JPH07508009A - Purification of kringle-containing proteins and especially t-PA - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Purification of kringle-containing proteins and especially t-PA Field of the invention The present invention provides a method for extracting kringle from a solution containing kringle-containing proteins. The present invention relates to a method for purifying glu-containing proteins.

Background of the invention Purification of proteins, especially those intended for pharmaceutical therapy, is a delicate process. That's about it. The starting material for purification is typically a biological fluid, such as plasma or will be serum or the protein can produce that protein. If it is produced by culturing a cell line that is cormorant. Regardless of the source of the starting material, it normally contains a large number of undesired proteins. and other materials from which the protein of interest is to be isolated. Probably. Traditional purification steps include filtration or centrifugation of dead cells, protein materials, etc. separation by precipitation and partitioning of the protein material, thereby A protein solution containing the desired protein is obtained, but it is still contaminated by other proteins. It's contaminated. This protein solution is then typically applied to a suitable purification column. For example, affinity chromatography or ion exchange chromatography - etc.

Affinity chromatography can be used for small-scale purification, e.g. Fully suitable. However, for commercial scale manufacturing it is mainly , not only because of the high cost of the column material but also because of the unsatisfactory chemical stability of the material. , lacking in charm. This column material becomes contaminated after a given use, and it can be recycled. It would increase manufacturing costs since new The material will have to be replaced more frequently.

Therefore, affinity chromatography is useful for large scale samples where large amounts of impurities are present. It is not particularly well suited for initial purification steps in models.

When purifying proteins on a large scale, it is important to use low-cost and chemical methods in the early stages. and physical stability are also amenable to using column materials. Also, this material cannot withstand high flow rates, e.g. 10-20 column volumes per hour. Must be. Such standards are typically achieved with ion exchange columns. It will be.

Traditional methods to elute proteins from ion exchange columns rely on their pH or is to charge ionic strength or both.

The proteins and impurities in question are then determined by i, o, their isoelectric point (p and will appear in different eluates. the protein in question and or therefrom The impurities to be separated have pIs in the same range, but such elution It can be non-specific.

This is because proteins with similar pH values elute together from the column to some extent. Because it will happen. Additional time-consuming and cost-increasing purification steps may therefore be necessary and risk increased degradation and loss of the desired protein. There will be danger.

EP 0256836B describes a method for purifying t-PA, which As a result, a mixture of t-PA with different molecular weights transforms the mixture into a hydroxyapatite. and successively various pH salt concentrations or both. The fractions are separated by elution. Therefore, this method has the above-mentioned deficiencies. I'm sure you'll suffer from some pain. The same method can be used for purification using t-PA or cation exchange columns. as described in EP 0261941A and eluted by salt gradient elution method. Applies to the methods listed. Japanese Patent Application No. 63-091080 discloses an adsorption carrier. on a body, e.g. controlled pore glass (CPG), silica gel or diatomaceous earth. By adsorbing t-PA and eluting it with a solution containing ω-amino acids. A method for purifying t-PA is described. Adsorption matrix is ion exchange Substantially different from the material. In adsorption chromatography, the matrix The retarding forces on the surface are mainly due to the surface energy and van der Waals forces, and their separation is due to polar and steric factors. Depends on the cause. In ion exchange chromatography, the retention power is mainly , is an electrostatic attraction, and its separation depends on the ionic nature of its components.

A problem encountered with known methods is that the protein in question is removed from the ion-exchange column. changing the pH and ionic strength to any substantial degree during the elution step of the The desired protein can be selectively eluted from the ion exchange column without This could be solved if the eluent that can be eluted makes it possible to . If this is possible, only the protein of interest will be eluted, while the impurities will be , will remain on the column.

The present invention surprisingly provides that a particular type of amino acid, the so-called ω-amino acid , based on the fact that it has been shown to have this desirable property. 1ys- Plasminogen, a derivative of plasminogen, S-5epharose in high yield with ε-amino caproic acid (6-AHA) It was more particularly shown that it could be selectively eluted from the column.

Summary of the invention In its broadest aspect, the invention provides proteins that include kringle-containing proteins. The Kringle-containing proteins that can bind to ω-amino acids are purified from the stock solution. A method for preparing a column comprising the steps of: a) the kringle-containing protein is a column material; Apply the protein solution to the ion exchange column under conditions such that it binds to: b) without changing its 9H and ionic strength in any substantial degree, ω - selectively elute kringle-containing proteins with amino acids; and C) A further suitable purification step removes the kringle-containing eluate from step b). isolating the protein.

In a narrower aspect, the invention provides plasminogen or plasminogen inducers. The present invention relates to a method for purifying a conductor, such as lys-plasminogen.

Brief description of the drawing The invention will be further explained with reference to the drawings. Here, FIG. 1 shows 0.5M NaCl Elution profile of Iys-plasminogen from S-5epharose by shows the rules: Figure 2 shows the 1ys-platinum of S-3epharose in 3mM 6-AHA. Showing the elution profile of Sminogen: Figure 3 shows Iys-plasminase in 0.5M NaCl and 3mM 6-AHA. Figure 4 shows non-reduced 5O3-PAGE of eluate fractions from the eluate of 0. Analysis of the dialysis eluate from the eluate of 1ys-plasminogen in 5M NaCl Elution profile from affinity column (ricin-3hepharose) It is: Figure 5 shows the translucency from the eluate of Iys-plasminogen with 3mM 6-ANA. Elution of the precipitate eluate from the affinity column (lysine-3hepharose) The profile is: Figure 6 shows the syn-5 hepharose color shown in Figures 4 and 5, respectively. Non-reducing 5OS-PAGE of various elution fractions from the sample elution profile: do Figure 7 shows lys-3 hepharose column. Results from plasmin analysis of plasminogen are shown.

Before describing the present invention, it is important to define certain terms used below. It can help you solve the problem.

Plasminogen: Plasminogen generally refers to human glU-plasmin. means nogen.

Plasminogen derivative: This term refers to one of the five kringle domains. at least 1, preferably more than 3, and most preferably all 5 Various plasminogens including 1ys-plasminogen with a glu domain will cover degraded or mutated forms. The mutation is the desired one or more within a native molecule with the purpose of modifying the properties of that molecule in a manner containing substitutions of amino acid residues or deletions or additions of amino acid residues. means an amino acid with a free amino group.

Kringle-containing protein: Kringle-containing protein is, for example, t-PA and proteins containing one or more so-called kringle structures known from plasminogen. It means quality.

Selective elution Two expressions “selective elution” refers to a method that contains undetectable amounts of undesired proteins. This refers to the elution of the desired protein.

Includes biological or aqueous solutions containing bacteria.

Biological solution: This is plasma, blood obtained by extraction of organs of human or animal origin. means a supernatant, a fraction thereof, or a solution.

Substantially constant pH is defined as pH compared to the pH in the buffer before addition of the ω-amino acid. ±I pH units, preferably ±0.5 pH units.

Substantially constant ionic strength means that the ionic strength is The ionic strength in the buffer can be varied within a range of ±20%, preferably ±10% of the pH. It means that it can be converted into

detailed description The purification method of the present invention can be used to purify kringle-containing products from biological solutions, such as plasma and serum. Although used for protein purification, it is preferably Such products from culture media or solutions obtained by the destruction of cells that produce substances intracellularly. can be used for the purification of proteins. Kringle-containing protein Kringle-producing cells encode natural or kringle-containing proteins Transformed or transfected cell lines capable of expressing inserted DNA can be.

Ion exchange columns are well known in the art. Suitable ion exchange color The systems are S-5epharose @ and Fractogel SOs.

The ω-amino acid preferably contains a carbon between its carboxylic acid and its ω-amino group. Those containing at least 4 carbon atoms in the chain. This carbon chain can be linear or cyclic. It can be in the form of Examples of suitable linear ω-amino acids are 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid (ε-amino caproic acid (6- AHA)), 7-aminohexanoic acid, 8-amine octanoic acid, lysine and algium It's nin. An example of a cyclic ω-amino acid is trans-4-aminomethyl cyclohexane. Xane carboxylic acid (tranexamic acid) ) and para-aminomethylbenzoic acid.

The method of the invention, in principle, binds to ω-amino acids and during elution the pH ion exchange color without any substantial change in ionic strength and/or ionic strength. For purification of any kringle-containing proteins that can be eluted from can be used for The greatest benefit of this is that the protein in question is Impurities in the form of other proteins that bind to Lamb may By not changing the concentration in any substantial degree, there will be no residue on the column. This means that there will be. The selective elution effect of ω-amino acids is due to the kringle Their binding to containing proteins is so strong that it l can be varied or by the amount that causes release from the column material. It lies in the fact that it is possible to induce some conformational changes in the child.

An important group of proteins that can be purified by nature are the so-called Kling L-containing proteins (e.g., L, Patthy, Bl.

od Coagulation and Fibrinolysis I (1 990), 153-166. ). These proteins are one or more Kringle structures, many of which contain lysine or lysine analogs. , for example, trans-4-aminomethyl cyclohexane carboxylic acid, and ε -Known to bind with amine caproic acid. This includes Ringer's Examples of proteinaceous proteins include European Patent Application No. 191.843; No. 207.589: No. 241.209; No. 233.013: No. 240.3 No. 34; No. 293.936: In No. 293.934 and No. 292.009 t-PA and t-PA analogs as described growth factOr and around the kringle domain and activation site It has various changes. Other examples are plasminogen, 1ys-lasmino apo-riboprotein, hepatocyte growth factor, FXII, prothrombin, uPA, and a recently discovered protein with putative tumor suppressor properties (Degen S, J, P, (1991), Abstract 20th Lindens Troem Lang Conference, Vingsted, Den mark, 164-166).

The present invention is mainly used in early purification steps where many impurities are present in large quantities. Although it is intended that some or most of the impurities be column exchange or affinity chromatography column purification or other suitable means. It may also be used in subsequent or final purification steps that have been removed by can.

The eluate from the ion exchange column is subjected to further suitable purification steps, e.g. dialysis. , purified by gel filtration and affinity chromatography.

The invention will be explained in more detail below with reference to the purification of plasminogen.

Human plasminogen is a single-chain glycoprotein with a molecular weight of approximately 90,000 have. It circulates in the plasma at a concentration of about 2 μM and 5 ephar human by binding to ose■ or by affinity chromatography on syn It can be purified from plasma. The natural form of plasminogen is N)It - has a terminal glutamic acid and is referred to as "glu-plasminogen".

This plasminogen molecule consists of 791 amino acids, 24 disulfide bridges, 5 triple-loop kringle” structure and serine protease domain. Motsu.

Several low molecular weight forms of human plasminogen are naturally occurring molecular proteins. Obtained as a result of decomposition and digestion. The nomenclature used for these molecules is , based on the sequence position of the amino-terminal amino acid. glue for a very short time - Exposure of plasminogen to plasmin is similar to glu-plasminogen. resulting in the cleavage of the amino-terminal 77 residues of the plasminogen molecule, i.e. 1ys-plasminogen, which has a molecular weight of approximately 83,000 and is similar to fibrin. It produces something that is very important for some properties of the degradation system.

The conversion of plasminogen to the active protease, plasmin, is a so-called plasminogen activators (P catalyzed by As)). Plasmin plays a major role in the circulation of vertebrates. It is thought to be a fibrin-degrading enzyme, but also in other extravascular proteolytic events. It can also play a useful role.

Plasminogen and Iys-plasminogen are thrombolytic agents988), See 165-175. ).

Human plasminogen can be purified from plasma by several methods. Ru. Recent methods for purification from human plasma are described in 1. Steteri, G., Deut. sch and E, T, Mertz, Fed, Proc, 29.64 7 (1979) and 5science, 170.1095-1096 (+970) based on affinity chromatography as described by.

The affinity matrix (lysine 5epharose @) is prepared by the covalent coupling of the α-amino group of syn to 5epharOSe. Ru. Plasma diluted with water was stored in phosphate buffer (pH 7,4) at room temperature. Equilibrated by or passed through a thin-5 epharose column, and then The system is washed with phosphate buffer. Next, plasminogen is 0.2Mε- Eluted by amino caproic acid (pH 7.4). This ε-amino capro The phosphoric acid was dissolved in 5ephadex equilibrated with phosphate buffer in the cold. The above gel filtration removes it from plasminogen.

The application of human proteins purified from plasma always has a certain risk of viral infection. Including risks. In particular, the risk of HIV and hepatitis infection is due to plasma-derived human proteins. has become a major concern in handling.

To avoid this risk, plasminogen can be produced using recombinant DNA technology. would be advantageous. Attempts to make plasminogen in transfected cells However, intracellular plasminogen activation and subsequent degradation may result in reasonable yields. clearly provides a barrier to the production of intact recombinant plasminogen in I made it clear.

This problem is caused by protease inhibition, which can inhibit plasminogen activation. was solved by expressing plasminogen together with the drug. European published patent issued See Application No. 319.944.

Lys-plasminogen or glu-plasminogen is therefore B) IK Intracellularly, Lys- or glu-plasminogen and α-2-plasmin can be produced by co-expression of inhibitor, α, -P1.

Glu-plasminogen or Lys-plasminogen is transfected with The cells were analysed by α-1-antitrypsin (AAT) in BHK cells or derivatives thereof. It can also be expressed at times. Co-expression with AAT is B) produced by IK cells. urokinase is inhibited by AAT cells. Urokinar Activation of plasminogen and plasmin-mediated proteolytic cleavage by , thus resulting in reduced and high levels of intact plasminogen.

Plasminogen was purified by one step of ion-exchange chromatography followed by glue-sin-5e. 2, comprising one step of affinity chromatography on pharose. It is purified from the culture medium by steps. More particularly, ion exchange columns (S-Sep harose) and then eluted with 0.5M NaCl. melt The output was transferred to an affinity chromatography column (Lysine-Seph). arose) and plasminogen was removed by elution with 6-AHA. isolated.

As mentioned earlier, transfected BH cells are In addition, it will express urokinase. of plasminogen and urokinase pl are 6.7-8.1 and 8.5, respectively.

Therefore, both plasminogen and urokinase bind to the ion exchange column. while the coexpressed plasminogen activation inhibitor is present in the void volume. It will be.

Although a high yield of plasminogen can be obtained by Kempo, the Activation may occur to some extent during the affinity chromatography step. It has been discovered that it is possible. This is probably due to inhibition of plasminogen activation. caused by the fact that the agent is removed in the preceding ion-exchange column purification step. be awakened.

Plasminogen and plasminogen active substances were collected on an ion exchange column (S-3e pharose), only a small amount of plasmino in the eluate -gen active substance (urokinase) activates plasminogen to plasmin causes and results in plasminogen products containing small amounts of plasmin be able to.

The use of 6-AHA in the first ion-exchange purification step surprisingly Ensure selective elution of LYS-plasminogen without co-elution of kinase. It was revealed that Plasminogen production in the eluate with urokinase Unintended activation to rasmin is therefore avoided, and by the salt gradient method A purer product is obtained compared to elution.

Experiment part Lys-plasminogen as described in European Patent Application Circular No. 319.914 Prepared by a method similar to .

Urokinase (uk) secreted by BHK cells together with plasminogen It is inhibited by coexpressed α-1-antitrypsin. The presence of this active substance , as evidenced by the development of the logarithmic product in a chromogenic assay for plasmin. I can do it. The presence of UK in the culture medium is due to the “fibrin layering technique”. overlay technique)” (Granelli-Piperno and Re1ch: J, Exp, Med, +48. p, 233 -34.1978).

Divide the culture medium sample into two equal fractions.

The comparison is made from the ion exchange column, S-9epharose, in the first stage. Perform between elutions with either 6-ANA-buffer or 0.5M NaCl. cormorant. Each eluate was further dialyzed and purified on lysine-3 epharose. refine.

Equilibrium buffer y -: 0.02M NaH2POa, 0.04M Na C1,0,02% Tween 80゜3mg/I　0 Aprotinin: pH 5.0° Wash buffer l/equilibration buffer.

Washing buffer 2 = Equilibrium buffer containing 0.15 M NaCl + pH ・5.5° Elution Buffer 3 ・Contains 0.50M NaCl and aprotinin Equilibration buffer without; pH = 5.5° sample for plasmin measurement. After aspiration, aprotinin was added to each sample.

The column was 5 ml, applied 750 ml of culture medium, and the flow rate was 125 ml. It was 1/hour.

The elution profile is shown in FIG. 1, top.

The elution profile is quite steep. Lys-plasminogen is the second peak It exists within.

In Figure 1, the underlined numbers (112 and 3) indicate when the various buffers were added. (1. Equilibration buffer, 2. Washing buffer (0,15M) NaCl, pH 5,5) and 3. Elution buffer y (0.5M NaC1゜pH5 ,5)). The underlined numbers (22,,,30) are in 5OS-PAGEC Figure 3). represents the fraction number used in

Experiment 2 S-9epharose, elution with 6-AHA (certification number 9] 0724) Experiments identical to those described above. However, the column is eluted in two stages, and the first elution is , 0.02M NaHx POa, 0.18M NaC1.0.02% Elution buffer containing Tween 80 and 3mM 6-ANA, pH 5.5 Do more.

A second elution is performed as in experiment 1.

The elution profile is shown in FIG. Lys-plasminogen is in the second peak exist. The third beak represents various impurities.

In Figure 2, the underlined numbers (I, 2.3 and 4) indicate when various buffers were added. (1. Equilibration buffer, 2. Washing buffer y - (0,5 M NaCl, pH 5,5), 3. ff1a Batsu y I (0,18M Na C1゜6-ANA, pH 5,5) and 4. Elution buffer y-1 [(0,5M NaCl pH 5,5)). The underlined numbers (16,,,42) are 5O3 - Represents the bipartite number used in PAGEC Figure 3).

Figure 3 shows non-reducing 5DS-PAGE of fractions from elution from both experiments.

A large number of macromolecules are eluted with some aprotinin.

This lane number represents: (Experiment I) 1. Molecular weight standard 2. Plasma 1us-Plasmibogen (control) 3. Assay 910723 Culture medium 4 used in fraction diagram 1 No. 23 5. Fraction diagram 1 No. 24 6. Fraction diagram 1 No. 25 7. Fraction diagram 1 No. 26 (Experiment 2) 8. Culture medium 9 used in assay 910724, fraction diagram 2 No. 16 10, Fraction diagram 2 No. 22 11, Fraction diagram 2 No. 23 12, Fraction diagram 2 No. 26 +3. Fraction diagram 2 No. 30 14, Fraction diagram 2 No. 42 15. Molecular weight standard.

Elution with 3mM 6~AHA yields pure product (Iy s-plasminogen), while elution with NaCl (lanes 4-7 ) is found to result in a product contaminated with some high molecular weight impurities. .

isolated (by activation by urokinase in the eluate from the ion exchange column) To test whether the collected 1ys-plasminogen contains a small amount of plasmin. Further purification was performed by lysine-3 epharose. This purification step , a, O, will remove aprotinin.

Therefore, both eluates were dialyzed against the equilibration buffer given below and the lysine - Purified by affinity chromatography on 5 epharose .

Column size: 5ml, flow rate: 50ml/hour Equilibrium buffer: 0. I M Nag HPO4, 0.02% Tween 80.3mg/l April.

pH7.4゜ Wash buffer l/equilibration buffer.

Washing Buffer 2 Contains 0.50M NaCl and does not contain aprotinin Equilibrium buffer.

The elution was carried out using 0.0.0% containing 0.02% Tween 80. IM Nag) IPOa and and 6-ANA from 0 to 5 m&l, each with a gradient of 50 m1. It was.

Figure 4 shows the elution flow of the dialysis eluate from experiment 1 (NaCl).

The numbers under the underline (l, 2.3 and 4) represent the following stages: 1, sample suitability; for, 2. Washing buffer l, 3. Wash buffer 2 and 4. Start of elution gradient.

The numbers above the underline (8, 1O112) are used in 5DS-PAGE (Figure 6) represents the fraction number.

Figure 5 shows the elution flow of the dialysis eluate from experiment 2 (6-AHA).

The numbers under the underline (l, 2.3 and 4) represent the following stages: 12 sample adaptations. for, 2. Washing buffer l, 3. Wash buffer 2 and 4. Start of elution gradient.

In some cases, a shoulder is visible, similar to Figure 4. Literature, plasmino Two sugar-added forms of the gene have different affinities for 3epharOSe. It is also known for its nitty-gritty.

Figure 6 is a 5DS-PAGE of the elution fraction from ricin-5 epharose. This lysine protein was present only through two purification steps. This shows that lasminogen is a rather pure product.

This lane number represents; 1. Molecular weight standard 2. Plasma 1us-Plasmibogen3, (1 figure not based on 6-AHA! Sample applied on Figure 4 (from test number 910723) 4,=3 5. Fraction diagram 4 No. 7 6. Fraction diagram 4 No. 9 7. Fraction diagram 4 No. 11 8, applied on Figure 5 (from 1 Figure 2 Certification No. 910724 by 6-AHA) sample 9,=8 10, Fraction diagram 5 No. 7 11. Fraction diagram 5 No. 8 12. Fraction diagram 5 No. 9 +3. Fraction diagram 5th 1θ number 14. Molecular weight standard.

FIG. 7 shows the present invention (A), that is, 6 in the S-3 epharose purification step. - Purification according to elution with AHA and when elution was performed with NaCl Figure 2 shows results from plasma analysis of purified 1ys-plasminogen. this The analysis was performed with chromogenic substrate 52251. . This test is a standard procedure. Here, p-NA is converted to 52251 (H-D-V) by the enzymatic action of plasmin. at-Leu-Lys-pNA), thereby allowing Develop the yellow color measured by All samples were tested with the same protein before assay. diluted to a high quality concentration. Plasmin is converted to 5-sepharose by 6-ANA. It is clearly seen that no color was detected in the product eluted from the sample (no color development). Ru. When elution was carried out with NaCl, the product contained plasmin and Iys-propylene. It shows that some activation of rasmin occurred in the eluate.

1.2.　3゜ S-3epharose 1.2. 3. 4゜ S-3epharose 5DS-PAGE, IOZ gel non-reduced sample 12.3 4 Lysin-3epharose FF1, 2.3. 4゜ Lysin-3 epharose FF5DS-PAGE analysis non-reduced sample minutes Elution with 6-AH△ + Human plasminogen 1φ-fraction 5 11 Fraction 7 11 Fraction 8 1→-fraction 9 4 Hi Fraction 12 Elution with NaCl + Human plasminogen →← Fraction 5 →1 fraction 7 10-Fraction 9 10-Fraction 11 4 Hi Fraction 13 Continuation of front page (51) Int, C1,' Identification symbol Internal reference number CI 2 N 9/64 9152-4B // A 61 K 38/46 (C12N 9/64 C12R1:91) (81) Designated countries EP (AT, BE, CH, DE.

DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE) , AU, BB, BG, BR, CA, CZ, FI, HU, JP, KP, K R, LK, MG, MN, MW, No, NZ, PL, RO, RU, SD .

SK, UA, US FI (72) Inventor Petersen, Lars Christian Denmark, Deko -2970 Halsholm, Habebye 4

Claims (7)

[Claims] 1. In this way, kringle-containing proteins that can bind to ω-amino acids A method for purifying a protein solution containing a kringle-containing protein, the method comprising: The following steps: a) such that the kringle-containing protein binds to the column material; applying the kringle-containing protein solution to an ion exchange column under conditions; b) ω- without changing its pH and ionic strength to any substantial degree. eluting the kringle-containing protein with amino acids; and c) A further suitable purification step removes the kringle-containing material from the eluate from step b). isolating the protein, A method comprising: 2. ω-amino acids are 4-aminobutyric acid, 5-aminopentanoic acid, 6-amino Hexanoic acid, 7-aminoheptanoic acid, 8-aminooctanoic acid, lysine, arginine and t-aminomethylcyclohexane-1-carboxylic acid. , the method of claim 1. 3. Claims that the kringle-containing protein is t-PA or a t-PA analog The method according to item 1 or 2. 4. Claim that the protein is plasminogen or a plasminogen derivative The method according to item 1 or 2. 5. According to claim 4, the plasminogen derivative is Iys-plasminogen. Method described. 6. The preceding claim wherein the ω-amino acid is 6-aminohexanoic acid (6-AHA) The method described in any of the above. 7. The eluate from step b) is subjected to affinity chromatography. , a method according to any of the preceding claims.