JP7230115B2 - Post-translationally modified α-thrombin - Google Patents

Description

Methods are provided that allow analysis and quantification of α-thrombin, homogeneously glycosylated α-thrombin, and/or thrombin-degrading polypeptides in liquid protein solutions. In particular, analytical and quantitative methods are provided that employ a single chromatographic step. Also provided are methods that allow efficient and reliable purification of α-thrombin and/or homogeneously glucosylated α-thrombin without thrombin-degrading polypeptides. The present invention can also be used to purify and/or quantify β-thrombin.

Thrombin is a serine protease widely used in clinical applications in several commercial products. It is a common component of surgical dressings and has been used in combination with fibrinogen and other proteins in hemostatic systems such as fibrin glues, adhesives and sealants. Fibrin sealants typically contain a fibrinogen component and a thrombin component. When both components are mixed (e.g. when applied to a bleeding wound or surgical incision), thrombin cleaves the fibrinogen peptides from fibrinogen, thus allowing the latter to produce an insoluble fibrin polymer/sealant. do.

Concentrated (eg, greater than 500 IU/mL) purified thrombin in aqueous liquid form may exhibit decreased activity during long-term storage, primarily as a result of autolysis. Evaluation of thrombin degradation is therefore an essential physicochemical analytical tool for determining thrombin stability.

Mammalian α-thrombin is composed of two disulfide-linked polypeptide chains, A and B. The B chain is post-translationally modified (eg, by glycosylation) to exhibit the proteolytic activity of thrombin on fibrinogen and other proteins. α-Thrombin autolyzes to β-thrombin and γ-thrombin polypeptide derivatives, which can be partially identified by gel electrophoresis and Western blot.

Thrombin autolysis is a major challenge in the production and storage of thrombin, especially at high concentrations. Methods known in the art for identifying thrombin-degrading polypeptides (β-thrombin and γ-thrombin derivatives) provide insufficient separation (denaturation separation) between thrombin and its degrading polypeptides, and / or it is labor intensive. Therefore, quantification is not accurate and/or impossible.

Background art includes:
Boissel JP et al. "Covalent structures of beta and gamma automatic derivatives of human alpha-thrombin". J Biol Chem. 1984 May 10;259(9):5691-5697, Chang JY. "The structures and proteolytic specificities of autolysed human thrombin". BiochemJ. 1986 Dec 15;240(3):797-802, Karlsson G.; "Analysis of human alpha-thrombin by hydrophobic interaction high-performance liquid chromatography". Protein Expr Purif. 2003 Jan;27(1):171-174, EP 0443724 and WO 2004/103519.

Boissel et al. describe the use of CEX-HPLC followed by analysis by RP-HPLC to separate different thrombin-degrading polypeptides. Chang describes HPLC analysis of pure thrombin fractions separated by SEC chromatography and further analyzed using RP-HPLC. The above method has the disadvantage of requiring at least two separation steps to quantify thrombin and separate it from other proteins.

Karlsson describes hydrophobic interaction chromatography (HIC) to separate thrombin degradation products.

EP 0443724 discloses a method for preparing virus-safe thrombin, but this method is denaturing and does not show separation between different thrombin degradation products or between different α-thrombin post-translational variants. .

WO2004/103519 discloses a method for separating charged molecules, such as proteins, according to their isoelectric point (pI), wherein the system and buffer employed to isolate charged molecules including compositions.

To analytical methods for quantifying α-thrombin or β-thrombin and methods for purifying active, native α-thrombin or β-thrombin from protein solutions, which overcome the drawbacks of the above techniques. , there is still an unmet need.

A one-step chromatographic method for the quantification of α-thrombin and/or homogeneous post-translationally modified α-thrombin in solution is provided, the solution containing α-thrombin and α-thrombin degrading polypeptides (β- thrombin and/or γ-thrombin polypeptides), post-translationally modified α-thrombin species, or at least one of another protein.

Methods for purifying α-thrombin from protein solutions by providing good separation of native, e.g. undegraded, functionally active α-thrombin and/or homogeneous post-translationally modified α-thrombin is also provided, the solution comprising α-thrombin and at least one of α-thrombin-degrading polypeptides (β-thrombin and/or γ-thrombin polypeptides), post-translationally modified α-thrombin species, or another protein Including one.

Also provided are methods for purifying homogeneous α-thrombin glycoforms from solutions containing heterogeneously glycosylated α-thrombin species.

To purify and/or quantify β-thrombin in a solution containing β-thrombin and at least one of α-thrombin, such as a post-translationally modified α-thrombin species, γ-thrombin, or another protein A method is also provided.

As used herein, the term "at least one of" is both conjunctive and disjunctive in function. For example, the phrase "at least one of A, B, or C" means A only, B only, C only, A and B together, A and C together, B and C together, or A, B, and It means "together with C".

The homogeneous post-translationally modified α-thrombin can be homogeneously glycosylated α-thrombin, or homogeneously glycosylated and homogeneously sialylated α-thrombin.

Homogeneous α-thrombin glycoforms according to the present application may be “homogeneously glycosylated α-thrombin” or “homogeneously glycosylated and homogeneously sialylated α-thrombin species”.

Typically, glycoforms are isoforms of a protein that differ only with respect to the number and/or type of glycans or polysaccharides attached. Glycoproteins are often composed of several different glycans and vary in the attached sugars.

The terms “glycan” and “polysaccharide” often refer to carbohydrate moieties of complex carbohydrates such as glycoproteins. Glycans can be homo- or heteropolymers of monosaccharide residues and can be linear or branched. Glycans may carry sugars with or without a negative charge.

The method includes contacting the solution with an anion exchanger. The method provides reliable and reproducible performance, provides highly purified and active α-thrombin and/or homogeneous post-translationally modified α-thrombin, and α-thrombin, homogeneous post-translationally modified Allows accurate quantification of α-thrombin and/or its degradation polypeptides. The method also allows quantification and/or purification of β-thrombin alone.

In one aspect, a method is provided for purifying α-thrombin from a solution comprising α-thrombin and at least one of an α-thrombin-degrading polypeptide or another protein, the method comprising: 1-a solution; with an anion exchanger and α-thrombin is purified by anion exchange chromatography (AEX) using 2-differential elution conditions to convert α-thrombin-degrading polypeptides (e.g., β-thrombin and/or from γ-thrombin polypeptides) and/or from at least one of the other proteins; collecting the 3-α-thrombin fraction, thereby obtaining purified α-thrombin; including.

In some embodiments, the method uses differential elution conditions to extract α-thrombin from α-thrombin-degrading polypeptides (eg, β-thrombin and/or β-thrombin) by anion exchange chromatography (AEX). or γ-thrombin polypeptide) and at least one of another protein.

In one embodiment, α-thrombin is from human blood or plasma sources. In another embodiment, α-thrombin is from a recombinant source.

As used herein, the term "separation" typically refers to isolating a specific compound from a solution containing the specific compound and another compound.

In one aspect, a method is provided for purifying homogeneously glycosylated α-thrombin from a solution comprising heterogeneously glycosylated α-thrombin, the method comprising: 1--a solution is purified by an anion exchanger; and 2 - separating homogeneously glycosylated α-thrombin from heteroglycosylated α-thrombin by anion exchange chromatography (AEX) using differential elution conditions 3- Recovering the homogeneously glycosylated α-thrombin fraction, thereby obtaining purified homogeneously glycosylated α-thrombin.

In one aspect, for purifying homogeneously glycosylated α-thrombin from a solution comprising heterogeneously glycosylated α-thrombin and at least one of an α-thrombin degrading polypeptide or another protein is provided. In another aspect, the homogeneously glycosylated α-thrombin is purified from a solution comprising at least one of the heterogeneously glycosylated α-thrombin, the α-thrombin degrading polypeptide, or another protein. A method is provided for The method comprises the steps of 1 - contacting the solution with an anion exchanger and 2 - isolating homogeneously glycosylated α-thrombin by anion exchange chromatography (AEX) using differential elution conditions. 3- Recovering the homogeneously glycosylated α-thrombin fraction, thereby obtaining purified homogeneously glycosylated α-thrombin.

In some embodiments, after the step 1 contacting step, a washing step is performed using an isocratic buffer/solution.

In one embodiment of the invention, the method comprises the steps of loading the anion exchanger with a thrombin-containing solution, washing with an isocratic solution, discarding the washed fraction, and applying an inert such as a pH gradient. Eluting the desired α-thrombin fraction using an isocratic solution. The use of isocratic solutions is typically associated with the use of mobile phases of constant composition in liquid chromatography.

The "desired α-thrombin fraction" typically includes, for example, homogeneous post-translationally modified α-thrombin, such as homogeneously glycosylated α-thrombin or homogeneously glycosylated and homogeneously sialylated It refers to any α-thrombin present in a solution intended for purification and/or quantification, including α-thrombin that has been purified.

In one aspect, a method is provided for purifying homogeneous α-thrombin glycoforms from a solution comprising heterogeneously glycosylated α-thrombin species, the method comprising:
contacting the solution with an anion exchanger;
separating homogeneous α-thrombin glycoforms from heterogeneous species by anion exchange chromatography using differential elution conditions;
recovering a homogenous α-thrombin glycoform fraction,
thereby obtaining a purified homogeneous α-thrombin glycoform.

In one embodiment, the method also includes quantifying the purified homogeneous α-thrombin glycoform.

In another aspect, a one step or single step chromatography method for quantifying α-thrombin in a solution comprising α-thrombin and at least one of an α-thrombin degrading polypeptide or another protein is provided. There is provided a method comprising separating α-thrombin from at least one of an α-thrombin degrading polypeptide or another protein by anion exchange chromatography by differential elution conditions; Collecting the thrombin fraction and quantifying α-thrombin. In another aspect, a one step or single step chromatography method for quantifying α-thrombin in a solution comprising α-thrombin and at least one of an α-thrombin degrading polypeptide or another protein is provided. Provided is a method for converting α-thrombin into α-thrombin-degrading polypeptides or other collecting the α-thrombin fraction; and quantifying α-thrombin.

In some embodiments, the α-thrombin is from a mammalian, eg, human or porcine plasma source, or a recombinant protein.

The chromatographic methods disclosed herein can be performed using any technique known to those of ordinary skill in the art. For example, high performance liquid chromatography devices, fast protein liquid chromatography (FPLC), and/or stand-alone columns with or without attached detectors may be employed.

In one embodiment, an anion exchange high performance liquid chromatography method is used. High performance liquid chromatography (HPLC; also called high pressure liquid chromatography) typically relies on a pump to force a pressurized liquid solvent containing a sample mixture through a column packed with a solid adsorbent material. Technique. Each component in the sample interacts with the adsorbent material slightly differently, resulting in separation of the components. HPLC is distinguished from conventional (“low pressure”) liquid chromatography by the very high operating pressures (50-350 bar). Some models of mechanical pumps in HPLC instruments are capable of mixing multiple solvents together in time-varying ratios, creating a gradient of composition in the mobile phase. Various detectors such as ultraviolet (UV), photodiode array (PDA), or mass spectrometry are popular. Detection can be performed using a UV absorbance detector at 190-400 nm (A _190nm -A _400nm ). In one embodiment, absorbance is measured at about A _{280 nm} when amines are included in the elution buffer.

Typically, performing a chromatographic separation, eg, HPLC, consists of at least the following steps: contacting, eg loading, the sample/mixture with the equilibrated column (“loading”). After loading, a washing step may be performed. After this step, the separated components are eluted from the column. This can be either isocratically (without altering the buffer composition when compared to the loading and/or equilibration step) or gradients (at least one of buffer properties, e.g. salt concentration, polarity, pH). change one). In one embodiment, elution is performed using a linear gradient. In a next step, the column can be regenerated (“column regeneration”), i.e. at the highest concentration of altered properties (salt concentration, polarity, pH) to elute any residual material from the column. Give the ingredients additional time. Regeneration can alternatively be performed by altering other buffer properties (not altered during the elution step). A final step (“column equilibration”) may be an equilibration step that allows the column to return to its original state, in which it is suitable for further use. The described process can alternatively be performed using an FPLC device and/or a stand-alone column. Chromatographic separation is described in Hidayat Ullah Khan (2012). It is well known in the art, as described in The Role of Ion Exchange Chromatography in Purification and Characterization of Molecules, Ion Exchange Technologies, Chapter 14, 331-334.

Advantageously, the method according to the invention provides good peak separation of native α-thrombin from its degraded polypeptides and/or from other proteins in the thrombin solution. Typically, in chromatographic methods, "good separation"/"good peak separation" means that peaks detected as representative of component elution do not overlap, i.e., detector response is baseline between peaks. Back to the level, it is considered an efficient separation of the components. The term "good peak separation" is also meant to include "sufficient separation" in which a clear distinction between elution peaks appears, but the detector response does not fully return to baseline levels between peaks. .

Separation/separability effectiveness can be assessed visually. Alternatively, or in addition, resolution (Rs), which is the extent to which a chromatographic column separates components from each other, can be defined mathematically, and resolution is defined by a selected peak and and the peak retention time of the peak multiplied by the constant 1.18 and divided by the sum of the peak widths of 50% of the peak height. The term "retention time" refers to the interval between the time of injection and the time of detection of the peak apex (uppermost point of the peak) as representative of elution.

Generally, a resolution level of 2 or more is considered good separation of the components and allows good quantification of the peaks. A resolution of 1.5 or greater (less than 2) is considered "sufficient resolution" to allow separation and/or quantification.

In one embodiment of the method, the resolution between the α-thrombin peak and its degraded polypeptide ranges from about 1.5 to about 8.

In one embodiment of the method, the resolution between the α-thrombin peak and other proteins in the thrombin solution is higher than 8.

In one embodiment of the method, the resolution between different α-thrombin species peaks ranges from about 1.5 to about 8.

In one embodiment, the resolution between different α-thrombin degrading polypeptides (β-thrombin and γ-thrombin) is lower than 1.5, eg equal to 0. In one embodiment, β-thrombin and γ-thrombin elute in the same peak. In another embodiment, certain β-thrombin forms elute in distinct peaks, e.g., resolution between β-thrombin and other components in solution of about 1.5 to about 8. . Accordingly, in one aspect, the invention also provides a method for purifying β-thrombin from a solution comprising β-thrombin and at least one of α-thrombin, γ-thrombin, or another protein. , the method is
β-thrombin is separated from α-thrombin, γ-thrombin, and/or another protein by anion exchange chromatography using differential elution conditions and contacting the solution with an anion exchanger. and recovering the β-thrombin fraction, thereby obtaining purified β-thrombin.

The term "β-thrombin fraction" is typically collected after elution of a loaded anion exchanger (e.g. a loaded column) with a buffer under differential elution conditions. refers to the fraction that was

In another aspect, the invention provides a one-step chromatography for quantifying β-thrombin in a solution containing β-thrombin and at least one of α-thrombin, γ-thrombin, or another protein. A method is provided which comprises contacting a solution with an anion exchanger and differential elution conditions to convert β-thrombin, α-thrombin, γ-thrombin, and/or β-thrombin by anion exchange chromatography to or separating from at least one of the other proteins; and quantifying β-thrombin.

In some embodiments, the method further comprises identifying the separated β-thrombin, α-thrombin, and/or γ-thrombin containing fraction. In some embodiments, the method further comprises quantifying α-thrombin and/or γ-thrombin.

In some embodiments, the method extracts β-thrombin from at least one of α-thrombin, γ-thrombin, and another protein by anion exchange chromatography using differential elution conditions. including the step of separating from one.

In some embodiments, the chromatographic method is an anion exchange high performance liquid chromatography method. In some embodiments, differential elution conditions include pH gradients generated, for example, by using eluants comprising amines or mixtures of amines. In some embodiments, the anion exchanger is made of non-porous particles.

In another aspect, the invention includes purified β-thrombin, isolated β-thrombin, and purified/isolated β-thrombin as described herein obtainable by the method of the invention. Provide a formulation/kit.

In some embodiments, the method allows isolation and recovery of a homogenous post-translationally modified α-thrombin fraction. In some embodiments, the homogeneous post-translational modification is homogeneous glycosylation. In some embodiments, homogeneous post-translational modifications are homogeneous glycosylation and sialylation. In some embodiments, the separated/collected α-thrombin fraction is homogeneous glycosylated α-thrombin. In some embodiments, homogeneous post-translationally modified α-thrombin is represented by a single glycoform. In some embodiments, the separated/recovered α-thrombin glycoforms are homogeneously glycosylated and/or sialylated. In some embodiments, the homogeneity of the isolated, separated, and/or recovered post-translationally modified α-thrombin, e.g., homogeneous α-thrombin glycoforms, is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% %, or the level of at least 100% identity. For example, less than 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100%, 50-55%, 50-60%, 50-65%, 50-70% , 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 50-99%, 50-100%, 55-60%, 55-65%, 55-70% , 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 55-99%, 55-100%, 60-65%, 60-70%, 60-75% , 60-80%, 60-85%, 60-90%, 60-95%, 60-99%, 60-100%, 65-70%, 65-75%, 65-80%, 65-85% , 65-90%, 65-95%, 65-99%, 65-100%, 70-75%, 70-80%, 70-85%, 70-90%, 70-95%, 70-99% , 70-100%, 75-80%, 75-85%, 75-90%, 75-95%, 75-99%, 75-100%, 80-85%, 80-90%, 80-95% , 80-99%, 80-100%, 85-90%, 85-95%, 85-99%, 85-100%, 90-95%, 90-99%, 90-100%, 95-99% , 95-100% identity, and any range between the disclosed percentages. In yet another embodiment, α-thrombin is unmodified, eg, not glycosylated.

In some embodiments, the protein solution contains another protein, an α-thrombin degrading polypeptide (eg, a β-thrombin polypeptide and/or a γ-thrombin polypeptide), α-thrombin that is not post-translationally modified (unmodified α -thrombin), or post-translationally modified α-thrombin.

In some embodiments, the solution comprises a mixture of unmodified and post-translationally modified α-thrombin.

In some embodiments, the solution comprises heterogeneous post-translationally modified α-thrombin, including different glycoform species of α-thrombin.

In some embodiments, the solution contains another protein or protein fragment, which can be, for example, a protein added to the solution. In some embodiments, the protein is, for example, human serum albumin (HSA). In some embodiments of the method, the solution comprises at least one of an α-thrombin degrading polypeptide or another protein.

In some embodiments of the method, the solution comprises at least one of α-thrombin degrading polypeptide or HSA.

In some embodiments of the method, the solution comprises at least one of α-thrombin-degrading polypeptide and HSA.

In some embodiments, the method includes loading the solution onto an anion exchange column. In some embodiments, the method includes contacting the solution with an anion exchanger in batch form.

As used herein, "batch method," "batch mode," and "batch mode" generally refer to techniques in which a solution is contacted with a resin, typically in a single-stage adsorption procedure. . A "single-stage adsorption procedure" refers to a procedure in which all of the purification process components (eg, resin and solution) are incubated together, eg, in a stirred tank, batch reactor, or vessel, and adsorption is performed in a continuous fashion. The resin-bound fraction can then be recovered by additional steps of centrifugation and/or filtration.

In some embodiments, the exchanger/column is equilibrated from a pH of 10.5 to about pH 7.0 (eg, a pH of 9.1) prior to contacting, eg, loading. Equilibration may be performed using a buffer(s) suitable for equilibrating the exchanger at a pH of 10.5 to about pH 7.0 (eg, pH of 9.1).

In one embodiment, the buffer comprises a mixture of amines. In some embodiments, the amine mixture used for equilibration is piperazine, triethanolamine, bis-trispropane, 1-methylpiperazine, bicine, bis-tris, diethanolamine, diethylamine, 1-histidine, imidazole, pyridine , tricine, triethanolamine, and/or tris.

In some embodiments, the amine mixture used for equilibration consists of piperazine, triethanolamine, bis-trispropane, and 1-methylpiperazine. In some embodiments, the concentration of amine in the equilibration buffer ranges from about 1 to about 100 mM, such as from about 10 to about 20 mM or about 20 mM.

The flow rate during contacting, eg, loading, can range from about 0.1 to about 1.4 mL/min. In some embodiments, conditions that allow separation between α-thrombin-degrading polypeptides, α-thrombin, homogeneous α-thrombin glycoforms, and/or other proteins include an anion exchanger/column. Including applying differential elution conditions, such as exposure to pH gradient conditions for elution.

In some embodiments, differential elution conditions comprise applying a pH gradient, eg, stepwise or continuously (eg, linear). Typically, a "continuous gradient" is defined as a gradient in which the composition of the eluent is varied gradually, continuously, and constantly, whereas a "step gradient" is defined as a Includes Instant Change.

In some embodiments, the length of the gradient ranges from 5 minutes to 100 minutes, or 5 minutes to 60 minutes. In another embodiment, the length of the gradient is longer than 25 minutes, such as longer than 30 minutes, or longer than 35 minutes. In some embodiments, the length of the gradient ranges from 25 minutes to over 35 minutes, or from 25 minutes to over 30 minutes.

In one embodiment, elution is performed with the same buffer used for equilibration of the anion exchanger.

In some embodiments, the linear pH gradient is from about pH 10.5 to about pH 2.0, such as from about pH 9.1 to about pH 3.4.

In some embodiments, a pH gradient is generated using an eluent comprising an amine or mixture of amines. In some embodiments, a linear pH gradient is generated using an eluent buffer containing a mixture of amines. In some embodiments, the amine mixture used in differential elution conditions is piperazine, triethanolamine, bis-tris propane, 1-methylpiperazine, bicine, bis-tris, diethanolamine, diethylamine, 1-histidine. , imidazole, pyridine, tricine, triethanolamine, and/or tris. In some embodiments, the amine mixture used in differential elution conditions consists of piperazine, triethanolamine, bis-trispropane, and 1-methylpiperazine. In some embodiments, the concentration of each amine in the buffer ranges from about 1 to about 100 mM. In some embodiments, the concentration of each amine in the buffer is about 20 mM.

In some embodiments, a linear pH gradient is generated using two eluent buffers containing mixtures of amines. In some embodiments, a linear pH gradient is generated using two eluent buffers containing the same mixture of amines. Typically, the pH of the elution buffer depends on the ratio between buffers A and B during elution. In some embodiments, Buffer A has a pH of about 9.1 and Buffer B has a pH of about 3.4, and the concentration of Buffer A is reduced from about 40% to about 60%. and the concentration of Buffer B increases from about 60% to about 40%. In some embodiments, Buffer A has a pH of about 9.1 and Buffer B has a pH of about 3.4, and the concentration of Buffer A is reduced from about 100% to about 0%. and the concentration of Buffer B increases from about 0% to about 100%. In some embodiments, Buffer A has a pH of about 9.1 and Buffer B has a pH of about 3.4, and the concentration of Buffer A is reduced from about 90% to about 0%. and the concentration of Buffer B increases from about 10% to about 100%. In some embodiments, the % increase in Buffer B per minute ranges from about 0.1% to about 10%, or from about 3.5% to about 4.5%. In some embodiments, the % increase in Buffer B per minute is selected from the group consisting of about 3.5%, 3.75%, 4%, 4.25%, or 4.5%. In some embodiments, the % increase in Buffer B per minute is about 3.5%.

In some embodiments, the elution conditions are from about 0.1 to about 1.4 mL/min, or from about 0.25 to 1.0 mL/min, or from 0.5 mL/min to 0.8 mL/min, or from about Including a flow rate of 0.8 to about 1.0 mL/min. In some embodiments, elution conditions comprise a flow rate of about 1 mL/min.

In some embodiments, elution conditions include the following steps: about 0.1% to about 10%, about 0.5% to about 10%, or about 3.5% to about 4.5% per minute 90% to 100% Buffer B with a linear increase/slope of % Buffer B.

In some embodiments, elution conditions include the following steps: about 0.1% to about 10%, about 0.5% to about 10%, or about 3.5% to about 4.5% per minute 0% to 100% Buffer B with a linear increase/slope of % Buffer B.

In some embodiments, the elution conditions comprise the following steps: 90%-100% Buffer B with a linear increase/slope of about 3.5% Buffer B per minute.

In some embodiments, the elution conditions comprise the following steps: 0% to 100% Buffer B with a linear increase/slope of about 3.5% Buffer B per minute.

In some embodiments of the method, the anion exchanger is a weak or strong anion exchanger. In some embodiments of the method, the anion exchanger consists of quaternary ammonium positively charged groups. In some embodiments of the method, the anion exchanger is based on polymeric beads of about 1 to about 1000 μm, eg, 5 μm. In some embodiments of the method, the polymeric beads consist of poly(styrene/divinyl/benzene). In some embodiments of the method, the anion exchanger consists of non-porous or porous particles, eg, particles having pores in the range of about 120-1000 angstroms (Å). In some embodiments of the method, the anion exchanger consists of non-porous particles. In some embodiments of the method, the anion exchanger consists of monodisperse particles.

In some embodiments of the method, an anion exchange column is used that has at least one of the following characteristics: a width in the range of 1.7-10 mm (eg, 4.6 mm), and a width of 10-10 mm; A length in the range of 250 mm (eg 250 mm).

Some embodiments of the method use an anion exchange column having a width in the range of 1.7-10 mm (eg, 4.6 mm) and a length in the range of 10-250 mm (eg, 250 mm). be.

In some embodiments of the method, the method consists of a one-step chromatographic method, eg, a type of chromatographic method without additional chromatographic and/or separation step(s).

In some embodiments, the purification method is performed by anion exchange high performance liquid chromatography, fast protein liquid chromatography (FPLC), and/or by a stand-alone column with or without a connected detector. be.

In some embodiments, the method is for analytical purposes.

In certain embodiments, provided herein is purified α-thrombin obtainable by the methods provided herein.

In another aspect, provided herein is an isolated homogeneous post-translationally modified α-thrombin. In some embodiments, α-thrombin is from a mammalian plasma source, such as a human or porcine plasma source. In another aspect, provided herein is isolated homogeneous post-translationally modified α-thrombin from a mammalian blood or plasma source.

In some embodiments the post-translational modification is glycosylation. In some embodiments, post-translational modifications are glycosylation and sialylation. In some embodiments, homogeneous post-translationally modified α-thrombin is represented by a single/specific glycoform. In some embodiments, the α-thrombin glycoform is additionally sialylated. In some embodiments, the α-thrombin glycoform is homogeneously sialylated. In some embodiments, the isolated homogeneous post-translationally modified α-thrombin is homogeneously glycosylated α-thrombin. In some embodiments, the isolated homogeneous post-translationally modified α-thrombin is represented by one specific glycoform. In some embodiments, the isolated homogeneous post-translationally modified α-thrombin is homogeneously sialylated α-thrombin.

In yet another aspect, provided herein are formulations comprising purified α-thrombin or isolated homogeneous post-translationally modified α-thrombin disclosed herein. In some embodiments, purified α-thrombin or isolated homogeneous post-translationally modified α-thrombin is obtained by the methods disclosed herein. In some embodiments of the formulation, the α-thrombin is from a mammalian plasma source. In some embodiments, α-thrombin is from blood or plasma sources. In some embodiments, the formulation includes a pharmaceutically acceptable carrier or diluent. The formulations disclosed herein can be frozen or lyophilized.

In another aspect, the surface of a subject is subjected to hemostatic treatment, sealing, graft fixation, including applying a formulation comprising purified α-thrombin, homogeneous post-translationally modified α-thrombin, or β-thrombin to the surface. Provided herein are methods of providing wound healing and/or anastomosis. The formulation may be applied in a solution containing fibrinogen. The surface may be a bleeding or non-bleeding site. A subject may be a human subject.

In another aspect, the present invention provides the isolated homogeneous post-translationally modified α-thrombin disclosed above for hemostatic treatment, sealing, graft fixation, wound healing, anti-adhesion, and/or anastomosis. , purified α-thrombin, or formulations containing β-thrombin.

In another aspect, a container such as an ampoule, a vial, and/or a syringe, and optionally an application device, containing purified α-thrombin, homogeneous post-translationally modified α-thrombin, or β-thrombin disclosed above. , and/or instructions for use are provided.

In another aspect, a kit is provided comprising, as a first component, a container comprising the isolated homogeneous post-translationally modified α-thrombin or the purified homogeneous post-translationally modified α-thrombin glycoform disclosed above. .

In some embodiments, the kit includes, for example, a container containing gelatin as a second component. The kit may further contain fibrinogen. In some embodiments, the kit includes, for example, a container containing fibrinogen as a second component. A kit may include at least one container and at least one label. Suitable containers include, for example, ampoules, vials, syringes, and tubes. The container can be made of glass, metal, or plastic, for example.

These and other aspects and embodiments of the invention will become apparent by reference to the following detailed description of the invention and drawings.

All embodiments disclosed herein relating to purification and/or quantification of α-thrombin also relate to purification and/or quantification of β-thrombin.

Magnification of representative chromatograms of areas of elution peaks obtained using reversed-phase high-performance liquid chromatography (RP-HPLC) of several samples of HSA, thrombin solution, formulated thrombin, and water. indicates The injected samples were a) 30 μL of thrombin solution, b) 100 μL of formulated thrombin, c) 100 μL of 5 mg/ml HSA, and d) 100 μL of HPLC grade water as a blank sample. In all figures, sample delineation is shown from top to bottom based on the start of the chromatogram, and the injected sample (top to bottom) is listed on the chromatogram. Different sample runs are shown in one figure as superimposed overlays. In all graphs, the y-axis is expressed as absorbance units (AU) and the x-axis is expressed as minutes. Shown is an enlargement of a representative chromatogram of the area of the elution peak obtained using anion exchange high performance liquid chromatography (AEX-HPLC) and elution with a linear NaCl salt gradient at pH 8.0. The injected samples were a) 30 μL of thrombin solution, b) 100 μL of formulated thrombin, c) 100 μL of 5 mg/ml HSA, and d) 100 μL of buffer A as a blank sample. Representative chromatograms obtained for different samples injected on an AEX-HPLC and eluted using a linear NaCl salt gradient at pH 6.0 are shown. The injected samples were a) 30 μL of thrombin solution, b) 100 μL of formulated thrombin, c) 100 μL of 5 mg/ml HSA, and d) 100 μL of buffer A as a blank sample. Shown is a magnified view of a representative chromatogram of the area of the elution peak obtained using AEX-HPLC with a linear NaCl salt gradient at pH 7.5. The injected samples were a) 30 μL of thrombin solution, b) 100 μL of formulated thrombin, c) 100 μL of 5 mg/ml HSA, and d) 100 μL of buffer A as a blank sample. A representative chromatogram of the area of the elution peak obtained using AEX-HPLC with a linear NaNO ₃ salt gradient at pH 8.0 is shown. The injected samples were a) 30 μL thrombin solution, b) 100 μL formulated thrombin, and c) 100 μL 5 mg/ml HSA. Representative chromatograms of injected samples obtained using AEX-HPLC with a linear pH gradient from pH 9.1 to pH 3.4 are shown. The injected samples were a) 30 μL of thrombin solution, b) 100 μL of formulated thrombin, c) 100 μL of 5 mg/ml HSA, and d) 100 μL of buffer A as a blank sample. Elution was performed with an amine-based buffer. FIG. 7 is an enlarged view of the thrombin elution region of the chromatogram shown in FIG. 6; Magnifications of chromatograms obtained using AEX-HPLC and elution with a linear pH gradient at different flow rates of 0.25, 0.5, 0.75 and 1 mL/min are shown. The injected sample was 30 μL of thrombin solution for each flow rate tested. Effect of linear slope slope on separation/resolution (assessed by visual inspection). Different percentage increases in Buffer B per minute were evaluated: 4.5%, 4.25%, 4%, 3.75% and 3.5%. The injected sample was 30 μL of thrombin solution for each increment tested. Shown are overlaid chromatograms obtained using AEX-HPLC at a flow rate of 1.0 mL/min for α, β, and γ thrombin standards, a thrombin solution, and Buffer A as a blank sample. A different thrombin peak was identified for the thrombin solution by comparison with the thrombin commercial standard. Shown are the different thrombin peaks separated from the thrombin solution using an AEX-HPLC linear pH gradient from 100% buffer A to 100% buffer B with a slope of 3.5% B per minute. Elution peaks were collected and further characterized using Western blots as a qualitative tool. Shown are chromatograms of different thrombin species separated by HPLC-AEX. Thrombin solutions that had undergone sialic acid removal and untreated thrombin solutions were injected. The results show that sialic acid removal affects the charge of this thrombin species, which in turn affects the elution profile, leading to an overall shift of the peak to the left of the chromatogram (when compared to the untreated thrombin solution). ). Full-length chromatogram of an injected thrombin sample obtained using an AEX-HPLC linear pH gradient with Buffer B increments of 3.5% per minute and a flow rate of 1.0 mL/min. indicates The results show complete separation between human serum albumin, several α-thrombin peaks corresponding to different homogeneous post-translationally modified α-thrombin species, and acetyltryptophan. Magnified views of α-thrombin species and degraded polypeptide elution regions are shown.

The methods provided herein utilize, in part, for post-translational modifications (e.g., glycosylation and/or sialylation levels) of native α-thrombin by anion exchange chromatography (AEX). It is based on the discovery that the protein can be isolated/purified from heterogeneous protein solutions by means of The method according to the invention is also based on the discovery that α-thrombin or β-thrombin can be isolated/purified from solutions containing stabilizers such as other proteins, eg human serum albumin and/or bovine serum albumin.

It has surprisingly been found that the method according to the invention allows high-resolution purification and/or quantification of α-thrombin or β-thrombin in the presence of large amounts of other proteins relative to the thrombin concentration in solution. .

More specifically, the method according to the present invention provides high resolution, distinct and homogeneous post-translational modifications from heterogeneous solutions containing stabilizers such as large amounts of other proteins, e.g. human serum albumin, bovine serum albumin, etc. Allows purification and/or quantification of α-thrombin species (eg, homogeneous α-thrombin glycoforms).

In some embodiments, the other protein, eg, serum albumin, is present in solution at a concentration of about 0.4 to about 50 mg/ml, eg, about 5 to about 6.5 mg/ml. In some embodiments, the thrombin concentration in the solution ranges from about 100 to about 10000 IU/ml, such as from about 800 to about 1200 IU/ml, or about 0.3 mg/ml. In some embodiments, the ratio of thrombin (IU) to other protein (mg) ranges from about 1:10 to about 1:40, or from about 1:14 to about 1:27.

In particular, α-thrombin is purified from thrombin-containing solutions and/or by providing good peak separation of native post-translationally modified α-thrombin from its degraded polypeptides and other proteins in thrombin preparations. A one-step chromatographic method for quantification is provided herein. Also provided herein are tools for separating between α-thrombin, its degradation polypeptides, and other proteins (eg, HSA) in thrombin solutions/formulations.

"Native alpha-thrombin" refers, for example, to the intact, undegraded, and/or functional form of alpha-thrombin.

Traditionally, thrombin was purified and analyzed using reversed-phase chromatography, hydrophobic interaction chromatography, cation exchange chromatography, and/or SDS-PAGE, and Western blotting.

A method for purifying α-thrombin from a solution comprising α-thrombin and at least one of an α-thrombin-degrading polypeptide (eg, β-thrombin and/or γ-thrombin polypeptide) or another protein is provided herein, the method comprises contacting a solution with an anion exchanger and using differential elution conditions, e.g., a pH gradient, to convert α-thrombin by anion exchange chromatography. , separating from at least one of the α-thrombin degrading polypeptide or another protein, and recovering the α-thrombin fraction, thereby obtaining purified α-thrombin.

Homogeneous post-translationally modified α-thrombin species (eg, homogeneous α-thrombin glycoforms), heterogeneous post-translationally modified α-thrombin species (eg, heterogeneously glycosylated α-thrombin species), and optionally Optionally also provided herein is a method for purifying from a solution comprising at least one of an α-thrombin degrading polypeptide or another protein, the method comprising the step of contacting the solution with an anion exchanger. and a homogeneous post-translationally modified α-thrombin species from another α-thrombin post-translationally modified species and optionally α-thrombin degrading polypeptides and/or separating from other proteins and recovering the homogeneous post-translationally modified α-thrombin fraction, thereby obtaining a purified homogeneous post-translationally modified α-thrombin species.

The terms "purifying," "to purify," and the like refer to the removal, single It refers to separating or separating. The α-thrombin-containing solution may also contain α-thrombin-degrading polypeptides (β-thrombin and/or γ-thrombin), other proteins, and/or other α-thrombin post-translational species. The β-thrombin-containing solution may also contain α-thrombin, γ-thrombin, another protein, and/or α-thrombin post-translational species.

The term "contacting" refers to the solution of the positively charged groups in such a manner that a binding interaction occurs between the positively charged group and any binding partner present in solution, such as α-thrombin or β-thrombin. It refers to any kind of binding action that brings the anion exchanger into sufficiently close contact with the containing anion exchanger. The solution can be incubated with the anion exchanger for a period of time sufficient to allow contact and/or binding between the positively charged groups and α-thrombin or β-thrombin, eg, 1 minute or more.

The term "α-thrombin fraction" typically refers to the fraction collected after elution of a loaded anion exchanger (e.g. a loaded column) with buffer under differential elution conditions. Point. In one embodiment, the recovered α-thrombin fraction consists exclusively of α-thrombin. In another embodiment, the recovered α-thrombin fraction consists of one homogeneous α-thrombin species. In another embodiment, the recovered α-thrombin fraction consists of a homogenous α-thrombin glycoform fraction.

The term "purified α-thrombin" typically refers to the removal of α-thrombin from α-thrombin-degrading polypeptides and/or other proteins present in the starting thrombin-containing solution using anion exchange chromatography methods. Refers to the α-thrombin preparation obtained after isolation of thrombin. As used herein, the term "purified α-thrombin" refers to the purification of homogeneous post-translationally modified α-thrombin from a heterogeneous post-translationally modified α-thrombin solution using an anion exchange chromatography method. It also refers to homogeneous post-translationally modified α-thrombin, eg homogeneously glycosylated and/or sialylated α-thrombin preparations obtained after isolation of . The term "purified homogeneous α-thrombin glycoform" typically uses anion-exchange chromatography methods to isolate the α-thrombin-degrading polypeptide, heterogeneous glycosyl, present in the starting thrombin-containing solution. It refers to a homogenous α-thrombin glycoform preparation obtained after isolation of α-thrombin glycoform from alpha-thrombin species and/or another protein.

In one embodiment, purified α-thrombin is native protein free of degraded polypeptides.

In one embodiment, the purified α-thrombin is a homogeneous post-translationally modified α-thrombin species. In another embodiment, the purified α-thrombin is an unmodified α-thrombin species. In another embodiment, purified α-thrombin is a homogeneous α-thrombin glycoform.

A purified α-thrombin preparation may consist of homogeneous post-translationally modified species isolated from a solution containing various post-translationally modified α-thrombin. The starting thrombin solution may also contain unmodified α-thrombin species.

The isolated post-translationally modified α-thrombin can be glycosylated, or glycosylated and sialylated. The degree of glycosylation and/or sialylation can vary between different species. Antennas can be branched to varying degrees, from bifurcated to pentafurcated. Sialic acid is neuraminic acid (5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2-urosonic acid), such as N-acetylneuraminic acid or N-glycolylneuraminic acid. It can be any derivative.

"α-Thrombin" means unmodified α-thrombin, homogeneous or heterogeneous α-thrombin, homogeneous post-translationally modified α-thrombin, such as homogeneously glycosylated α-thrombin or homogeneously glycosylated and Homogeneously sialylated α-thrombin, or homogeneously sialylated α-thrombin, and heterogeneously post-translationally modified α-thrombin, such as heterogeneously glycosylated or glycosylated and sialylated α - may contain thrombin; α-Thrombin can be from mammalian blood and/or plasma sources, such as human, bovine, or porcine plasma sources, or from recombinant sources.

In some embodiments, the "another protein"/"other protein" is human serum albumin (HSA), or any other protein included in the thrombin formulation, e.g., for stabilization of the formulation. . "Another protein" is different from α, β, and γ-thrombin. "Another protein" can be prothrombin, immunoglobulins, HSA, and many other proteins that can be found in blood or plasma. Another protein can be a protein fragment. In some embodiments, another protein is a stabilizer such as human serum albumin and/or bovine serum albumin.

The term "anion exchange chromatography" refers to a separation technique in which molecules are separated based on their net charge. Anion exchangers are named for their ability to attach or bind anions or negatively charged particles. Anion exchangers are well known in the art (Practical Protein Chromatography edited by Kenney and Fowell Volume 11; Chapter 16, 249-258; Humana Press, 1992). In an anion exchanger, the resin is positively charged and molecules bind when the buffer pH is above the isoelectric point of the protein. The term "isoelectric point" refers to the pH at which molecules have no net charge. In media with a pH below the isoelectric point the molecules have a net positive charge, above which the molecules have a net negative charge. The terms "anion exchanger" and "anion exchange matrix" are used interchangeably herein.

As used herein, the terms "support" and "resin" include a carrier or any matrix used to attach, immobilize, support or stabilize positively charged groups. Supports are described in Hermanson GT, Mallia AK and Smith PK 1992 "Immobilization Affinity Ligand Techniques" pp. 1-45 Academic Press, Inc.; well known in the art, as described in San Diego, USA.

A support for carrying out the method of the invention can be made of any material capable of binding a molecule comprising a positively charged group, ie a molecule comprising a positively charged chemical group. Solid supports include matrices, columns, coverslips, chromatography materials, filters, microscope slides, test tubes, vials, vials, ELISA supports, glass or plastic surfaces, chromatography membranes, sheets, particles, magnetic beads. Examples include, but are not limited to, beads, gels, powders, fibers, and the like.

In one embodiment of the invention, the support is in the form of a chromatographically compatible material. In another embodiment of the invention the support is in the form of a chromatographic membrane. Supports may comprise organic artificial/synthetic polymers such as agarose, sepharose, acrylic beads, cellulose, controlled pore glass, silica gel, hydrophilic materials such as dextran, hydrophobic materials, or polyacrylamide or polystyrene based materials. . Typical materials/polymers are Sephacryl® (Pharmacia, Sweden), Ultragel® (Biosepara, France) TSK-Gel Toyopearl® (Toso Corp., Japan), HEMA (Alltech Ass. (Deer-field, Ill., USA), Eupergit® (Rohm Pharma, Darmstadt, Germany), and azlactone-based materials (3M, St. Paul, Minn., USA). ).In one embodiment, the support comprises Agarose® or Sepharose®.These materials are commercially available.Typically, anion exchange resins or polymers are It is an insoluble matrix (or support structure), usually in the form of microbeads made from an organic polymer matrix.

In some embodiments of the method, the beads are sized from about 1 to about 1000 μm, or such as 5 μm. Beads can be non-porous or porous particles. The beads may be monodisperse (ie, substantially uniform in size) particles. In one embodiment, the beads range from 1.7 μm to 10 μm.

Anion exchangers can be weak or strong anion exchangers. A weak anion exchanger generally refers to an exchanger containing a weak base, while a strong anion exchanger generally contains a strong base that can sustain its charge over a wider pH range. Refers to the exchanger.

In some embodiments of the method, the positively charged group is ammonium, alkylammonium, dialkylammonium, trialkylammonium, quaternary ammonium, alkyl group, H ⁺ , Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , amino functional groups, and combinations thereof.

Resin beads can be suspended in a suitable medium and the resulting slurry can be used, for example, in a chromatography column, referred to herein as "column purification." Alternatively, columns can be purchased in pre-packed form.

"Column purification" and "column chromatography" generally refer to techniques in which a solution (mobile phase) can be moved at a certain flow rate through a column containing a packed resin, and the individual components Or some component is adsorbed by the resin (stationary phase), ie by the chromatographic material. Unbound material can be recovered from the other side of the column after the mixture has passed through it. By using certain elution conditions, it is possible to alter the binding between different compounds and the stationary phase, leading to elution of specific purified compounds from the column one by one. Column purification is well known in the art, as described in Practical Protein Chromatography edited by Kenney and Fowell Volume 11; Chapter 16, 249-258; Humana Press, 1992.

Typically, a slurry of resin is poured into the column. After it has settled, the column is pre-equilibrated in buffer before the protein mixture/solution is applied. Alternatively, pre-packed columns can be purchased. Unbound protein appears in the flow-through and/or subsequent buffer washes. Proteins bound to the resin can be retained and eluted with salt or pH/polarity adjustment. The term "unbound material"/"unbound fraction" typically refers to, for example, the same buffer used for equilibration and/or the buffer used to load the column with a thrombin-containing solution. (“binding buffer”) refers to the fraction discarded after washing the loaded column. A "heterogeneous solution" is used as the elution condition. A “heterogeneous solution” is typically, for example, a solution and/or condition that differs from that used to load, wash, and/or equilibrate the column, and/or refers to a solution different from the solution used in the step of Elution conditions employ shifts in the composition of the mobile phase, thus altering factors in the binding environment created by the binding buffer.

Generally, equilibration is performed until pH and/or conductivity and/or UV index are stable. In one embodiment, equilibration is performed with ≧5 column volumes of buffer. In one embodiment, equilibration is performed with 1-5 column volumes of buffer.

The term "elution conditions" refers to heteroconcentration conditions, e.g., solutions and/or conditions different from those used to load and/or equilibrate the column and/or solutions used in previous steps. refers to the use of different solutions and/or conditions. The solutions and/or conditions used at the start and/or end of the elution step (e.g., gradient elution) are identical to the solutions and/or conditions used during the wash, load, and/or regeneration steps. obtain. The term "elution conditions" can also refer to gradient elution in which changes in salt concentration and/or pH/polarity occur. Elution conditions are such that proteins and degraded polypeptides are separated and differentially eluted. The method according to the invention comprises at least one elution step with a heterogeneous solution. Elution conditions typically involve increasing salt concentration and/or changing pH/polarity. It has been found herein that using a pH gradient for elution is efficient.

In some embodiments of the method, the method consists of one chromatographic step, ie, a single chromatographic step.

Typically, the terms "one-step chromatographic method" or "one chromatographic step" or "one-step anion-exchange chromatography" mean, without additional chromatographic and/or separation step(s), α-thrombin, homogeneous or heterogeneous α-thrombin, homogeneous post-translationally modified α-thrombin, such as homogeneously glycosylated α-thrombin, or by an anion exchanger directly on the sample material Homogeneously glycosylated and homogeneously sialylated α-thrombin, unmodified α-thrombin, heterogeneously post-translationally modified α-thrombin, such as heterogeneously glycosylated or glycosylated and sialylated Refers to methods that allow the purification and/or quantification of α-thrombin and/or thrombin-degrading polypeptides or β-thrombin.

In one embodiment of the invention, column purification is utilized. In another embodiment, an anion exchange high performance liquid chromatography method is used. The column can be regenerated after elution of solution components for repeated use. Total run time from loading to regeneration can range from 30 to 120 minutes, eg, about 46,55 minutes. In one embodiment, the gradient separation takes 28.6 minutes.

Separation according to the present invention is performed by employing differential elution conditions. The term "differential elution conditions" refers to the separation of α-thrombin from its degrading polypeptides and/or from other proteins, homogenous post-translationally modified α-thrombin species, thrombin degrading polypeptides, Separation of protein and/or heterogeneous post-translationally modified α-thrombin species, separation of homogeneous α-thrombin glycoforms from heterogeneous post-translationally modified α-thrombin species, eg glycosylated α-thrombin species separation of homogeneous α-thrombin glycoforms from at least one of an α-thrombin degrading polypeptide, another protein, or a heterogeneous post-translationally modified α-thrombin species, and/or β-thrombin; Refers to conditions that allow separation from α-thrombin, γ-thrombin, and/or another protein.

Elution conditions may involve changes in salt concentration and/or pH of the elution buffer. In one embodiment, differential elution conditions include pH changes, eg, pH gradients. In one embodiment, the resins used according to the invention are suitable for working in the pH range according to the invention. In one embodiment, the resin is suitable for exposure to organic materials (methanol and/or acetonitrile).

Column volumes can range from about 0.03 to about 53 mL. In one embodiment of the invention, the column volume is about 4.1 mL, such as 4.15 mL. In another embodiment, most of the peaks are collected within one column volume.

In additional embodiments, the method is an analytical method, eg, a physico-chemical analytical method, and may be performed as a one-step chromatographic method.

In some embodiments, the method further comprises identifying the separated α-thrombin, β-thrombin, and/or γ-thrombin containing fraction.

In some embodiments, the method further comprises identifying different post-translationally modified α-thrombins. Differential glycosylation can be analyzed using mass spectroscopy, capillary electrophoresis, by using different HPLC methods, or by any other method known in the art.

In one embodiment, different fractions/peaks are visually identified after injecting the sample set. Typically, the peak profile is robust, so each peak can be easily identified. In another embodiment, different thrombin peaks are identified by injecting α, β, and γ thrombin standards into the HPLC and identifying correlated peaks for thrombin solutions. In another embodiment, the different thrombin peaks are run against alpha, beta, and gamma thrombin standards and/or based on the known molecular sizes of alpha-, beta-, and gamma-thrombin, Identified by Western blot analysis.

Provided herein is a one-step chromatographic method for quantifying α-thrombin in a solution comprising α-thrombin and at least one of an α-thrombin-degrading polypeptide or another protein, wherein the The method comprises separating α-thrombin from at least one of an α-thrombin degrading polypeptide or another protein by anion exchange chromatography by differential elution conditions; collecting and quantifying α-thrombin.

As used herein, homogenous post-translationally modified α-thrombin in solution comprising heterogeneous post-translationally modified α-thrombin and optionally at least one of an α-thrombin degrading polypeptide or another protein ( For example, a one-step chromatographic method for quantifying homogenous glycoforms is provided, which employs differential elution conditions to generate homogenous post-translationally modified α-thrombin in solution by anion exchange chromatography. recovering a homogeneous post-translationally modified α-thrombin fraction; and quantifying the homogeneous post-translationally modified α-thrombin. Provided herein is a one-step chromatographic method for quantifying α-thrombin in a solution comprising α-thrombin and at least one of an α-thrombin-degrading polypeptide or another protein, wherein the The method comprises the steps of contacting the solution with an anion exchanger and differential elution conditions to convert α-thrombin into at least one of an α-thrombin degrading polypeptide and/or another protein in anion exchange chromatography. separating from one and quantifying α-thrombin.

In some embodiments, the method separates α-thrombin from at least one of an α-thrombin degrading polypeptide and another protein by anion exchange chromatography by differential elution conditions. including.

In some embodiments, the method further comprises quantifying one or more degraded polypeptides, eg, β-thrombin and/or γ-thrombin polypeptides.

Also herein, homogeneous post-translationally modified α-thrombin in a solution comprising heterogeneous post-translationally modified α-thrombin and optionally at least one of an α-thrombin degrading polypeptide or another protein Also provided is a one-step chromatographic method for quantifying Homogeneous post-translational modifications in anion exchange chromatography by contacting the solution with an anion exchanger and differential elution conditions separating α-thrombin from heterogeneous post-translationally modified α-thrombin and optionally from at least one of α-thrombin degrading polypeptides and/or other proteins; and quantifying α-thrombin. In one embodiment, the solution comprises at least one of an α-thrombin degrading polypeptide (β-thrombin and/or γ-thrombin) and/or another protein.

In some embodiments, the solution further comprises at least one of an α-thrombin-degrading polypeptide or another protein, and the method comprises homogenous post-translationally modified α-thrombin, α-thrombin-degrading polypeptide and/or also separating from at least one of the other proteins. In some embodiments, the method comprises separating homogeneous post-translationally modified α-thrombin from at least one of an α-thrombin-degrading polypeptide and another protein.

Quantitation can be performed, for example, by calculating the integral, for example, by measuring the area under the peak of the chromatogram. Peaks can be quantified either by integrating the peak and comparing the peak area to the total eluted area, or by estimating the peak height. Areas or heights can be converted to absolute numbers if standards are used, or relative peak areas can be evaluated.

In some embodiments of the method, the separating step comprises applying differential elution conditions. In some embodiments, the elution conditions comprise applying a pH gradient. In some embodiments, elution conditions comprise applying a linear pH gradient. Typically, a linear pH gradient is defined as a gradient that changes pH gradually and evenly over time. In some embodiments, the pH gradient is from about pH 9.1 to about pH 3.4. In some embodiments, a linear pH gradient is generated using an eluent comprising an amine or mixture of amines. In some embodiments, the eluent comprises a mixture of amines. In some embodiments, amine-based buffers include piperazine, triethanolamine, bis-tris propane, 1-methylpiperazine, and mixtures thereof. In some embodiments, the concentration of each amine in the buffer ranges from about 1 to about 100 mM, such as from about 10 to about 20 mM or about 20 mM. Buffers with similar properties suitable for creating a pH gradient, such as phosphate buffers with different pH values, can be used. Alternative compounds, not listed here, can be used to construct a suitable buffer system for the elution of thrombin from the anion exchanger.

The results show that elution using AEX-HPLC and a linear gradient from pH 9.1 to pH 3.4 provides good resolution between HSA, acetyltryptophan, α-thrombin degrading polypeptide and α-thrombin. Therefore, in one embodiment, a linear pH gradient elution step from pH 9.1 to pH 3.4 is used as differential elution conditions. In one embodiment, the HPLC comprises a 5 min loading step at a flow rate of 0.80 mL/min, a 20 min linear pH gradient elution step at a flow rate of 0.80 mL/min, wherein the linear pH gradient is the same mixture of amines. was generated by using two elution buffers containing, the concentration of buffer A decreased from 90% to 0%, buffer B increased from 10% to 100%, and the increase of buffer B was 4.5%. In another embodiment, the HPLC includes a 15 minute column equilibration step at a flow rate of 0.80 mL/min. In another embodiment, the HPLC includes a 5 minute column regeneration step at a flow rate of 0.80 mL/min.

In some embodiments, the temperature during the elution step ranges from about 10°C to about 50°C, eg, about 25°C.

In some embodiments, the flow rate during the linear pH gradient elution step is 0.25, 0.5, 0.75, and 1 mL/min. The results show that the resolution between α-thrombin and its degraded polypeptide increased with increasing flow rate, with the best resolution achieved at a flow rate of 1.0 mL/min. Thus, in one embodiment, the flow rate during the linear pH gradient elution step is higher than 0.75 mL/min, eg, about 1.0 mL/min.

The results indicate that elution of proteins from columns with a wider pH range results in better separation between peaks. Thus, in one embodiment, a linear pH gradient elution step is generated by using two elution buffers consisting of the same mixture and concentration of amines, with the gradient concentration of Buffer A decreasing from 100% to 0%. , Buffer B increases from 0% to 100%, and the increase in Buffer B is about 4.5% per minute. In such embodiments, the linear pH gradient elution step can be about 22 minutes.

The total run time from column loading of the thrombin solution to the maximum column regeneration step (including, for example, loading step, linear gradient elution step, column regeneration step, and column equilibration step) ranges from 30 to 120 minutes, such as Total run times can be in the range of 46-61 minutes, such as about 46, 51, 56, and 61 minutes, and the elution step can be in the range of 20-35 minutes, such as about 20, 25, 30, and 35 minutes. could be. The results show that at total run times of 56 and 61 min (30 and 35 min long gradient elution steps) additional peaks eluting in regions distinct to the thrombin peak were separated when compared to shorter run times. indicates that Therefore, in one embodiment, a linear gradient elution step longer than 25 minutes is performed.

The results show that elution with linear pH gradients of 4.5%, 4.25%, 4%, 3.75%, and 3.5% per minute is effective in separating different thrombin peaks, and 3.5% per minute. A % increase indicates having the best separation profile. The tilt slope can be affected by increasing the percent of Buffer B per minute when more than one buffer is used as the elution buffer. Typically, a lower percent increase in Buffer B per minute results in a shallower slope, thereby increasing protein elution, compared to a higher percent increase in Buffer B per minute. Affects profile. Thus, in some embodiments, the elution is a 3.5-4.5% Buffer B slope per minute, for example, 100% Buffer A to 100% Buffer B at a 3.5% slope. A linear pH gradient of .

In some embodiments, the starting solution comprising α-thrombin (purified and/or quantified) further comprises another protein and is substantially devoid of degradation polypeptides (e.g., the solution is containing less than 10% w/w of β-thrombin and/or γ-thrombin).

In some embodiments, the starting solution comprising α-thrombin further comprises degrading polypeptides, eg, β-thrombin and/or γ-thrombin. In some embodiments, the starting solution comprising α-thrombin further comprises the degrading polypeptide without another protein.

In some embodiments, the α-thrombin-containing starting solution further comprises a degrading polypeptide (eg, β-thrombin and/or γ-thrombin), and another protein.

In some embodiments, the starting solution comprising β-thrombin further comprises α-thrombin and lacks γ-thrombin and/or another protein. In some embodiments, the starting solution comprising β-thrombin further comprises γ-thrombin and lacks α-thrombin and/or another protein. In some embodiments, the starting solution comprising β-thrombin further comprises another protein and lacks α-thrombin and/or γ-thrombin. In some embodiments, the starting solution comprising β-thrombin further comprises α-thrombin and γ-thrombin and lacks another protein. In some embodiments, the starting solution comprising β-thrombin further comprises α-thrombin and another protein and lacks γ-thrombin. In some embodiments, the starting solution comprising β-thrombin further comprises γ-thrombin and another protein and lacks α-thrombin. In some embodiments, the starting solution comprising β-thrombin further comprises α-thrombin, γ-thrombin, and another protein.

The starting solution may contain heterogeneous post-translationally modified α-thrombin and/or unmodified α-thrombin. In some embodiments, the starting solution contains another protein. In some embodiments, the starting solution lacks another protein.

The thrombin concentration in the solution ranges from about 2 to about 10000 IU/mL, from about 100 to about 10000 IU/mL, from about 2 to about 4000 IU/mL, from about 800 to about 3000 IU/mL, or from about 800 to about 1200 IU/mL. Possibly, the total protein concentration can range from about 0.3 to about 55 mg/ml, from about 0.3 to about 10 mg/ml, or from about 1 to about 7 mg/ml. In some embodiments, 1000 IU/ml is equal to 0.3 mg/ml. The solution may have a pH ranging from about 6.9 to about 7.1 and may contain 5.0-6.5 mg/mL human serum albumin (HSA) and other stabilizers such as acetyltryptophan.

A solution containing α-thrombin, for example, has a thrombin activity in the range of 800-1200 IU/ml, a total protein concentration of about 5.7-6.5 mg/ml, and a pH of 5.0-6 at pH 6.9-7.1. A thrombin formulation or formulated thrombin (eg, a thrombin solution containing excipients and/or stabilizers) having .5 mg/ml human serum albumin (HSA), eg, a solution containing a pharmaceutical agent. The thrombin formulation may contain other stabilizers such as acetyltryptophan. In one embodiment, the HSA used in the formulation of thrombin contains the stabilizer acetyltryptophan.

As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition. Examples of excipients include, but are not limited to, human albumin, mannitol, sodium acetate, and water for injection. Human albumin in solution can range from about 2 to about 8 mg/ml. Mannitol can range in concentration from about 15 to about 25 mg/ml. Sodium acetate can also be added to the solution in the range of about 2 to about 3 mg/ml.

In one embodiment, the thrombin solution comprises about 3000 IU/ml thrombin, about 1 mg/ml total protein concentration, 20 mM sodium acetate at pH 6.9-7.1. In another embodiment, the thrombin formulation comprises thrombin in the range of 800-1200 IU/ml, total protein concentration of about 5.7-6.5 mg/ml, and pH 6.9-7.1 of 5.0-6. Contains 5 mg/ml human serum albumin (HSA).

The solution may contain calcium chloride. The calcium chloride concentration in the solution can range from about 2 to about 6.2 mg/ml, or from about 5.6 to about 6.2 mg/ml, such as a concentration of 5.88 mg/ml.

Thrombin clotting activity can be measured, for example, directly by the procedure of the European Pharmacopoeia Assay (0903/1997), by measuring the distance traveled on an incline (or drop test model), and/or by any other method known in the art. can be measured by the method of

Thrombin activity is determined using a coagulation analyzer equipped with a mechanical endpoint detection system to detect clot formation, such as the Diagnostica Stago ST4 Coagulation Analyzer, or a device that measures changes in turbidity due to fibrin clot formation. obtain.

Another method by which thrombin activity can be measured is to use chromogenic or fluorogenic peptide substrates for thrombin. Often in this method solubilized thrombin is mixed with excess chromogenic or fluorogenic substrate. Thrombin cleaves the substrate, releasing a chromophore or fluorophore that can be monitored with a spectrophotometer or fluorometer. Examples of chromogenic or fluorogenic substrates are β-Ala-Gly-Arg-p-nitroanilide diacetate and Z-Gly-Pro-Arg-AMC [Z = benzyloxycarbonyl; AMC = 7-amino- 4-methylcoumarin]. The rate of released chromophore or fluorophore can be correlated to the activity of thrombin.

Thrombin can be prepared from blood compositions. The blood composition can be whole blood or a blood fraction, ie a fraction of whole blood such as plasma. The source of thrombin can be autologous, thus manufactured from the patient's own blood, from pooled blood or fractions. A thrombin solution may be prepared from human or mammalian plasma. In one embodiment, thrombin can be prepared by recombinant methods in prokaryotic cells.

In one embodiment, the thrombin solution can be formulated as a sterile solution (pH 6.8-7.2) containing highly purified human thrombin. A thrombin formulation may contain human thrombin (800-1200 IU/mL), calcium chloride, human albumin, mannitol, sodium acetate, and water for injection. In one embodiment, thrombin is activated with calcium chloride after chromatographic purification of prothrombin from cryodepleted plasma, eg, as described in US Pat. No. 5,143,838, incorporated herein. Manufactured by

In another aspect, provided herein is a one-step analytical method for quantifying α-thrombin in a formulated thrombin (eg, pharmaceutical product) comprising α-thrombin and another protein (eg, human serum albumin). Provided is a method comprising the steps of: contacting formulated thrombin with an anion exchanger; separating α-thrombin from other proteins by anion exchange chromatography by differential elution conditions; and quantifying thrombin. In some embodiments, differential elution conditions include pH gradients generated, for example, by using eluants comprising amines or mixtures of amines. In some embodiments, an anion exchange high performance liquid chromatography method is used. In some embodiments, the anion exchanger is made of non-porous particles. In some embodiments, the formulated thrombin further comprises undesirable α-thrombin degrading polypeptides (β-thrombin and/or γ-thrombin polypeptides) and the method separates α-thrombin from the degrading polypeptides. Including process. In some embodiments, the formulated thrombin does not contain degradation polypeptides.

As used herein, the indefinite articles "a" and "an" mean "at least one" or "one or more" unless the context clearly indicates otherwise. .

As used herein, the terms "comprising," "including," "having," and grammatical variations thereof are intended to identify the feature, step, or component being described. , does not exclude the addition of one or more additional features, steps, components, or groups thereof.

When the term "about" precedes a numerical value, the term "about" is intended to indicate ±10%.

"Thrombin" or "thrombin polypeptide" is a mammalian serine protease that is part of the blood clotting cascade and converts fibrinogen into insoluble chains of fibrin as well as catalyzing other coagulation-related reactions. In humans, prothrombin is encoded by the F2 gene and the resulting polypeptide is proteolytically cleaved within the coagulation cascade by Factor Xa or other serine proteases along with a cofactor (FVa) to generate thrombin. Thrombin serves, among other things, as the active ingredient in some hemostatic products. For example, fibrin sealants typically contain a fibrinogen component and a thrombin component. When both components are mixed (eg when applied to a bleeding wound), thrombin cleaves fibrinogen to form a fibrin polymer with hemostatic properties. Fibrin sealant is typically a blood product obtained from a commercial source or from a particular local transfusion center. Commonly used ingredients for the preparation of fibrin glue are fibrinogen, thrombin, factor VIII, factor XIII, fibronectin, vitronectin, and von Willebrand factor (vWF). Fibrin sealants are typically formed by enzymatic reactions involving fibrinogen, thrombin, and factor XIII, among others. The terms "fibrin sealant" and "fibrin glue" are interchangeable.

Human thrombin is a 295 amino acid protein consisting of two polypeptide chains A and B joined by disulfide bonds. The B-chain of α-thrombin is involved in thrombin's proteolytic activity on fibrinogen and other proteins and in its autolytic activity leading to β-thrombin and γ-thrombin degrading polypeptides. Cleavage of the B chain at the Arg106-Tyr107 bond yields a 70 amino acid B1 fragment and a 188 amino acid β-thrombi (B2) form. γ-Thrombin is generated by further cleavage of the β-thrombin B2 chain at the Lys190-Gly191 bond. Typically, these proteolytic forms of thrombin have a reduced ability to convert fibrinogen to insoluble chains of fibrin than native α-thrombin.

The human α-thrombin B chain is further post-translationally modified, e.g., by glycosylation, possibly more potent and/or when compared to the unmodified form and/or when compared to other forms of post-translational modification. or lead to stable thrombin forms (for other glycosylated proteins, see Ricardo J. Sola and Kai Griebenow. "Glycosylation of Therapeutic Proteins: An Effective Strategy to Optimize Efficacy". BioDrugs. ). Mature α-thrombin has a single N-linked glycosylation site on its "heavy chain". Sialic acid, also called neuraminic acid, is important for glycoprotein bioavailability, function, stability, and metabolism. The glycosylated form of α-thrombin (mature native human α-thrombin, amino acid residue N416) can be further sialylated with 1-5 sialic acid residues. Thus, α-thrombin may contain different degrees/levels of sialylation, eg α-thrombin may have varying amounts of N-acetylneuraminic acid (NANA) residues (sialic acid) at the glycosylation site. The degree of sialylation can affect protein potency and stability. Typically, the higher the sialylation level, the higher the potency and the higher the stability.

The results show that separation of α-thrombin by anion exchange chromatography allows separation of many α-thrombin peaks containing different amounts of NANA. The results show that treatment of thrombin with N-acetylneuraminidase, an enzyme capable of removing NANA residues from the ends of glycans, affects the elution profile of thrombin, leading to an overall increase in the peaks to the left of the chromatogram. results in a shift (when compared to untreated thrombin). Thus, the method of the invention can be used to purify and/or quantify different α-thrombin glycoforms that differ, for example, in NANA content. In one embodiment, the method according to the invention can be used to purify/isolate different homogeneous α-thrombin species containing substantially identical profiles of NANA using AEX-HPLC. In another embodiment, the method according to the invention is used to purify/isolate homogeneous post-translationally modified α-thrombin from a protein solution and/or from a solution comprising heterogeneous post-translationally modified α-thrombin. can be

A "post-translational modification" is a step in protein biosynthesis. Proteins are made by ribosomes that translate mRNA into polypeptide chains. Polypeptide chains undergo post-translational modifications, such as cleavage, folding, and other processes before they mature into the final protein product.

After translation, post-translational modifications of an amino acid affect protein function by adding it to other biochemical functional groups, altering the chemical properties of the amino acid, or altering its structure (e.g., formation of disulfide bridges). Expand the range of Modifications can be glycosylation, phosphorylation, ubiquitination, methylation, nitrosylation, acetylation, lipidation. Typically, modifications control protein behavior, eg, activation and deactivation of enzymes.

に付加される７６個のアミノ酸からなる８－ｋＤａポリペプチドである。ポリユビキチン化タンパク質は、ユビキチン化タンパク質の分解及びユビキチンの再利用を触媒する２６Ｓプロテアソームにより認識される。ヒストンメチル化及び脱メチル化は転写のためのＤＮＡの利用能に影響を及ぼすため、メチル化は、エピジェネティック調節の周知の機構である。アミノ酸残基は、修飾の作用を増加させるために、単一のメチル基又は複数のメチル基に抱合され得る。 Glycosylation typically has significant effects on protein folding, conformation, distribution, stability, and activity. Glycosylation involves the addition of sugar moieties to proteins ranging from simple monosaccharide modifications of nuclear transcription factors to highly complex branched polysaccharide modifications. Phosphorylation plays an important role in regulating many cellular processes, including cell cycle, growth, apoptosis, and signaling pathways. Methylation, which is the transfer of a one-carbon methyl group to nitrogen or the transfer of oxygen to an amino acid side chain (N- and O-methylation, respectively), increases the hydrophobicity of proteins and, when bound to carboxylic acids, , can neutralize the negative amino acid charge. In ubiquitination, ubiquitin binds to the target protein's lysine via the C-terminal glycine of ubiquitin.

It is an 8-kDa polypeptide consisting of 76 amino acids appended to the . Polyubiquitinated proteins are recognized by the 26S proteasome, which catalyzes the degradation of ubiquitinated proteins and the recycling of ubiquitin. Methylation is a well-known mechanism of epigenetic regulation, as histone methylation and demethylation affect the availability of DNA for transcription. Amino acid residues may be conjugated to a single methyl group or multiple methyl groups in order to increase the effect of the modification.

"Unmodified α-thrombin" refers to α-thrombin that has not undergone post-translational modifications, eg, non-glycosylated and/or therefore non-sialylated α-thrombin.

“Homogeneous post-translationally modified α-thrombin” refers to substantially identical forms of α-thrombin, eg, with respect to glycosylation and/or sialylation levels. Homogeneity between different α-thrombin molecules is expressed by having the same post-translational modifications, eg, the same glycosylation, although each thrombin molecule can have different levels and/or forms of other modifications. α-Thrombin can be glycosylated and/or sialylated forms of α-thrombin. In one embodiment, homogeneous α-thrombin is homogeneously glycosylated. In another embodiment, the homogeneous α-thrombin is homogeneously sialylated. Glycosylated α-thrombin can have 0-5 sialic acid residues. In some embodiments, the homogeneous post-translationally modified α-thrombin is sialylated α-thrombin having 1, 2, 3, 4, or 5 sialic acid residues.

As used herein, the different/heterogeneous post-translationally modified α-thrombin populations of α-thrombin and unmodified α-thrombin are also known as “different α-thrombin species”. Heterogeneous post-translationally modified α-thrombin may have different glycosylated and/or sialylated forms.

As used herein, "α-thrombin glycoform" refers to homogeneously glycosylated and/or sialylated α-thrombin species.

In some embodiments, α-thrombin prepared using the methods described herein is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85% , to a level of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity Homogeneous. For example, less than 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100%, 50-55%, 50-60%, 50-65%, 50-70% , 50-75%, 50-80%, 50-85%, 50-90%, 50-95%, 50-99%, 50-100%, 55-60%, 55-65%, 55-70% , 55-75%, 55-80%, 55-85%, 55-90%, 55-95%, 55-99%, 55-100%, 60-65%, 60-70%, 60-75% , 60-80%, 60-85%, 60-90%, 60-95%, 60-99%, 60-100%, 65-70%, 65-75%, 65-80%, 65-85% , 65-90%, 65-95%, 65-99%, 65-100%, 70-75%, 70-80%, 70-85%, 70-90%, 70-95%, 70-99% , 70-100%, 75-80%, 75-85%, 75-90%, 75-95%, 75-99%, 75-100%, 80-85%, 80-90%, 80-95% , 80-99%, 80-100%, 85-90%, 85-95%, 85-99%, 85-100%, 90-95%, 90-99%, 90-100%, 95-99% , 95-100% identity, and any range between the disclosed percentages.

The present disclosure provides methods for isolating homogeneous populations/species of α-thrombin, eg, homogeneous post-translationally modified α-thrombin. Further provided are purified homogeneous populations of α-thrombin, as well as formulations comprising isolated homogeneous post-translationally modified α-thrombin and a pharmaceutically acceptable carrier or diluent.

In yet another aspect, provided herein are formulations comprising purified α-thrombin or isolated homogeneous post-translationally modified α-thrombin disclosed herein. In some embodiments, purified α-thrombin or isolated homogeneous post-translationally modified α-thrombin is obtained by the methods disclosed herein. In some embodiments, purified α-thrombin or isolated homogeneous post-translationally modified α-thrombin can be obtained by the methods disclosed herein. In some embodiments of the formulation, the α-thrombin is from a mammalian plasma source. In some embodiments, the formulation includes a pharmaceutically acceptable carrier or diluent. The formulations disclosed herein can be frozen or lyophilized.

A formulation containing purified α-thrombin or homogeneous post-translationally modified α-thrombin can be applied to the surface of a subject. The formulation may be applied in a solution containing fibrinogen. The formulations may be used, for example, in hemostasis, tissue fixation, graft fixation, wound healing, and anastomosis.

In another aspect, provided herein are formulations comprising the purified β-thrombin disclosed herein. In some embodiments, purified β-thrombin is obtained by the methods disclosed herein. A formulation containing purified β-thrombin can be applied to the surface of a subject. The formulation may be applied in a solution containing fibrinogen. The formulations may be used, for example, in hemostasis, tissue fixation, graft fixation, wound healing, and anastomosis.

The term "purified β-thrombin" typically refers to alpha-thrombin, gamma-thrombin, and/or another protein present in the starting thrombin-containing solution using anion exchange chromatography methods. Refers to the β-thrombin preparation obtained after isolation of β-thrombin. "Isolated" generally when referring to "isolated homogenous post-translationally modified α-thrombin" or "isolated β-thrombin" refers to a molecule or compound that does not occur in nature. is separate and distinct from the whole organism in which it is found, and/or is sufficiently free of other molecules so that the molecule can be used for its intended purpose.

A "pharmaceutically acceptable carrier or diluent" is one that is compatible with the constituents in the formulation and that provides reasonable benefits/risks without undue toxicity, irritation, allergic reactions, or other complications. Refers to proportionate reagents, compounds, materials, compositions, diluents suitable for use in contact with human and animal tissue. Pharmaceutically acceptable carriers suitable for use with the formulations disclosed herein can be liquid, semi-solid and solid materials. The carrier can be a sponge, film, cast, surgical dressing, or bandage.

In another aspect, a subject in need of hemostatic treatment, sealing, graft fixation, wound healing, anti-adhesion, and/or anastomosis comprises applying to the subject an effective amount of a formulation according to the present invention. A method is provided herein. The terms "therapeutically effective amount" or "effective amount" refer to the dose necessary to prevent or treat (alleviate some or all symptoms) of a disease, disorder, or condition. Effective amounts can be determined based on any changes during the course of the disease in response to administration of the formulation. Effective doses can vary depending on the age and weight of the subject, the disease and its severity (eg, early stage or late stage), and other factors that can be recognized by those skilled in the art.

In another aspect, provided herein is a method for screening compounds for potential use in stabilizing thrombin activity in an aqueous liquid thrombin formulation, the method comprising adding a test compound to , incubating with a solution comprising α-thrombin, and after incubation, α-thrombin and/or degrading polypeptides (e.g., β-thrombin and/or γ-thrombin polypeptides) according to the methods disclosed herein. and identifying one or more suitable test compounds that have potential use in stabilizing thrombin activity, the preferred compounds having an initial α-thrombin content and Comparatively, a compound that maintains α-thrombin content at a level of about 70% to about 100% and/or from about 0% to about when compared to the level of degraded polypeptide in the absence of the test compound. Compounds that reduce the level of degraded polypeptides by 30%.

"Stabilization of thrombin activity" refers to, for example, reduction of thrombin autolysing activity. "Stabilization of thrombin activity" means as an aqueous thrombin solution, e.g., a concentrated thrombin solution, e.g. It can also refer to maintaining thrombin activity when superconserved without significantly impairing thrombin's biological activity toward heterologous substrates, including its ability to convert fibrinogen to fibrin. "Room temperature" is intended to include temperatures from about 20°C to about 25°C, or from 22°C to about 25°C. "Thrombin activity" refers to thrombin-mediated conversion of heterologous substrates, including proteins, e.g., conversion of fibrinogen to fibrin, as well as conversion of factor VIII to factor VIIIa, factor XI to factor XIa. conversion of factor XIII to factor XIIIa, and conversion of factor V to factor Va. A "heterologous substrate" is a substrate other than thrombin, preferably a protein substrate. In some embodiments, thrombin activity refers to the conversion of fibrinogen to fibrin.

The term "stabilized" maintains thrombin activity/potency within a liquid thrombin formulation, e.g., at a level of about 70% to about 100% (e.g., about 90-100%) compared to the initial thrombin activity. means that

In some embodiments, the compound(s) inhibit thrombin autolysis by about 70% to about 100%, about 70% to about 95%, about 70% to about 90%, or about 70% to about 80% inhibition and retention of about 70% to about 100%, about 70% to about 95%, about 70% to about 90%, or about 70% to about 80% of thrombin biological activity.

The term "test compound" or "test substance" is a chemically defined compound or mixture of compounds whose ability to stabilize thrombin is defined by the methods of the present invention. These compounds or mixtures of compounds are described by Dave A. et al. Parkins and Ulla T. Lashmar "The formulation of biopharmaceutical products". PSTT Vol. 3, No. 4 April 2000, and other excipient(s)/stabilizers known in the art.

The term "initial α-thrombin content" is defined, for example, immediately after thawing a frozen thrombin formulation, immediately after reconstituting thrombin powder, and/or under conditions that allow autolysis (e.g., 800 IU/ml to 10,000 IU /ml or higher, e.g., storage at -18°C or less for >2 years, 2-8°C for >1 month, and/or room temperature for >1 day). refers to the activity of thrombin on fibrinogen measured in the thrombin liquid formulation of .

In some embodiments, the incubation time is greater than 1 day (eg, at room temperature), greater than 2 years at -18°C or lower, and/or 2-8°C as an aqueous thrombin solution, such as a concentrated thrombin solution. for more than a month at

The term "degradation polypeptide" refers to β-thrombin and/or γ-thrombin polypeptides.

The term "surface" can refer to the external surface of the skin that is visible to the naked eye, and surfaces of internal body parts that are part of the internal structure of an organism. External surfaces include, but are not limited to, facial skin, throat, scalp, chest, back, ears, neck, hands, elbows, hips, knees, and other skin areas. Examples of internal body parts include body cavities or anatomical openings exposed to the external environment, as well as genital regions including nostrils, lips, ears, uterus, vagina and ovaries, lungs, anus, spleen, liver, and heart muscle. include, but are not limited to, the viscera of The surface may be a bleeding or non-bleeding site.

The formulations and kits disclosed herein are useful for tissue and organ graft fixation, for sealing surgical wounds, in vascular surgery including providing hemostasis, for anti-adhesion, and for arterial, gastrointestinal , and for tracheal anastomosis.

As used herein, "subject" includes animals of human and mammalian origin. In one embodiment, the subject is a surgical or wound patient.

Purified α-thrombin and/or purified β-thrombin may be used in hemostatic products. α-Thrombin or β-thrombin can be used in combination with fibrinogen to form a fibrin sealant.

Fibrinogen can be prepared from the initial blood composition. The blood composition can be whole blood or a blood fraction, ie a product of whole blood such as plasma. In one embodiment of the invention, the fibrinogen component consists of a plasma-derived protein solution, a biologically active ingredient (BAC), which includes tranexamic acid and/or arginine, lysine, their pharmaceutically acceptable It may further comprise stabilizers such as salts, or mixtures thereof. The BAC may be derived from cryoprecipitate, particularly concentrated cryoprecipitate.

The term "cryoprecipitate" refers to a blood component obtained from frozen plasma prepared from whole blood. Cryoprecipitate can be obtained by thawing frozen plasma at low temperatures, typically between 0 and 4° C., resulting in the formation of a precipitate containing fibrinogen and factor XIII. The precipitate can be collected, for example, by centrifugation and dissolved in a suitable buffer such as a buffer containing 120 mM sodium chloride, 10 mM trisodium citrate, 120 mM glycine, 95 mM arginine hydrochloride. The solution of BAC may further contain factor VIII, fibronectin, von Willebrand factor (vWF), vitronectin, etc., as described, for example, in US Pat. No. 6,121,232(B) and WO9833533. can contain. Preferably, the BAC composition may include stabilizers such as tranexamic acid and arginine hydrochloride. Typically, the amount of fibrinogen in the BAC ranges from about 40 to about 60 mg/mL. The amount of tranexamic acid in the solution of BAC can be from about 80 to about 110 mg/mL. The amount of arginine hydrochloride can be about 15 to about 25 mg/mL.

If necessary, the solution is buffered to a physiologically compatible pH value. The buffer may consist of glycine, sodium citrate, sodium chloride, calcium chloride, and water for injection as solvent. Glycine may be present in the composition in an amount of about 6 to about 10 mg/mL, sodium citrate may range from about 1 to about 5 mg/mL, and sodium chloride may range from about 5 to about 9 mg/mL. Well, calcium chloride can be at a concentration of about 0.1-0.2 mg/mL.

In another embodiment, the concentration of plasminogen and plasmin in the BAC composition is adjusted according to, for example, US Pat. 15 μg/ml or less, such as 5 μg/ml or less of plasminogen using the methods described in US Pat. In another embodiment of the invention, when the concentration of plasminogen and plasmin in the BAC composition is reduced, the composition does not contain tranexamic acid and aprotinin. The fibrinogen solution may be a BAC2 component (EVICEL®) or any other fibrinogen containing solution such as purified recombinant fibrinogen or cryoprecipitate produced from human plasma.

Fibrinogen can be of human or non-human sources, including autologous, pooled plasma. It is also possible that fibrinogen can be prepared by recombinant methods or chemically modified.

The following examples, which demonstrate certain embodiments of the invention, should not be construed as limiting the scope of the invention, but rather as contributing to the complete description of the invention.

For all examples, the following terms are used herein.
"Thrombin solution" refers to a solution of about 3000 IU/ml thrombin in 20 mM sodium acetate, pH 6.9-7.1, with a total protein concentration of about 1 mg/ml thrombin.

A "thrombin preparation" or "formulated thrombin" has a thrombin activity in the range of 800-1200 IU/ml, a total protein concentration of about 5.7-6.5 mg/ml, and a pH of 5.0 at 6.9-7.1. Formulated thrombin drugs EVITHROM® Thrombin, Topical (Human) (ETHICON, Inc.), or EVICEL® Fiber Sealant (ETHICON, Inc.) with ~6.5 mg/ml human serum albumin (HSA) .) refers to the thrombin component. HSA used in the formulation of thrombin contains the stabilizer acetyltryptophan.

In all of the embodiments below, the thrombin solution is not formulated with thrombin present (e.g., stabilizer-free) and not highly concentrated (approximately 3000 IU/ml); It was used as a 'control sample' containing a thrombin-degrading polypeptide because it tends to degrade faster (compared to thrombin present in 'thrombin formulations').

In the examples below, the tools were evaluated for their ability to provide separation between α-thrombin, its degradation polypeptides, and HSA, if present, and to quantify α-thrombin and its degradation polypeptides. rice field.

Generally, "good separation" is considered "baseline resolution" between peaks. "Baseline resolution" means sufficient separation of the analytes such that the peaks detected as representative of the elution of the analytes do not overlap, ie, the detector response returns to baseline levels between peaks.

By "sufficient separation" there is a clear difference between the elution peaks, but the detector response does not fully return to baseline levels between the peaks.

Poor separation is considered when overlapping peaks appear in the chromatogram.

Resolution/resolution levels were assessed visually unless indicated by a value. The resolution (R _s ), which is the extent to which a chromatographic column separates components from each other, is mathematically defined as follows, where the values are listed in the examples below: It is the difference between a peak and its previous peak retention time multiplied by a constant of 1.18 and then divided by the sum of the peak widths of 50% of the peak height.

A resolution level of 2 or more is considered "baseline resolution" and thus indicates good separation and allows good quantification of peaks. A resolution of 1.5 or greater (less than 2) is considered "sufficient resolution" to allow separation and quantification.

The terms "separation" and "separability" are used interchangeably with respect to the effectiveness of a chromatographic method.

Example 1: Reversed Phase High Performance Liquid Chromatography (RP-HPLC) of HSA, Thrombin Solution, and Formulated Thrombin
A standard procedure for separating proteins and their fragments employs HPLC equipment in reversed-phase mode. The basic principle of the RP-HPLC method is a device consisting of a dual pump, polar column and detector. Protein is injected into the device and retained on the column. Increasing the concentration of organic solvent releases the retained proteins and peptides from the column and elutes to a detector where a response is obtained based on the amount of protein eluted at a given time. .

In the following examples, RP-HPLC with a C4 column (Phenomenex, Jupiter, 00G-4167-B0, 4.6 x 250 mm) was used to measure the distance between α-thrombin, its degrading polypeptides, and HSA, if present. It was isolated and evaluated as a tool to quantify α-thrombin and its degradation polypeptides.

HPLC analysis was performed using a Waters Alliance Separation Module e2695 equipped with a 100 μL injection loop and a photodiode array (PDA) detector 2998 (scanning A _{190 nm} to A _{450 nm} ) connected to an integrated Waters Used with column oven.

The organic solvent/solution used for the separation was
Buffer A: HPLC grade water + 0.1% (v/v) trifluoroacetic acid (TFA),
Buffer B: was acetonitrile + 0.1% (v/v) trifluoroacetic acid (TFA).

Different solution gradients (ratio of buffer A to buffer B over time) were evaluated.

The injected samples were a) 30 μL thrombin solution, b) 100 μL formulated thrombin, c) 100 μL 5 mg/ml HSA, and d) 100 μL HPLC grade water as a blank sample.

In all experiments, different injection volumes were based on the fact that the thrombin in the thrombin solution was more concentrated when compared to formulated thrombin.

Prior to injection, all samples were filtered through a 0.45 μm polyvinylidene difluoride (PVDF) membrane (Millipore, to filter larger particles, eg aggregates). Samples were stored at 10° C. in the integrated sample compartment until injection into the HPLC.

FIG. 1 shows a magnified view of a representative chromatogram of the area of the elution peak.

In all figures, sample delineation is shown from top to bottom based on the start of the chromatogram, and the injected sample (top to bottom) is listed on the chromatogram. Different sample runs are shown in one figure as superimposed overlays.

Although there was separation between the main peaks of HSA and thrombin, overall good separation was not achieved. The peaks eluted from the column were so close together that reliable peak separation and/or quantification was not possible.

Additional experiments were performed in which conditions including temperature, column chemistry (different tested RP columns are listed below), mobile phase chemistry (methanol, etc.), and gradients were varied, but α-thrombin and its degradation were Resolution between polypeptides and/or other proteins such as HSA was not improved.

The following additional RP columns were tested for separation and quantification as described above: C4, 5 μm, 300A, 4.6×250 mm; Sepax BioC18, 3 μm, 300A, 4.6×150 mm; LiChroCART Sepax C8, 5 μm, 300 A, 4×250 mm; Waters XBridge C4, 3.5 μm, 300 A, 4×250 mm.

Therefore, RP-HPLC is not a suitable tool when "one step" or "single column separation" and/or quantification of α-thrombin in the presence of degrading polypeptides and/or HSA is desired. concluded.

Example 2: Anion Exchange High Performance Liquid Chromatography (AEX-HPLC) and Elution Using a Linear Salt Gradient and pH 8.0 The standard procedure for separating proteins and their fragments is HPLC in anion exchange mode. Adopt equipment. The basic principle of the AEX-HPLC method is a device consisting of a dual pump, polar column and detector. Protein is injected into the device and retained on the column. Changing the solvent properties (e.g., salt concentration, pH) releases the proteins and peptides retained on the column and elutes to the detector, where the amount of protein eluted at a given time is , the response is retrieved.

In this experiment, HPLC analysis using an anion exchange column separated between α-thrombin, its degradation polypeptides, and HSA, if present, and was a tool for quantifying α-thrombin and its degradation polypeptides. was evaluated as

AEX-HPLC analysis was performed using a Waters Alliance Separation Module e2695 equipped with a 100 μL injection loop, PDA detectors were used at A _{220 nm} and A _{280 nm} , and an integrated Waters column oven was used at 25°C. rice field. The column used was a Sepax 403NP5-4625 (Sepax Proteomix SAX-NP5 NP 4.6 x 250 mm 403NP5-4625). The columns (4.6 mm wide and 250 mm long) are based on 5 μm polymer beads. The beads are non-porous, monodisperse particles with quaternary ammonium chemistry.

For elution from AEX-HPLC, a linear salt gradient between Buffer A: 20 mM Tris (pH 8.0) in HPLC grade water and Buffer B: 20 mM Tris (pH 8.0) and 1 M NaCl in HPLC grade water. was used.

The injected samples were a) 30 μL of thrombin solution, b) 100 μL of formulated thrombin, c) 100 μL of 5 mg/ml HSA, and d) 100 μL of buffer A as a blank sample.

All samples were filtered through a 4 mm syringe filter with a 0.45 μm pore size PVDF membrane before injection. Samples were stored at 10° C. in the integrated sample compartment until injection into the HPLC. The run time was 37 minutes, the flow rate used was 0.8 mL/min, and the pressure was approximately 18000 kPa (2600 psi).

FIG. 2 shows a magnified view of a representative chromatogram of the area of the elution peak.

The results show that AEX-HPLC using an elution buffer of pH 8.0 and a linear salt gradient to 1 M NaCl did not provide sufficient separation and/or did not allow reliable quantification. The peaks eluted from the column were very close to each other and resolution was poor.

Example 3: AEX-HPLC and Elution Using a Linear Salt Gradient and pH 6.0 The previous example showed that at pH 8.0 and a linear salt gradient, the separation between α-thrombin, its degrading polypeptides, and HSA was showed limited.

In this example, elution using pH 6.0 phosphate buffer with an increasing gradient to 1 M NaCl was evaluated using the column, apparatus, and experimental setup described in Example 2.

For elution from AEX-HPLC, buffer A: HPLC grade 20 mM phosphate buffer in water (pH 6.0) and buffer B: HPLC grade 20 mM phosphate buffer in water (pH 6.0) and 1 M NaCl. A linear salt gradient between was used.

The injected samples were a) 30 μL of thrombin solution, b) 100 μL of formulated thrombin, c) 100 μL of 5 mg/ml HSA, and d) 100 μL of buffer A as a blank sample.

FIG. 3 shows representative chromatograms obtained for different samples.

The results show that AEX-HPLC using an elution buffer of pH 6.0 and a salt gradient to 1 M NaCl did not provide sufficient separation and/or did not allow reliable quantification.

Acetyltryptophan seen in the chromatogram is the stabilizer present in the HSA formulation.

Example 4: Elution using AEX-HPLC and a linear salt gradient and pH 7.5 The previous examples separated using elution buffers of pH 6.0 (Example 3) and 8.0 (Example 2). An elution buffer of pH 7.5 was tested, as it showed that .

HPLC analysis and conditions were performed as described in Example 2. Elution was performed using Tris buffer pH 7.5 with an increasing linear gradient of NaCl. The buffers used were Buffer A: 20 mM Tris (pH 7.5) in HPLC grade water and Buffer B: 20 mM Tris (pH 7.5) and 1 M NaCl.

The injected samples were a) 30 μL of thrombin solution, b) 100 μL of formulated thrombin, c) 100 μL of 5 mg/ml HSA, and d) 100 μL of buffer A as a blank sample. The results are shown in FIG. 4 (enlarged view). The results show that AEX-HPLC using an elution buffer of pH 7.5 and a salt gradient to 1 M NaCl did not provide sufficient separation and/or did not allow reliable quantification.

Example 5: Elution using AEX-HPLC and a linear NaNO ₃ salt gradient and pH 8.0 As an alternative to NaCl, NaNO ₃ (sodium nitrate) removes thrombin-degrading polypeptides from α-thrombin and the rest in solution. was evaluated for its ability to separate from proteins such as HSA.

A linear salt gradient was evaluated between Buffer A: 20 mM Tris (pH 8.0) in HPLC grade water and Buffer B: 20 mM Tris (pH 8.0)/1 M _NaNO3 . Columns, equipment, and experimental setup were as described in Example 2.

The injected samples were a) 30 μL thrombin solution, b) 100 μL formulated thrombin, and c) 100 μL 5 mg/ml HSA. The results are shown in FIG. As described above, the thrombin solution contained degraded polypeptides.

The results show that using _NaNO3 as an eluent greatly increases the separation between HSA, acetyltryptophan, and thrombin, but sufficient resolution was not achieved between thrombin and its degrading polypeptides. indicates that

Example 6: Elution using AEX-HPLC and a linear gradient from pH 9.1 to pH 3.4 As an alternative to using a salt gradient to achieve separation between related peaks eluted from the AEX-HPLC resin, an amine-based buffer pH gradients using liquids were evaluated. Detection was performed at A _{280 nm} due to the amine nature of the buffer.

Buffers A and B were 20 mM piperazine (Sigma Aldrich, P45907), 20 mM triethanolamine (Sigma Aldrich, T9534), 20 mM bis-tris propane (Sigma Aldrich, B4679), and 20 mM 1-methylpiperazine (Sigma Aldrich, 13000 ~ 1) was contained.

Buffers were adjusted to pH 9.1 (buffer A) and pH 3.4 (buffer B) by titration with HCl. Total run time was 46 minutes. In all experiments, a linear gradient was run between steps 2 and 3 (see Table 1 below) and the linear gradient run time was 20 minutes. During steps 2 and 3 material is eluted from the column.

The injected samples were a) 30 μL of thrombin solution, b) 100 μL of formulated thrombin, c) 100 μL of 5 mg/ml HSA, and d) 100 μL of buffer A as a blank sample.

Flow conditions and ratios between buffers are shown in Table 1. The pH of the elution buffer depends on the ratio between buffers A and B. In general, a typical HPLC run consists of at least the following steps:
Load the material onto the equilibrated column (the time between steps 1 and 2, "Load").

After this step, material is eluted from the column (between steps 2 and 3, "linear gradient"). This can be either isocratically (without changing the buffer composition when compared to the loading and/or equilibration step) or gradients (one of the buffer properties, e.g. salt concentration, polarity/pH ) can be implemented through In this example, elution was performed using a linear gradient.

In the next step, the column can be regenerated (between steps 3 and 4, "Column Regeneration"), i.e. altered properties (salt concentration, polarity, pH) to elute any remaining material from the column. at the highest concentration of , giving additional time to the remaining material.

The final step (between steps 5 and 6, "column equilibration") is an equilibration step to return the column to its original state suitable for further separations.

Conditions, columns, and equipment were as described in Example 2.

－工程１と２との間－装填－５分。
－工程２と３との間－直線勾配－２０分。緩衝液Ｂの増加は毎分４．５％であった。
－工程３と４との間－「カラム再生」－５分。
－工程５と６との間－「カラム平衡化」－１５分。

- Between steps 1 and 2 - Loading - 5 minutes.
- between steps 2 and 3 - linear gradient - 20 minutes. Buffer B increase was 4.5% per minute.
- Between steps 3 and 4 - "Column Regeneration" - 5 minutes.
- Between steps 5 and 6 - "Column equilibration" - 15 minutes.

In all of the tables below, the steps are characterized and numbered in the same manner.

Figure 6 shows a representative chromatogram of the injected sample. FIG. 7 is an enlarged view of the thrombin elution region of the chromatogram from FIG.

The results show that good resolution was obtained between HSA, acetyltryptophan and thrombin. Several thrombin peaks were obtained under the conditions described (best shown in Figure 6). Additional parameters were examined in the examples below to further enhance the resolution of the thrombin peak.

Example 7: Elution using AEX-HPLC and a linear pH gradient with different flow rates To obtain better separation between the different thrombin peaks, the temperature (at 25°C as in Examples 2-7) and pH gradient were kept constant. While maintaining, different flow rates were evaluated.

Buffers A and B were the same as in Example 6. The program (see Table 2 below) was run four times. Each time the flow rate was different: 0.25, 0.5, 0.75, and 1 mL/min.

The slopes evaluated are as shown in Table 2 below.

The injected sample was 30 μL of thrombin solution for each flow rate tested.

FIG. 8 shows a magnified view of the chromatogram of the flow screen that was performed.

Separation (visual inspection) between HSA, acetyltryptophan, and thrombin was not affected by increased flow rates (data not shown).

It was shown that the resolution between α-thrombin and its degraded polypeptide increased with increasing flow rate (Fig. 8). Best resolution was achieved at 1.0 mL/min, eg more peaks were observed.

Example 8: Elution Using AEX-HPLC and a pH Gradient from 100% Buffer A In this example, the AEX-HPLC method was run at a higher pH compared to the previous example for resolution. Initiating action was assessed. For this purpose a pH gradient with 100% Buffer A (see Table 3) instead of 90% (as used in Table 2) was used. In steps 1, 2, 5, and 6, the same set of samples was run in the manner described in Table 3 as a control, except that the percentage of buffer A was 90 and the percentage of buffer B was 10. It has been executed.

Buffers A and B were the same as in Example 6. The experimental set-up was the same as in Example 6 unless otherwise stated.

Resolution between peaks was assessed visually. Table 3 shows the slope and flow conditions.

－緩衝液Ｂの増加は毎分４．５５％であった。

- Buffer B increase was 4.55% per minute.

Results (data not shown) indicated that starting the gradient at a higher pH gave better resolution of the thrombin peak. Therefore, elution of proteins from columns with a wider pH range results in better separation between peaks.

In the examples below, a pH gradient of 100% Buffer A was used.

Example 9: Elution using AEX-HPLC and a linear pH gradient with increasing gradient run times Gradient increase (i.e., time increment to step 2 and 3) were evaluated. A run time of 46 minutes (as in Example 6) was also tested. Resolution was measured from each peak to the previous peak.

Unless otherwise stated, the experimental setup was the same as in Example 8 using the parameters listed in Table 3.

Thrombin solution (30 μL) was injected. Buffers A and B are the same as in Example 6. Tables 4, 5, 6, and 7 show the resolution achieved at retention times and total run times of 46, 51, 56, and 61 minutes, respectively. Retention time is the interval between the time of injection and peak apex (topmost point of peak) detection as representative of elution.

The results showed that at total run times of 56 and 61 min (gradient lengths of 30 and 35 min), additional peaks eluting in regions distinct to the thrombin peak were separated when compared to shorter run times. indicate.

Advantageously, gradient lengths greater than 25 minutes can be used to obtain additional peaks eluting in distinct thrombin regions.

A total run time of 56 minutes was used in the following examples.

Example 10 Effect of Linear Gradient on Separation Power Different linear slope gradients were evaluated for their ability to improve the separation of the thrombin peak (the gradient used between steps 2 and 3). The slope is affected by increasing the percentage of Buffer B per minute. A lower percentage increase in Buffer B per minute results in a shallower slope when compared to a higher percentage increase in Buffer B per minute, thereby affecting the elution profile of the protein. In contrast to Example 8, where the slope was affected by using different starting pHs, in this example the slope was affected by increasing pH values at different rates per minute (starting and The endpoint pH is equal for all samples).

Buffers A and B were the same as in Example 6. The experimental set-up was the same as in Example 6 unless otherwise stated. Thrombin solution (30 μL) was injected. Buffer A (30 μL) was used as a blank (not shown).

Percent increases in Buffer B per minute were evaluated: 4.5%, 4.25%, 4%, 3.75%, and 3.5%. For example, when a percentage of 4.5% per minute is used, after the first minute you will get 4.5% Buffer B per minute, after 2 minutes you will get 9% Buffer B per minute, Up to 100% Buffer B per minute is obtained, such as 13.5% Buffer B per minute after 3 minutes. At each minute, buffer A was used to bring the total solution to 100%.

A shallower slope typically results in an increase in execution time. Run times were as follows: 48, 49.5, 51, 52.7, and 54.6 minutes, respectively, for the Buffer B percentages listed.

Figure 9 shows the chromatograms obtained for the different gradients evaluated and visual inspection was performed to determine the resolution.

Results show that all slopes tested show satisfactory/sufficient separation between thrombin peaks, with an increase of 3.5% having the best separation (seen in magnified view, data not shown).

Example 11 Identification of Different Thrombin Peaks by Injecting Commercial Standards on AEX-HPLC To identify the thrombin peaks in the chromatograms, α, β, and A thrombin solution was injected as in Example 7 in addition to the γ-thrombin standard.

Standards (Haematological Industries; Human alpha-Thrombin, HTI HCT-0020, Human beta-Thrombin, HTI-0022, Human gamma-Thrombin, HTI-0021) were diluted to 0.3 mg/mL prior to injection. 30 μL of thrombin solution and 100 μL each of α-, β-, and γ-standards were injected into the HPLC. Buffer A was used as a blank.

Buffers A and B were the same as described in Example 7 and used in the program shown in Table 2.

FIG. 10 shows duplicate chromatograms. Based on the peaks obtained for the standards, correlating peaks of the thrombin solution can be identified, thereby confirming that separation between α-thrombin and its degradation polypeptides β and γ thrombin can be achieved. It was possible. In addition, it was noted that α-thrombin eluted as multiple peaks in the chromatogram.

Example 12: Identification of Thrombin Peaks Using Western Blot as a Qualitative Tool In the previous example, identification of thrombin peaks was performed on AEX-HPLC by injection of commercially available α, β, and γ thrombin standards. rice field.

To corroborate the above results, in this example the thrombin peak was collected from the injected thrombin solution and analyzed using commercially available standards based on α-thrombin and its degradation polypeptides of known size, β- and γ-thrombin. (as in Example 11) were further qualitatively characterized by Western blot.

To obtain sufficient amounts of β and γ thrombin, the thrombin solution is subjected to conditions that enhance thrombin autolysis for at least 12 hours at room temperature (about 20-25° C.), such as overnight, before injection into the HPLC. was incubated. The experimental set-up was similar to Example 10 and an increase of 3.5% B/min was used. 60 μL of sample (in quadruplicate) and 100 μL of Buffer A were injected.

Distinct peaks (shown and identified in Figure 11 and Table 8) were collected from four separate runs (due to the low amount of protein present in each peak) and pooled (according to visual identification and retention time). , was lyophilized due to the large amount recovered. Each lyophilized and pooled peak was reconstructed (in a lower volume of water compared to the initial volume due to the limited loading capacity of SDS-PAGE). The resulting pooled samples were separated by SDS-PAGE, transferred onto nitrocellulose sheets and immunoblotted against polyclonal anti-α-thrombin (data not shown). A mixture of α, β, γ was used as a control.

Peaks were identified based on the molecular weights of the bands obtained in Western blots and by comparison with a standard α, β, γ mix.

The results obtained in the Western blot show that the thrombin-cleavage polypeptide elutes in peaks 3, 5 and 5a. Peaks 1, 2, 4, 6, and 8 with similar molecular weights were identified as α-thrombin. The relative areas of the peaks labeled "not identified" were smaller compared to the "identified" peaks.

Without wishing to be bound by mechanism, α-thrombin was separated into several peaks in the HPLC-AEX system, probably representing several α-thrombin species differing in their net charge.

The results of Examples 11 and 12 advantageously show that complete separation between α, β, γ-thrombin, α-thrombin species, and HSA (HSA separation data not shown in this example) was achieved at 3.00 min/min. It shows that it can be obtained using an AEX-HPLC linear pH gradient from 100% Buffer A to 100% Buffer B with a 5% Buffer B slope. The composition of the buffer is as described in Example 6.

Example 13 Identification of α-Thrombin Species Separated by HPLC-AEX The purpose of this example was to characterize multiple peaks detected for α-thrombin in HPLC-AEX chromatography. We explored whether different species of α-thrombin are due to different post-translationally modified α-thrombin forms. There are some post-translational modifications. Glycosylation is one possibility. Since glycosylation affects the activity of proteins (Ricardo J. Sola and Kai Griebenow. "Glycosylation of Therapeutic Proteins: An Effective Strategy to Optimize Efficacy". BioDrugs. 2010, et al. (1):92-1; The examples focus on glycosylation.

Human α-thrombin has a single N-linked glycosylation site on its "heavy chain". α-Thrombin separated in HPLC-AEX chromatography has different sialylation levels on N-linked glycosylation sites, i.e., varying amounts of N-acetylneuraminic acid (NANA) (sialic acid) at the glycosylation sites. We explored the possibility that it corresponds to the α-thrombin it contains.

For this purpose, the thrombin solution was subjected to N-acetylneuraminidase treatment according to the manufacturer's instructions (Sigma Aldrich, N2876). N-acetylneuraminidase (NANase) is an enzyme that can remove NANA residues from the ends of glycans. Removal of these charged sugar residues results in the same level of overall charge for each of the glycosylated proteins.

In the next step, the NANase-treated thrombin solution was injected into the AEX-HPLC system as described in Example 12. An untreated thrombin solution was injected as a control.

The results (Figure 12) show that treatment of thrombin with NANase affects the elution profile, resulting in an overall shift of the peaks to the left of the chromatogram (when compared to untreated thrombin solution). Loss of the negative charge of the sialic acid residues increases the net protein charge of thrombin at a given pH, thereby allowing faster elution from the column. In view of these results, it can be concluded that many of the peaks are due to differences in NANA content.

Example 14 Purification of Homogeneous Post-Translationally Modified α-Thrombin from Protein Solutions In previous examples, it was found that α-thrombin can be separated into distinct peaks containing different amounts of NANA/sialylation levels. .

In this example, the aim was to use AEX-HPLC to isolate homogeneous α-thrombin species containing substantially identical profiles of NANA. The following conditions were used.

The column used was the same as in Example 2, Sepax 403NP5-4625, 4.6 mm wide x 250 mm long. 30 μL of thrombin solution, 100 μL of formulated thrombin, and 100 μL of buffer A (not shown) were injected.

Protein elution from the resin was performed with 20 mM piperazine (Sigma Aldrich, P45907), 20 mM triethanolamine (Sigma Aldrich, T9534), 20 mM bis-tris propane (Sigma Aldrich, B4679), and 20 mM 1-methylpiperazine (Sigma Aldrich, B4679). 13000-1) was used. Buffers were adjusted to pH 9.1 (buffer A) and pH 3.4 (buffer B).

A linear pH gradient with Buffer B increments of 3.5% per minute and the flow rate conditions shown in Table 9 were used.

FIG. 13 shows full length chromatograms of two eluted thrombin samples. FIG. 14 shows a close-up view of the α-thrombin species and degraded polypeptide elution regions.

Regarding the chromatogram of formulated thrombin, it can be seen that complete separation between HSA, several charged α-thrombin species (indicated by arrows), and acetyltryptophan can be achieved.

Regarding the chromatogram of the thrombin solution, it can be seen that a complete separation between some charged α-thrombin species (indicated by arrows) and the degraded polypeptides can be achieved.

These results demonstrate that different homogenous α-thrombin species can be separated from each other in thrombin-containing samples. The results also show that the quality of the separation makes it possible to purify homogeneous post-translationally modified α-thrombin from protein solutions and/or solutions containing heterogeneous post-translationally modified α-thrombin.

Example 15: Quantification of Homogeneous Post-Translational Modified α-Thrombin and Thrombin Degrading Polypeptides The previous examples demonstrated that α-thrombin peaks containing a homogeneous content of NANA can be well resolved by AEX-HPLC. show. Complete separation of peaks allows quantification of α, β, γ thrombin variants in thrombin-containing solutions by calculating the integrals of the relevant separated peaks (see Table 10). Conditions used for AEX-HPLC were as described in previous examples.

^＊面積はソフトウェアによって計算されたピーク下積分面積を指す。
^＊＊計算されたピーク合計面積からの相対面積。

^* Area refers to the integrated area under the peak calculated by the software.
^** Relative area from calculated peak total area.

It was shown that quantification of all α-thrombin species and degraded polypeptides was obtained.

The method can also advantageously be used to quantify the amount of α-thrombin from all proteins present in solution and/or to screen suitable formulations.

The results also show that one type of β-thrombin can be purified and quantified using the method of the invention.

While various embodiments have been described herein, many modifications and variations to those embodiments may be implemented. Also, although materials are disclosed for particular components, other materials may be used. The foregoing description and the following claims are intended to cover all such modifications and variations.

Any patent, publication, or other disclosure that is incorporated herein by reference, in whole or in part, is subject to any existing definitions, statements, or other disclosures set forth in this disclosure. are incorporated herein only to the extent not inconsistent with the disclosure of . As such, and to the extent necessary, the disclosure as explicitly set forth herein supersedes any conflicting statements incorporated herein by reference.

Citation or identification of any reference in this application shall not be construed as an admission that such document is available as prior art to the present invention.

Section headings are used herein to facilitate understanding of the specification and should not necessarily be construed as limiting.

[Mode of implementation]
(1) A one-step chromatographic method for quantifying α-thrombin in a solution comprising α-thrombin and at least one of an α-thrombin degrading polypeptide or another protein, comprising:
contacting the solution with an anion exchanger;
separating said α-thrombin from said at least one of said α-thrombin degrading polypeptide and/or said another protein by anion exchange chromatography by differential elution conditions;
quantifying said α-thrombin.
(2) The method of embodiment 1, further quantifying the one or more degraded polypeptides.
(3) The method of embodiment 1 or 2, wherein the separated α-thrombin is homogeneous post-translationally modified α-thrombin, whereby homogeneous post-translationally modified α-thrombin is quantified.
(4) The method of embodiment 3, wherein said homogeneous post-translationally modified α-thrombin is homogeneously glycosylated α-thrombin, whereby homogeneously glycosylated α-thrombin is quantified.
(5) The method of embodiment 4, wherein said isolated homogeneous post-translationally modified α-thrombin is homogeneous sialylated α-thrombin, whereby homogeneous sialylated α-thrombin is quantified.

(6) The method of any of embodiments 1-5, wherein said solution comprises said another protein, and said another protein is human serum albumin.
(7) A one-step chromatography method for quantifying homogeneous post-translationally modified α-thrombin in a solution containing heterogeneous post-translationally modified α-thrombin, comprising:
contacting the solution with an anion exchanger;
separating the homogeneous post-translationally modified α-thrombin from the heterogeneous post-translationally modified α-thrombin by anion exchange chromatography by differential elution conditions;
quantifying said homogenous post-translationally modified α-thrombin.
(8) the solution further comprises at least one of an α-thrombin-degrading polypeptide or another protein, and the method comprises combining the homogenous post-translationally modified α-thrombin with the α-thrombin-degrading polypeptide and 8. The method of embodiment 7, comprising also separating from said at least one of said further proteins.
(9) The method of any of embodiments 1-8, wherein said differential elution conditions comprise a pH gradient.
(10) The method of embodiment 9, wherein said pH gradient is generated by using an eluent comprising an amine or a mixture of amines.

(11) The method of any of embodiments 1-10, wherein the anion exchanger is made of non-porous particles.
(12) The method of any of embodiments 1-11, wherein said chromatography method is an anion exchange high performance liquid chromatography method.
(13) A method for purifying α-thrombin from a solution comprising said α-thrombin and at least one of an α-thrombin-degrading polypeptide or another protein, comprising:
contacting the solution with an anion exchanger;
separating said α-thrombin from said at least one of said α-thrombin degrading polypeptide and/or said another protein by anion exchange chromatography using differential elution conditions;
collecting the α-thrombin fraction;
and thereby obtaining purified α-thrombin.
(14) The method of embodiment 13, wherein said α-thrombin is from a human blood or plasma source.
(15) The method according to embodiment 13 or 14, wherein the recovered α-thrombin fraction is homogeneous post-translationally modified α-thrombin, thereby obtaining purified homogeneous post-translationally modified α-thrombin.

(16) The method of embodiment 15, wherein the recovered homogeneous post-translationally modified α-thrombin is homogeneous glycosylated α-thrombin, thereby obtaining purified homogeneous glycosylated α-thrombin.
(17) The method according to embodiment 16, wherein the recovered homogeneous post-translationally modified α-thrombin is homogeneous sialylated α-thrombin, thereby obtaining purified homogeneous sialylated α-thrombin.
(18) The method of any of embodiments 13-17, wherein said solution comprises said another protein, and said another protein is human serum albumin.
(19) The method according to any of embodiments 13-18, wherein said method consists of one chromatography step.
(20) A method for purifying homogeneous α-thrombin glycoforms from a solution containing heterogeneously glycosylated α-thrombin species, comprising:
contacting the solution with an anion exchanger;
separating said homogeneous α-thrombin glycoform from said heterogeneous species by anion exchange chromatography using differential elution conditions;
recovering a homogenous α-thrombin glycoform fraction;
thereby obtaining a purified homogeneous α-thrombin glycoform.

(21) The method of embodiment 20, wherein said purified homogeneous α-thrombin glycoform is further quantified.
(22) The method of any of embodiments 13-21, wherein said differential elution conditions comprise a pH gradient.
(23) The method of embodiment 22, wherein the pH gradient is generated by using an eluent comprising an amine or a mixture of amines.
(24) The method of any of embodiments 13-23, wherein said anion exchanger is made of non-porous particles.
(25) Isolated homogeneous post-translationally modified α-thrombin.

(26) The isolated homogeneous post-translationally modified α-thrombin according to embodiment 25, wherein said α-thrombin is from a human plasma source.
(27) The isolated homogeneous post-translationally modified α-thrombin according to embodiment 25 or 26, which is homogeneously glycosylated α-thrombin.
(28) The isolated homogeneous post-translationally modified α-thrombin of any of embodiments 25-27, wherein said α-thrombin is represented by one specific glycoform.
(29) The isolated homogeneous post-translationally modified α-thrombin according to embodiment 28, which is homogeneously sialylated α-thrombin.
(30) Purified α-thrombin obtainable by the method according to any one of embodiments 13-24.

(31) A formulation comprising the isolated homogeneous post-translationally modified α-thrombin according to any of embodiments 25-29 and/or the purified α-thrombin according to embodiment 30.
(32) Use of the formulation according to embodiment 31 for hemostasis, sealing, graft fixation, wound healing, anti-adhesion and/or anastomosis.
(33) as a first component, a purified homogeneous α-thrombin glycoform obtainable by a method according to any of embodiments 20-24, and/or a glycoform according to any of embodiments 25-29; A kit comprising a container containing isolated homogeneous post-translationally modified α-thrombin.
(34) A one-step chromatography method for quantifying β-thrombin in a solution comprising β-thrombin and at least one of α-thrombin, γ-thrombin, or another protein, comprising:
contacting the solution with an anion exchanger;
separating said β-thrombin from said at least one of said α-thrombin, γ-thrombin and/or said another protein by anion exchange chromatography by differential elution conditions;
quantifying said β-thrombin.
(35) The method of embodiment 34, wherein said chromatography method is an anion exchange high performance liquid chromatography method.

(36) A method for purifying β-thrombin from a solution comprising said β-thrombin and at least one of α-thrombin, γ-thrombin, or another protein, comprising:
contacting the solution with an anion exchanger;
separating said β-thrombin from said at least one of said α-thrombin, γ-thrombin and/or another protein by anion exchange chromatography using differential elution conditions; ,
collecting the β-thrombin fraction;
and thereby obtaining purified β-thrombin.
(37) The method of any of embodiments 34-36, wherein said differential elution conditions comprise a pH gradient.
(38) The method of embodiment 37, wherein the pH gradient is generated by using an eluent comprising an amine or a mixture of amines.
(39) The method of any of embodiments 34-38, wherein said anion exchanger is made of non-porous particles.
(40) Purified β-thrombin obtainable by the method according to any of embodiments 36-39.

(41) Isolated β-thrombin.
(42) A formulation comprising the purified β-thrombin according to embodiment 40 and/or the isolated β-thrombin according to embodiment 41.
(43) Use of the formulation according to embodiment 42 for hemostasis, sealing, graft fixation, wound healing, anti-adhesion and/or anastomosis.
(44) A kit comprising, as a first component, a container comprising purified β-thrombin according to embodiment 40 and/or isolated β-thrombin according to embodiment 41.
(45) A method for screening compounds for potential use in stabilizing thrombin activity in an aqueous liquid thrombin formulation, comprising incubating a test compound with a solution containing α-thrombin for a given time. and, after said incubation, quantifying α-thrombin and/or degrading polypeptides according to any of embodiments 1, 2, 6, or 9-12, and potential use in stabilizing thrombin activity. and identifying one or more suitable test compounds having a - A method, wherein the compound is a compound that maintains thrombin content and/or reduces the level of degraded polypeptide by about 0% to about 30% when compared to the level of degraded polypeptide in the absence of said test compound.

Claims (6)

A post-translationally modified α-thrombin containing a sialic acid residue at the site of glycosylation . Post- translationally modified α-thrombin according to claim 1, which is from a human plasma source. The post-translationally modified α-thrombin of claims 1 or 2, wherein said glycosylation site is an N-linked glycosylation site on the heavy chain. 4. The post-translationally modified α-thrombin of claim 3, having 1, 2, 3, 4, or 5 of said sialic acid residues at said N-linked glycosylation site. A formulation comprising post -translationally modified α-thrombin according to any one of claims 1 to 4 for use in hemostatic procedures, sealing, graft fixation, wound healing, anti-adhesion and/or anastomosis for, formulations . A kit comprising, as a first component, a container containing post -translationally modified α-thrombin according to any one of claims 1 to 4 , for use in hemostasis treatment, sealing, graft fixation, wound healing, A kit for use in anti-adhesion and/or anastomosis .

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