction thereof.
[7] The method according to any one of [1] to [5], wherein the HRG-containing solution is derived from a recombinant animal cell, a transgenic non-human animal or a part thereof, or a cell or tissue culture derived from human stem cells. the method of.
[8] A human HRG-containing composition derived from an animal producing plasmin and / or plasminogen or a cell or tissue thereof, wherein the increase in HRG degradation rate after incubation at 25 ° C. for 24 hours is 9.2% or less.
[9] The composition according to [8], wherein the amount of plasmin and / or plasminogen mixed is less than 100 ppm with respect to the total amount of protein.
[10] A pharmaceutical composition containing the composition according to [8] or [9].

According to the present invention, plasmin and / or plasminogen can be sufficiently suppressed even at room temperature by the two steps of immobilized metal affinity chromatography and anion exchange resin treatment as in the conventional case. The amount of the mixture can be effectively reduced. By further purifying, a high-purity HRG-containing composition that is substantially free of plasmin / plasminogen can be obtained.

It is a figure which shows the result of having examined the stability of HRG to which plasmin was added at various concentrations with time at 25 degreeC (A) or 4 degreeC (B). The vertical axis shows the decomposition rate of HRG. Immobilized metal affinity chromatography of a human plasma pool The purified solution was loaded onto an anion exchange resin (MonoQ: manufactured by GE Healthcare) so as to have various HRG load densities, and NaCl linear gradient elution was performed. It is a figure which shows the chromatogram. The elution peak immediately after the start of gradient elution is the HRG monomer. It is a figure which plotted the column retention time at the peak top of the HRG monomer confirmed in FIG. 2 and the column retention time at the start of elution of plasminogen / plasmin confirmed by Western blotting. When MonoQ was used as the anion exchange resin, it was confirmed that when HRG was adsorbed at a load density of 5.1 mg / mL or more, Mass OverLoad of HRG occurred at the time of column elution and the column retention time was shortened. From the chromatogram when the solution after immobilization metal affinity chromatography purification of the plasma pool was loaded into 7 kinds of anion exchange resins so as to have two load densities and gradient elution of NaCl was performed. It is the figure which plotted the column retention time (solid line) at the peak top of the calculated HRG monomer, and the column retention time at the start of elution of plasminogen / plasmin (broken line) calculated from Western blotting. It was shown that the likelihood of mass overload, that is, the difference in column retention time between low and high load densities, differs depending on the resin. It is a figure which shows the conductivity transition in various elution examples using a salt concentration gradient.

The present invention presents a human HRG-containing sample taken from an animal producing plasmin / plasminogen or its cells or tissues, and thus a human HRG-containing sample inevitably contaminated with an amount of plasmin / plasminogen that causes HRG degradation. Provided is a method for producing an HRG-containing composition in which plasmin and / or plasminogen is sufficiently reduced (hereinafter, also referred to as “method of the present invention”), using plasmin and / or plasminogen as a starting material. The method is characterized in that a solution containing the human HRG-containing sample or a processed product (partially purified product) thereof is treated with an anion exchange resin under the condition that HRG causes Mass OverLoad. In addition, in this specification, "human HRG" is simply an amino acid sequence of human HRG (for example, an amino acid sequence registered as RefSeq No. NP_000403 or its gene polymorphism registered in dbSNP) or some amino acids. Not only has a similar amino acid sequence substituted with, but also has a molecular structure substantially the same as that of the endogenous human HRG (ie, at four asparagine residues, undergoes sugar chain modification equivalent to that of human HRG, and five It should be understood as meaning a protein having (retaining an intramolecular disulfide bond).

The human HRG-containing sample used as a starting material for the method of the present invention was collected from an animal producing plasmin / plasminogen or a cell or tissue thereof (hereinafter, these are collectively referred to as "biomaterial"). There is no particular limitation as long as the amount of plasmin / plasminogen that causes the decomposition of HRG is unavoidably mixed. For example, in human body fluids (eg, blood components such as plasma and serum, spinal fluid, lymph, etc.), human plasma fractions and plasma fractionation deposits (eg, cone fractions I, II + III, IV, V). Supernatants and precipitates), recombinant animal cells into which a nucleic acid encoding human HRG has been introduced (eg, HEK293 cells, CHO cells, BHK cells, COS-7 cells, Vero cells, etc.), transgenic non-human animals or theirs. Cultures of some (eg, primary culture of cells or tissues, milk, eggs, etc.), cells or tissues derived from various human stem cells (eg, cells / tissues / organs that highly express HRG such as liver, kidneys, erythrocytes) (Example, culture supernatant) and the like can be mentioned. Recombinant animal cells into which a nucleic acid encoding human HRG has been introduced can be prepared, for example, according to the method described in WO 2013/183494, but any other method known per se is available.

Human HRG-containing samples collected from any of the above biomaterials can be subjected to anion exchange resin treatment as they are, but preferably a partially purified HRG-containing solution using one or more other protein separation techniques. It is preferably subjected to an anion exchange resin treatment. For example, other protein separation techniques include affinity chromatography, cation exchange chromatography, and metal fixation using a substance that specifically binds to HRG (eg, anti-HRG antibody, HRG-binding peptide, RNA aptamer against HRG, etc.) as a ligand. Chemical affinity chromatography (IMAC), size exclusion chromatography, heparin affinity chromatography and the like, but are not limited thereto. IMAC is preferred. The metal ion chelate adsorbent used in IMAC is a transition metal, such as a divalent ion of cobalt, copper, nickel, iron, or a trivalent ion of iron, aluminum, preferably a divalent ion-containing solution of cobalt or nickel. Is prepared by contacting with a matrix to which a ligand such as iminodiacetic acid group, nitrilotriacetic acid group, tris (carboxymethyl) ethylenediamine group and the like is attached and binding to the ligand. The matrix portion of the chelate adsorbent is not particularly limited as long as it is an ordinary insoluble carrier. More specifically, for the IMAC treatment, for example, the methods described in Non-Patent Document 2 and WO 2013/183494 can be used.

In the method of the present invention, HRG columns an untreated human HRG-containing solution taken from a biological material or a human HRG-containing solution partially purified using another protein separation technique (eg, eluate after IMAC treatment). It is brought into contact with an anion exchange resin so as to have a load density that causes Mass OverLoad in the process of elution, and HRG is adsorbed on the ion exchanger. Here, the "load density" means the mass of HRG loaded per volume of the anion exchange resin (adsorbent). For example, when 5 mL of a solution containing HRG at a concentration of 1 mg / mL is loaded on 1 mL of an ion exchanger, the load density becomes a 5 mg / mL adsorbent.

As mentioned above, "Mass OverLoad" is a phenomenon described as a non-linear region in Langmuir's adsorption isotherm, that is, a change in retention time and a change in peak shape that occurs at a protein concentration exceeding the number of adsorption sites. Is. Under normal load density, that is, under conditions where HRG does not cause Mass OverLoad during column elution, plasmin / plasminogen adsorbed on the column is also within the range of salt concentration that elutes most of the HRG adsorbed on the anion exchange resin. Since it is eluted, plasmin / plasminogen cannot be removed efficiently. Therefore, in order to efficiently remove plasmin / plasminogen from the HRG-containing solution by the anion exchange resin treatment, the anion exchange resin treatment is performed under the condition that HRG causes Mass OverLoad at the time of column elution to shorten the column retention time. , It is necessary to elute from the column faster than plasmin / plasminogen. In the present invention, at least in part, HRG is a protein that easily causes Mass OverLoad, and the HRG-containing solution is brought into contact with an anion exchange resin to adsorb HRG so that the load density is such that HRG causes Mass OverLoad when the column is eluted. By doing so, the column retention time of HRG can be shortened, and most of the HRG loaded on the ion exchanger can be efficiently eluted while most of plasmin / plasminogen is retained in the column. It is based on the discovery of.

The "load density that causes Mass OverLoad" is different from the type of anion exchange resin (for example, the type of ligand, the ligand density, the material of the base material, the size of the micropores, etc. Most of the ion exchange resins currently available have a column retention time and plasmin / plus that maximize the elution of HRG monomers when the HRG load density is about 2 mg / mL or less. There is almost no difference from the column retention time when the minogen begins to elute. On the other hand, when the load density of HRG exceeds 2 mg / mL, Mass OverLoad of HRG occurs, and most of HRG elutes by the time when plasmin / plasminogen begins to elute, resulting in plasmin / plasminogen. By recovering the eluate up to just before the start of elution, an HRG solution with a significantly reduced plasmin / plasminogen concentration in the eluate can be obtained with a high recovery rate. Preferably, the load density of the HRG-containing solution is 3 mg / mL adsorbent or higher, more preferably 4 mg / mL adsorbent or higher, even more preferably 5 mg / mL adsorbent or higher, even more preferably 10 mg / mL. Adsorbent or higher (eg, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mg / mL adsorbent or higher). As the load density is increased, the amount of the anion exchange resin used can be reduced and the manufacturing cost can be further reduced. Therefore, the upper limit of the load density is not particularly limited, and the anion exchange resin to be used can be loaded. It may be less than or equal to the maximum value. The load density is, for example, 150 mg / mL adsorbent or less (eg, 150, 100, 75, 50, 39, 38, 37, 36, 35, 34, 33, 32, 31 or 30 mg / mL adsorbent or less). Is. If the load density of HRG becomes too high, the retention time of plasmin / plasminogen may be shortened, but those skilled in the art can efficiently elute HRG within the range where plasmin / plasminogen does not start elution. Dissolution conditions can be set as appropriate.

The anion exchange resin used in the method of the present invention can sufficiently reduce the amount of plasmin / plasminogen mixed in the eluate as long as HRG can cause Mass OverLoad by selecting appropriate elution conditions. As long as it can be obtained, any anion exchange resin known per se can be used. Here, "the amount of contamination is sufficiently reduced" means that the amount of plasmin / plasminogen mixed in the eluent increases the HRG decomposition rate after incubating the eluate at 25 ° C. for 24 hours by 9.2%. The amount of plasmin / plasminogen mixed in the HRG-containing solution obtained by the following or at least a simple subsequent addition step is reduced to such an extent that the condition for increasing the HRG decomposition rate after the incubation is satisfied. To say that you are. Here, the "HRG decomposition rate" can be measured by the method described in "Measurement of HRG decomposition rate" in Examples described later, and the "increase in HRG decomposition rate" is the HRG decomposition rate at the start of incubation and 24. It is defined as the difference in HRG degradation rate after time incubation.

More specifically, as a guideline for sufficiently reducing the amount of plasmin / plasminogen mixed in the anion exchange resin treatment, the amount of plasmin / plasminogen mixed in the eluate after the treatment is the total protein amount. On the other hand, it is at least less than 650 ppm. At least the amount of plasmin / plasminogen in the final purified product should be less than 650 ppm, since the storage stability of HRG is significantly reduced when the amount of contamination is 650 ppm or more. Preferably, in order to reduce the increase in HRG degradation rate after incubating the eluate at 25 ° C. for 24 hours to 9.2% or less, the amount of plasmin / plasminogen mixed is 500 ppm or less, more preferably 400 ppm. Below, it is more preferably 300 pm or less, even more preferably 200 ppm or less, and particularly preferably 100 ppm or less (eg, 100, 90, 80, 70, 65 ppm or less). As shown in the examples below, if an anion exchange resin that easily causes Mass OverLoad of HRG is selected, any condition (eg, load density, eluate concentration (conductivity), eluate pH, treatment temperature) Even below, the amount of plasmin / plasminogen mixed in the eluate can be reduced to less than 650 ppm, and depending on the conditions, it can be reduced to 100 ppm or less (for example, 65 ppm or less). In addition, even if the amount of plasmin / plasminogen mixed after the anion exchange resin treatment exceeds 100 ppm, other simple purification steps (eg, ultrafiltration, gel filtration chromatography, etc.) are performed thereafter. By adding, the amount of plasmin / plasminogen mixed in the final purified product can be reduced to 100 ppm or less (for example, 65 ppm or less).

A wide variety of anion exchange resins for industrial use are currently on the market, but as described above, the ligand type, ligand density, base material material, micropore size, etc. are different for each resin. Therefore, the adsorption isotherm of HRG and anion exchange resin also differs depending on the resin. Therefore, in order to construct a highly robust manufacturing process that can reduce the amount of plasmin / plasminogen mixed in the eluate to at least less than 650 ppm with respect to the total protein amount, mass overload of HRG occurs. It is desirable to select an easy ion exchange resin. Examples of such an ion exchange resin include a strong anion exchange resin and a weak anion exchange resin, and for example, polystyrene-crosslinked divinylbenzene is used in the gel matrix as a strong anion exchange resin having a quaternary amino group. GE Healthcare Mono Q, SOURCE 15Q, SOURCE 30Q, Thermo Fisher Scientific POROS ™ HQ, GE Healthcare Q Sepharose Fast Flow, Q Sepharose High Performance, Capto using highly crosslinked agarose in the gel matrix Q ImpRes, BIO-RAD Macro-Prep® High Q using methacrylate for the gel matrix, Pall Corporation Mustang® Q, 3rd grade amino using PES membrane for the matrix Examples of the weak anion exchange resin having a group include DEAE Sepharose Fast Flow and Capto DEAE manufactured by GE Healthcare, which use highly crosslinked agarose in the gel matrix, but the present invention is not limited to this. The ion exchange resin is mainly granular, but may have a fibrous or film-like morphology.

The elution of HRG retained in the anion exchange resin can be carried out by passing the eluent through the anion exchange resin under the condition that plasmin / plasminogen is not completely eluted from the column. Here, the "condition that does not completely elute from the column" means a condition that the amount of plasmin / plasminogen mixed in the eluate after treatment is at least 650 ppm with respect to the total amount of protein. do. In efficient plasmin / plasminogen removal by Mass OverLoad of HRG, the salt concentration (conductivity) of the eluent satisfying the above conditions, the pH of the eluent, the treatment temperature, etc. vary depending on the type of anion exchange resin. However, when gradient elution is performed using the anion exchange resins listed as examples of the usable anion exchange resins, elution of plasmin / plasminogen is started if the pH and temperature conditions described below are within the range. The column retention time at the time was sufficiently higher than the column retention time at the time when the HRG monomer was maximally eluted within the above preferable load density range, and as the load density increased, the plasmin / plasminogen The column retention time of HRG does not change significantly, whereas the elution peak of HRG shifts to an earlier time. Here, "plasmin / plasminogen starts elution" means that plasmin / plasminogen is above the detection limit in the method described in "Calculation of column retention time" in Examples described later. .. By recovering HRG eluted earlier than plasmin / plasminogen by causing Mass OverLoad, an HRG solution with a significantly reduced plasmin / plasminogen concentration in the eluate can be obtained with a high recovery rate. Can be done. For example, in the test results shown in FIG. 3 using Mono Q manufactured by GE Healthcare as an anion exchange resin, when the load density of HRG is about 5 to about 15 mg / mL, plasmin / plasminogen is about 66 minutes. Elution is started at the time when the column retention time is reached, whereas the HRG has a column retention time at the peak top of elution of about 54 to about 64 in the load density range of about 5 to about 19 mg / mL. The width is a minute, and it becomes shorter depending on the load density. Therefore, for example, by recovering the column eluate within the range of the column holding time of about 54 to about 66 minutes, an eluate in which the amount of plasmin / plasminogen mixed is sufficiently reduced can be obtained. Even when other anion exchange resins are used, for example, the load density of HRG as shown in FIG. 4, the column retention time at the top of the elution peak of HRG, and the time when plasmin / plasminogen starts elution. It is possible to use an eluate in which plasmin / plasminogen is not completely eluted from the column and HRG is efficiently eluted by investigating the relationship with the column retention time.

An eluent having a constant salt concentration may be used throughout the elution step (stepwise elution method), but in a preferred embodiment, a gradient elution method can also be used. As the gradient elution method, for example, a non-linear gradient (curve gradient) elution method, a linear gradient elution method, a multi-segment linear gradient elution method, or a combination thereof can be used. As used herein, "non-linear gradient" also includes curve gradients performed under a stepwise program. The eluent gradient is, for example, 0 (ie, stepwise) to 1000 column volumes (CV) with conductivity ranging from 0 mS / cm to about 80 mS / cm at a linear velocity of 5000 cm / hour or less. It can be raised by the amount of water, and can be appropriately set according to the type of salt of the eluent and the like. In one embodiment, for example, when increasing the NaCl concentration from 20 mM to 1 M in 50 mM Tris buffer, the linear velocity is 150 cm / hour and the conductivity is from about 4 mS / cm to about 80 mS / cm. Within the range, it can be increased over 0-50 CV.
Elution fractions at each salt concentration can be fractionated to collect and collect fractions in which HRG elutes and plasmin / plasminogen does not elute.

The pH of the eluate varies depending on the type of anion exchange resin used, and can be appropriately selected, for example, in the range of pH 6 to 10, preferably pH 7 to 9.

The temperature conditions during the anion exchange resin treatment are not particularly limited as long as the amount of plasmin / plasminogen mixed in the eluate is sufficiently reduced, and are, for example, 0 to 60 ° C, preferably 4 to 40 ° C. More preferably, it can be appropriately selected within the range of 4 to 30 ° C.

As described above, HRG can be efficiently recovered while sufficiently reducing the amount of plasmin / plasminogen mixed. The recovery rate of HRG in the anion exchange resin treatment using Mass OverLoad is 50% or more, preferably 60% or more, more preferably 70% or more, further preferably 80% or more, still more preferably 90% or more, particularly preferably. Can be cited as 95%. The lower limit of the recovery rate is not particularly limited, but may be, for example, 99% or less, 95% or less, 90% or less.

The eluate obtained as described above can be subjected to a further purification step, if necessary. For example, such purification steps include, but are not limited to, gel filtration chromatography, cation exchange chromatography, diafiltration, virus filtration and the like.

In the human HRG-containing composition thus obtained, the amount of plasmin / plasminogen mixed is sufficiently reduced. Therefore, the present invention is also referred to as an HRG-containing composition derived from a biological sample producing plasmin / plasminogen, in which the amount of plasmin / plasminogen mixed is sufficiently reduced (hereinafter, also referred to as "composition of the present invention"). .)I will provide a. Here, "the amount of contamination is sufficiently reduced" is synonymous with the above, but preferably, the increase in HRG decomposition rate after incubation at 25 ° C. for 24 hours is 9.2% or less, and plasmin / plasminose to some extent. It means that the amount of gen mixed in is reduced. More specifically, the amount of plasmin / plasminogen mixed is at least less than 650 ppm, preferably 500 ppm or less, more preferably 400 ppm or less, still more preferably 300 ppm or less, even more preferably 200 ppm or less. Particularly preferably, it is 100 ppm or less (eg, 100, 90, 80, 70, 65 ppm or less). If the amount of plasmin / plasminogen mixed can be reduced to 100 ppm or less (for example, 65 ppm or less), the degradation of HRG by the protease can be almost ignored.
According to the method of the present invention, IMAC can be obtained by appropriately selecting the type of anion exchange resin, the load density of HRG and the elution conditions (salt concentration of eluent, pH of eluent, treatment temperature, linear velocity, etc.). The amount of plasmin / plasminogen mixed can be reduced to 100 ppm or less (for example, 65 ppm or less) only by the two steps of the anion exchange resin treatment. Even if these steps alone cannot reduce the amount to 100 ppm or less (for example, 65 ppm or less), it can be easily achieved to 100 ppm or less (for example, 65 ppm or less) by further adding the above-mentioned other purification steps. be able to.

The compositions of the present invention are high purity (eg, relative to total protein) of human HRG proteins (as described above, "human HRGs" herein have substantially the same molecular structure as endogenous human HRGs). The proportion of HRG protein is 80% or more, preferably 90% or more, more preferably 95.0 to 99.9%), and the amount of plasmin / plasminogen mixed is such that the degradation of HRG by the protease is negligible. Therefore, it is extremely useful for producing a human HRG-containing pharmaceutical composition having long-term storage stability.

The compositions of the present invention can be used for therapeutic and / or prophylactic applications of various diseases based on the known pharmacological activity of human HRG. Such diseases include, for example, diseases caused by neutrophil activation and / or inflammatory diseases associated with neutrophil activation (eg, sepsis, but also WO 2013 / 183494 and various diseases described in JP-A-2016-53022 are included), cytokine storms, systemic inflammatory response syndrome (SIRS) (see WO 2016/104436), and the like.

The composition of the present invention may be administered by itself, or may be administered as a pharmaceutical composition in an appropriate shape and form together with other pharmaceutically acceptable additives. The pharmaceutical composition used for administration may contain one or more selected from the composition of the present invention and a pharmacologically acceptable carrier, diluent or excipient. Such pharmaceutical compositions are provided in dosage forms suitable for oral or parenteral administration.

As the composition for parenteral administration, for example, injections, suppositories, intranasal administrations and the like are used, and the injections are intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, drip injection and the like. Dosage forms such as agents may be included. Such injections can be prepared according to known methods. As a method for preparing an injection, for example, HRG can be prepared by dispersing, dissolving, suspending, mixing, kneading, emulsifying or emulsifying in a sterile aqueous liquid or an oily liquid usually used for injections. As the aqueous solution for injection, for example, a physiological saline solution, an isotonic solution containing monosaccharides (glucose, fructose, etc.), disaccharides (maltos, trehalose, etc.) and other adjuvants are used, and appropriate dissolution is used. Auxiliary agents such as alcohols (eg ethanol), polyalcohols (eg propylene glycol, polyethylene glycol), nonionic surfactants [eg polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)] May be used in combination with such. As the oily liquid, for example, sesame oil, soybean oil and the like are used, and benzyl benzoate, benzyl alcohol and the like may be used in combination as a solubilizing agent. The prepared injection solution is preferably filled in a suitable ampoule. In addition, the injection may be a time-dissolving type preparation in which the above liquid preparation is freeze-dried by a conventional method and then dissolved in an appropriate solvent (for example, distilled water for injection) at the time of use. ..
The suppository used for rectal administration may be prepared by blending or mixing the composition of the present invention with a conventional suppository base.

Compositions for oral administration include solid or liquid dosage forms, specifically tablets (including sugar-coated tablets and film-coated tablets), pills, granules, powders, powders and capsules (including soft capsules). , Syrups, emulsions, suspensions, poultices and the like. Such a composition is produced by a known method and may contain one or more of carriers, diluents or excipients commonly used in the pharmaceutical field. Examples of carriers and excipients for tablets include monosaccharides (dextrin, galactose, fructose, etc.), disaccharides (lactose, maltose, trehalose, etc.), oligosaccharides (fructo-oligosaccharide, isomaltooligosaccharide, galactooligosaccharide, etc.), Polysaccharides (starch, cellulose, dextrin, isomaltodextrin, etc.), sugar alcohols (mannitol, xylitol, maltose, etc.), magnesium stearate, etc. are used.

It is convenient that the parenteral or oral pharmaceutical composition described above is prepared in a dosage form of a dosage unit suitable for the dose of the active ingredient. Dosage forms of such dosage units include, for example, tablets, pills, capsules, injections (ampoules), and suppositories. Human HRG is typically contained at 0.1-5000 mg per dosage form, particularly preferably 5-1000 mg for injectables and 10-2500 mg for other dosage forms.

The dose of the above-mentioned drug containing the composition of the present invention varies depending on the administration subject and its gender, age, target disease, symptom, administration frequency, administration route, etc., but for example, human HRG is usually used as a single dose. About 0.0001 to 200 mg / kg body weight can be intravenously administered once or multiple times a day to several months. In the case of other parenteral administration and oral administration, an equivalent amount can be administered. If the symptoms are particularly severe, the dose may be increased according to the symptoms.

The above-mentioned pharmaceutical composition may contain other active ingredients in addition to the composition of the present invention as long as the combination with human HRG does not cause an unfavorable interaction. Examples of the active ingredient include compounds having a known therapeutic effect on the disease, depending on the target disease, and those skilled in the art can easily select such a compound.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

[Method]
The following assay method was used to obtain the data used in the present invention.
(Measurement of protein concentration)
The protein concentration was measured by the Bradford method using the Pierce ™ Coomassie (Bradford) Protein Assay Kit manufactured by Thermo Fisher Scientific.

(Measurement of HRG decomposition rate)
The decomposition rate (%) of HRG was calculated from the data of SDS-PAGE, InstantBlue (registered trademark) Protein Stain staining. NuPAGE 4-12% Bis-Tris Gel manufactured by Thermo Fisher Scientific was used as the electrophoresis gel, and the XCell SureLook ™ minicell electrophoresis system was used as the electrophoresis tank. An SDS-treated HRG protein solution was applied to the gel in a reduced state at 2 μg / lane, and electrophoresis was performed at 200 V for 42 minutes. After staining with Expedeon's Instant Blue (registered trademark) Protein Stain, the decomposition rate (%) was calculated from the band Density of the image captured using the BIO-RAD's densitometer GS-900.

(Calculation of column retention time)
Western blotting was used as a sample for the column retention time of plasminogen and plasmin monomers using anion exchange chromatography elution fractions. Anti-Plasminogen antibody (Cat: ab154560) manufactured by abcam was used as the primary antibody, and Goat Anti-Rabbit IgG H & L (HRP) (Cat: ab205718) manufactured by abcam was used as the secondary antibody. Detection was performed using ECL Prime Western Blotting Detection Reagent manufactured by GE Healthcare, and elution initiation fraction of plasminogen and plasmin monomer was identified using Amasham imager 600 manufactured by GE Healthcare. The starting point of fractionation of the specified fraction was defined as the column retention time at the starting point of elution of plasminogen and plasmin monomers.

(Quantification of HRG monomer)
Western blotting was used for the quantification of HRG monomer. Self-prepared human plasma-derived HRG monomer for standard protein, Human HPRG Antibody (Cat: AF1869) manufactured by R & D Systems for primary antibody, and abcam Donkey Anti-Goat IgG H & L (HRP) preadsorbed (Cat) for secondary antibody. : ab97120) was used. Detection was performed using GE's ECL Prime Western Blotting Detection Reagent, and quantification was performed from the calibration curve of self-prepared human plasma-derived HRG monomers prepared using GE's Amasham imager 600.

(Quantification of plasminogen / plasmin)
The quantification of plasminogen and plasmin was carried out by the ELISA method. A human Plasminogen ELISA Kit (PLG) (Cat: ab108893) manufactured by abcam was used.

[Example 1]
Anion exchange resin using a solution (total protein concentration 3.0 mg / mL, HRG monomer concentration 1.7 mg / mL, plasminogen / plasmin concentration 60.6 μg / mL) after immobilization metal affinity chromatography purification of a human plasma pool as a sample. (Mono Q: manufactured by GE Healthcare) Load under 6 conditions where the sample load is 1, 3, 5, 7, 9, or 11 mL for 1 mL, and the linear velocity is 150 cm / hour and the gradient volume is 25 mL. A linear gradient elution with a salt concentration of 20 mM to 510 mM was carried out. The result is shown in FIG. The elution peak immediately after the start of gradient elution in the chromatogram was the HRG monomer, and as the load density was increased, the column retention time and peak shape change peculiar to Mass OverLoad were observed. This indicates that HRG is prone to Mass OverLoad.

The column retention time at the peak top of the HRG monomer confirmed in FIG. 2 and the column retention time at the elution initiation fraction of plasminogen and plasmin confirmed by Western blotting were plotted. The result is shown in FIG. When 5.1 mg or more of HRG is adsorbed per 1 mL of anion exchange resin, Mass OverLoad of HRG occurs at the time of column elution, and by using this Mass OverLoad, plasmin, which is a decomposing substance of HRG, and plasmin, which is a precursor, are used. The gen content could be reduced efficiently.

[Example 2]
Immobilized metal affinity chromatography of a human plasma pool Using a purified solution (total protein concentration 3.0 mg / mL, HRG monomer concentration 1.4 mg / mL, plasmin / plasminogen concentration 55.0 μg / mL) as a sample, manufactured by GE Healthcare. Anion exchange resin SOURCE 30Q, Capto Q ImpRes, Q Sepharose High Performance manufactured by GE Healthcare so that the column volume is 1 mL (height 1 cm) on the anion exchange columns Mono Q 5/50 GL and Tricorn 5/50 column. , Thermo Fisher Scientific anion exchange resin POROS (trademark) HQ, POROS (trademark) XQ, TOYOPEARL SuperQ-650S manufactured by Toso Co., Ltd. Was loaded, and the HRG fraction was eluted with a salt concentration linear gradient with a NaCl concentration of 20 mM to 1 M at a linear velocity of 150 cm / hour. FIG. 4 shows the column retention time at the top of the elution peak of the HRG monomer in the chromatogram, and the column retention time at the start of elution of the plasminogen and plasmin monomers calculated by Western blotting. In addition, the total protein concentration of the eluted fraction was measured by the Bradford method, and the plasminogen / plasmin protein concentration was measured by the ELISA method, and the percentage of plasminogen / plasmin to the total protein amount was calculated. The results are shown in Table 1. TOYOPEARL SuperQ-650S, POROS ™ XQ was confirmed to be an anion exchange resin that does not easily cause Mass OverLoad in the data shown in Fig. 4, but it was actually an HRG solution purified using these anion exchange resins. It was confirmed that the plasmin and plasminogen contained therein were 5000 ppm or more with respect to the total protein amount, and the removal efficiency was low (Table 1). On the other hand, it was confirmed that Mono Q and Capto Q ImpRes, which are anion exchange resins that easily cause Mass OverLoad, can efficiently remove plasmin and plasminogen by using Mass OverLoad (Table 1).

[Example 3]
Immobilized metal affinity chromatography of a human plasma pool Using a purified solution (total protein concentration 3.2 mg / mL, HRG monomer concentration 1.4 mg / mL, plasmin / plasminogen concentration 52.2 μg / mL) as a sample, manufactured by GE Healthcare. Anion exchange column Mono Q 5/50 Load the sample on the GL column under two conditions with a sample loading of 5.5 mL or 11 mL, and under the conditions of pH 7.0 to 9.0, 4 ° C or 25 ° C, and linear velocity of 150 cm / hour. The HRG fraction was eluted. For elution, a non-linear gradient (curve gradient) elution method using a salt concentration gradient and a linear gradient elution method were used. A curve gradient under stepwise program control was used as the non-linear gradient. The total protein concentration of the eluted fraction was measured by the Bradford method, and the plasminogen / plasmin protein concentration was measured by the ELISA method, and the percentage of plasminogen / plasmin to the total protein amount was calculated. The results are shown in Table 2.

When the non-linear gradient elution method and the linear gradient elution method are used, plasmin and plasminogen in the purified HRG solution are 650 ppm with respect to the total protein amount at any pH of 7.0 to 9.0. The amount of contamination could be reduced to 20 ppm or less depending on the conditions. It was also confirmed that plasmin and plasminogen can be efficiently removed by using Mass OverLoad not only under 4 ° C conditions but also under 25 ° C conditions. The transition of conductivity in some of the above elution methods is shown in FIG.

[Example 4]
Thermo Fisher Scientific Co., Ltd. used a solution of human plasma pool after immobilization metal affinity chromatography purification (total protein concentration 3.0 mg / mL, HRG monomer concentration 1.7 mg / mL, plasmin / plasminogen concentration 60.6 μg / mL) as a sample. Anion exchange resin POROS ™ HQ packed with a 20 cm high column loaded with 11 mL of sample per 1 mL of anion exchange resin and at linear speeds of 400 cm / hour and 800 cm / hour, NaCl concentration. The HRG fraction was eluted with a linear gradient of 20 mM to 265 mM. The eluted fractions were concentrated by ultrafiltration and further purified using a GE gel filtration chromatography column HiLoad 26/600 Superdex 200 prep grade equilibrated with PBS (-). The total protein concentration after each purification step was measured by the Bradford method, and the plasminogen / plasmin protein concentration was measured by the ELISA method, and the percentage of plasminogen / plasmin to the total protein amount was calculated. The results are shown in Tables 3 and 4.

It was confirmed that even under the conditions of this example, the plasmin and plasminogen contents can be efficiently removed so as to be 160 ppm or less with respect to the total protein amount by using Mass OverLoad in the anion exchange chromatography step. (Table 3). Furthermore, it was confirmed that plasmin and plasminogen could be further removed to 65 ppm or less based on the total protein amount after the gel filtration chromatography step (Table 4).

[Test Example 1]
Human HRG obtained by performing purification using immobilized metal affinity chromatography, anion exchange chromatography, and gel filtration chromatography using a plasma fractionation intermediate rich in HRG obtained by the corn fractionation method as a raw material. The storage stability of the contained composition was tested to evaluate the effectiveness of the plasmin / plasminogen removal effect. To the HRG-containing composition having a total protein concentration of 2 mg / mL containing 77 ppm plasmin / plasminogen, 65 ppm, 650 ppm or 6500 ppm plasmin was added and incubated at 4 ° C or 25 ° C for 24 hours. The decomposition rate of HRG was measured over time. The results are shown in FIG. Under all temperature conditions, the storage stability of HRG was significantly reduced when the plasmin addition concentration was 650 ppm or more relative to the total protein content. Therefore, it was clarified that it is important to reduce the amount of plasmin / plasminogen mixed so as to be 650 ppm or less with respect to the total protein amount in order to improve the stability of HRG.

Although the present invention has been described with emphasis on preferred embodiments, it will be apparent to those skilled in the art that preferred embodiments can be modified. The present invention is intended that the invention can be practiced in ways other than those described in detail herein. For example, in the isothermal adsorption type linear region similar to HRG, other human proteins that are difficult to separate from plasmin / plasminogen (for example, plasma proteins such as blood coagulation factors and animal cells such as monoclonal antibodies are used. For industrially recombinantly produced human protein), a solution containing the protein and plasmin / plasminogen was subjected to anion exchange resin treatment using Mass OverLoad to obtain plasmin / plasminogen. A target protein-containing composition with a reduced amount of contamination can be prepared. Furthermore, even if the contaminating protein is a protease other than plasmin / plasminogen, the method of the present invention can be applied even when the target protein can be separated from the protease by Mass OverLoad.
Accordingly, the present invention includes all modifications contained within the spirit and scope of the appended claims.

The contents of all publications, including the patents and patent application specifications mentioned herein, are incorporated herein by reference in their entirety to the same extent as expressly stated. ..

According to the present invention, plasmin and / or plasminogen can be sufficiently suppressed even at room temperature by the two steps of immobilized metal affinity chromatography and anion exchange resin treatment as in the conventional case. It is possible to reduce the amount of the mixture. Therefore, the human HRG-containing composition obtained by the present invention is extremely useful in the production of a pharmaceutical preparation having excellent long-term storage stability of HRG as compared with the conventional human HRG-containing composition.

Claims (10)

A human histidine-rich glycoprotein (HRG) -containing solution from an animal or cell or tissue that produces plasmin and / or plasminogen in an anion exchange resin under conditions where HRG causes Mass OverLoad. A method for producing a human HRG-containing composition having a reduced amount of plasmin and / or plasminogen, which comprises treating. The process is the following step:
(1) A step of contacting and adsorbing an HRG-containing solution with an anion exchange resin so that HRG has a load density that causes Mass OverLoad in the process of column elution, and (2) HRG adsorbed on the anion exchange resin. The method according to claim 1, further comprising a step of eluting with an eluent under the condition that the plasmin / plasminogen is not completely eluted. Claim 1 or 2, wherein the plasmin and / or plasminogen is reduced such that the increase in HRG degradation rate after incubating the HRG-containing composition at 25 ° C. for 24 hours is 9.2% or less. the method of. The method according to claim 1 or 2, wherein the amount of plasmin and / or plasminogen mixed is less than 650 ppm with respect to the total amount of protein. The method according to claim 1 or 2, wherein the amount of plasmin and / or plasminogen mixed is less than 100 ppm with respect to the total amount of protein. The method according to any one of claims 1 to 5, wherein the HRG-containing solution is derived from human plasma or a fraction thereof. The method according to any one of claims 1 to 5, wherein the HRG-containing solution is derived from a recombinant animal cell, a transgenic non-human animal or a part thereof, or a cell or tissue culture derived from human stem cells. A human HRG-containing composition derived from an animal or cell or tissue producing plasmin and / or plasminogen, wherein the increase in HRG degradation rate after incubation at 25 ° C. for 24 hours is 9.2% or less. The composition according to claim 8, wherein the amount of plasmin and / or plasminogen mixed is less than 100 ppm with respect to the total amount of protein. A pharmaceutical composition containing the composition according to claim 8 or 9.

Images