CZ328594A3 - PURIFICATION OF CRINGL-CONTAINING PEPTIDES, PARTICULARLY t-PA - Google Patents

Abstract

Kringle containing proteins capable of binding to omega -amino acids can selectively be eluted from an ion exchange column by means of omega -amino acids.

Description

Purification of kringl containing proteins, particularly t-PA

Technical field

The present invention relates to a method for purifying kringl proteins from a protein solution comprising the kringl containing proteins. According to this method, kringl-containing proteins having the ability to bind to ω-amino acids are selectively eluted from the ion exchange column by these ω-amino acids.

BACKGROUND OF THE INVENTION

Purification of proteins, in particular proteins considered to be useful in therapy, is a sensitive process. The starting materials for purification will typically be biological fluids such as plasma or serum, cell lysates or culture media when the protein is prepared by culturing a cell line capable of producing the protein. Regardless of the source of the starting material, this material normally contains numerous unwanted proteins and other materials from which the desired protein must be separated. Traditional purification steps include separating cell debris or similar materials by filtration or centrifugation, precipitating the proteinaceous material, and separating the proteinaceous material to yield a protein solution that contains the desired protein but still contaminated with other proteins. This protein solution is then typically applied to suitable purification columns, such as affinity chromatography or ion exchange chromatography columns or the like.

Affinity chromatography is suitable for small-scale purification, for example for research purposes. However, it is less attractive for large-scale commercial preparation due to the high cost of the column material, but also to the unsatisfactory chemical stability of the material. After a certain period of use, the column material will be contaminated and, because it cannot be regenerated, must often be replaced with new material which increases the cost of production.

Thus, affinity chromatography columns are not particularly suitable for initial large-scale purification steps when many impurities are present.

For large-scale protein purification, it is preferred that in the initial stages a column material of low cost and good chemical and physical stability is used. This material should also be able to withstand high flow rates, for example 10 to 20 column volumes per hour. Typically, ion exchange columns meet these criteria.

The traditional method of eluting proteins from an ion exchange column is by changing the pH or ionic strength or both. The desired product and impurities then appear in various eluates according to, inter alia, their isoelectric point (pI). If the protein of interest and the impurities to be separated have a pI within the same range, this elution may be non-specific since proteins with similar pI values will elute to a certain degree from the column together. Thus, additional purification steps may be required with additional time consumption and cost increases, as well as the risk of increased degradation and loss of the desired protein.

European patent 0 256 836 B discloses a t-PA purification method in which a mixture of t-PA particles of different molecular weights is separated after the mixture is contacted with hydroxyapatite and the different fractions are successively eluted at different pH, concentrations salts or both. Thus, this process has the above drawbacks. The same is true of the process described in European Patent Application 0 261 941 A, in which t-PA is purified on a cation exchange column by elution with a salt gradient. Japanese Patent Application No. 63-091080 discloses a method of purifying t-PA by adsorbing t-PA on an adsorption support, such as controlled pore glass (CPG), silica gel or kieselguhr, eluting with a solution containing ω-amino acid. The adsorption matrix differs substantially from the ion exchange material. In the adsorption chromatgoraphia, the delay forces on the matrices are predominantly surface energy, and the Van der Waals forces and division depend on polar and steric factors. In ion exchange chromatography, the delay forces are predominantly electrostatic forces and the separation depends on the ionic nature of the components.

The problem encountered in known methods can be solved if the protein of interest can be eluted from the ion exchange column with an eluting agent that is capable of selectively eluting the desired protein from the ion exchange column without changing the pH and ionic strength at any substantial degree during elution. If possible, only the protein of interest elutes, while the impurities remain on the column.

The present invention is based on the fact that it has surprisingly been shown that certain types of amino acids, the so-called ω-amino acids, have desirable properties. In particular, it has been shown that lys-plasminogen, a derivative of plasminogen, can be selectively eluted from the S-Sepharose e-aminocaproic acid (6-AHA) column in high yields.

SUMMARY OF THE INVENTION

In its broadest aspect, the present invention relates to a method of purifying kringl-containing proteins that are capable of binding to ω-amino acids from a solution of proteins containing kringl-containing protein. The method is characterized in that it comprises the following steps:

(a) the protein solution is applied to an ion exchange column under conditions such that the protein containing kringl binds to the column material;

(b) the protein containing kringl is selectively eluted by the action of the ω-amino acid without substantially altering the pH or ionic strength; and

c) isolating the kringl-containing protein from the eluate of step b) by further suitable purification steps.

In a narrower aspect, the invention relates to a method of purifying plasminogen or a plasminogen derivative, such as lys-plasminogen.

The invention will be described in the following by means of the following figures.

Figure 1 shows the elution profile of lys-plasminogen from S-sepharose by elution with 0.514 NaCl.

Figure 2 shows the elution profile of lys-plasminogen from S-sepharose by elution with 3ml4 6-AHA.

Figure 3 shows the non-reducing SDS-PAGE of the eluted fractions eluting with lys-plasminogen by treatment with 0.514 NaCl and 3 mM 6-AHA.

Figure 4 shows the elution profile from the affinity column (lysine-sepharose) of the dialysed eluate in the elution of lys-plasminogen eluting with 0.514 NaCl.

Figure 5 shows the elution profile from an affinity column (lysine-sepharose) of the dialysed eluate in eluting lys-plasminogen eluting with 3 mM 6-AHA.

Figure 6 shows the non-reducing SDS-PAGE of the various eluted fractions from elution on the lysine-sepharose columns shown in Figures 4 and 5.

Figure 7 shows the result of analysis of plasmin of lys-plasminogen further purified on a lysine-sepharose column.

Before introducing the invention, it may be useful for understanding to define some of the terms used herein.

The term plasminogen commonly known human glu-plasmino gene.

The term plasminogen derivatives includes differently degraded or mutated forms of plasminogen, including lys-plasminogen, which retain at least one of the five kringle domains, preferably more than three, and most preferably all five, kringle domains. A mutation involves replacing one or more amino acid residues from a natural molecule or deleting or adding an amino acid residue to modify the properties of the molecule in a desirable manner.

The term ω-amino acids means amino acids with a free amino acid attached to the furthest carbon atom relative to the carboxyl group.

The term proteins containing kringl means those proteins which contain one or more so-called kringle structures known, for example, in t-PA and plasminogen.

The term selective elution means eluting the desired protein with undetectable amounts of undesired proteins.

The term protein solution means a biological solution or an aqueous solution containing numerous proteins, including the desired protein.

The term biological solutions means plasma, serum or fractions thereof or solutions obtained by extraction of organs of human or animal origin.

The term substantially constant pH will mean variations in the range of ± 1 pH unit, preferably ± 0.5 pH units compared to the pH in the buffer prior to the addition of α-amino acid.

A substantially constant ionic strength will mean that the ionic strength can vary within ± 20%, preferably ± 10% of the ionic strength in the buffer prior to the addition of the ω-amino acid.

Although the purification method disclosed herein may be used to purify kringl containing proteins from biological solutions such as plasma and serum, it may preferably be used to purify these proteins from the culture medium or solution obtained by breaking cells that intracellularly produce the protein. The cells that produce the kringl-containing protein may be natural cells or transformed or transfected cell lines that are capable of expressing the inserted DNA sequence encoding the kringl-containing protein.

The ion exchange column is well known to those skilled in the art. Suitable ion exchange columns include S-Sepharose column (S-Sepharose ' ^R)) and Fractogel S0 _third

The ω-amino acids are preferably amino acids with at least 4 carbon atoms in the carbon chain between the carboxyl group and the ω-amino group. The carbon chain may be linear or cyclic. Examples of suitable linear ω-amino acids are 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid (ε-aminocaproic acid (6-AHA)), 7-aminoheptanoic acid, 8-aminooctanoic acid, lysine and arginine. Examples of cyclic ω-amino acids include trans 4-aminomethylcyclohexanecarboxylic acid (tranexamic acid) and para-aminomethylbenzoic acid.

The method of the invention can in principle be used to purify any protein containing kringl that binds to the ω-amino acid and which is able to elute from the ion exchange column without any substantial change in pH and / or ionic strength during elution. A great benefit is that impurities in the form of other column bound proteins along with the desired protein remain on the column, because the pH or ionic strength does not change substantially. The selective elution effect of ω-amino acids may result in their binding to kringl-containing protein being so strong that it may change the p1 of the protein or cause some conformational grain molecules, causing release from the column material.

An important group of proteins that can be purified by the methods of the invention are proteins containing so-called kringl [see, for example, L. Patthy, Blood Coagulation and Fibrinolysis 1, 153 (1990).]. These proteins may contain one or more kringle structures, many of which bind to lysine or lysine analogs, such as trans -4-aminomethylcyclohexanecarboxylic acid and ε-aminocaproic acid. Examples of proteins containing such kringle structures include t-PA and t-PA analogs with various modifications of growth factor and kringle domains and surrounding activation sites as described in published European patent applications 191 843, 201 153, 207 589, 241 209, 233 013, 240 334, 293 936, 293 934 and 292 009. Other examples are plasminogen, lys-plasminogen, apo-lipoprotein α, hepatocyte growth factor, FXII, prothrombin, uPA and a novel protein discovered with anticipated tumor suppressor properties [Degen SJF : Abstract of the 20th Lindestrom Lang Conference, Vingstedt, Denmark, 164 (1991).].

Although this method is primarily intended for use in initial purification stages where the number of impurities is large, it can also be used in later or final purification stages where some or most of the impurities are already removed by prior purification by ion exchange or affinity support column chromatography. or by other suitable means.

The eluate from the ion exchange column is preferably further purified by other suitable purification steps such as dialysis, gel filtration and affinity chromatography.

The present invention will be explained in more detail below with respect to purification of plasminogen.

Human plasminogen is a single-stranded glycoprotein with a molecular weight of about 90,000. It circulates in plasma at a concentration of about 2μΜ. from human plasma can be purified by affinity chromatography on lysine bound to sepharose (Sepharose ®). The natural form of plasminogen has glutamic acid at the amino terminus and is called glu-plasminogen. The plasminogen molecule consists of a 791 amino acid with 24 disulfide bridges, 5 triple loop krill regions and a serine proteinase domain.

Several low molecular weight forms of human plasminogen have been obtained as a result of proteolytic cleavage of the natural molecule. The nomenclature used for these molecules is based on the amino acid position calculated from the amino terminus in the sequence. Exposure of glu-plasminogen to plasmin for a very short time leads to cleavage of 77 residues at the amino terminus of glu-plasminogen to form a plasminogen molecule, lys-plasminogen having a molecular weight of approximately 83,000, which is of great importance for some properties of the fibrinolytic system.

The conversion of plasminogen to active proteinase, plasmin, is catalyzed by so-called plasminogen activators, plasminogen activators (PAs). Plasmin is considered to be the primary fibrinolytic enzyme in the circulation of higher vertebrates, but may also play an important role in other miroovascular proteolytic events.

Plasminogen and lys-plasminogen can be used as a thrombolytic agent [see K. Anderle et al., Haemostatis 18, 165 (1988). ].

Human plasminogen can be purified from plasma in several ways. All previously known methods of purification from human plasma are based on affinity chromatography as described by DG Deutsch and ETMertz: Fed. Why. 29, 647 (1979) and Science 170: 1095 (1970). An affinity matrix (lysine-Sepharose ' ^R)) are prepared by covalently attaching α-amino group of L-lysine to Sepharose. The lysine-sepharose column equilibrated with phosphate buffer (pH 7.4) is then passed at room temperature through water diluted with phosphate buffer. The plasminogen was then eluted with 0.2 M ε-aminocaproic acid (pH 7.4). The ε-aminocaproic acid is removed from plasminogen by cold gel filtration on sephadex equilibrated with phosphate buffer.

The application of human plasma-purified proteins always involves a certain risk of viral infection. In the case of human plasma-derived proteins, this is particularly true of the risk of HIV infection and hepatitis.

To avoid this risk, it would be advantageous to produce plasminogen by recombinant DNA technology. However, attempts to produce plasminogen in transfected cells have shown that activation of intracellular plasminogen and subsequent degradation are an obstacle to the production of intact recombinant plasminogen in acceptable yields. This problem has been solved by coexpressing plasminogen with a proteinase inhibitor capable of inhibiting plasminogen activation, see European Published Patent Application No. 319,944.

Thus, lys-plasmiongene or glu-plasminogen can be produced by coexpression of lys- or glu-plasminogen and α-2-plasmin inhibitor, and _2- PI, in BHK cells. Glu-plasminogen or lys-plasminogen can also be co-expressed with α-1-antitrypsin (AAT) in transfected BHK cells or derivatives thereof. Co-expression with AAT ensures that urokinase produced by BHK cells is inhibited by AAT. Activation of plasminogen by urokinase and plasmin-mediated proteolytic cleavage is thereby reduced and high levels of intact plasminogen are achieved. Plasminogen is purified from the culture medium by a two step process comprising an ion exchange chromatography step followed by an affinity chromatography step on lysine-sepharose. In more detail, the ion exchange column (S-sepharose) is washed and then eluted with 0.5M NaCl. The eluate is passed through an affinity chromatography column (lysine-sepharose) and the plasminogen is isolated by elution with 6-AHA.

As mentioned above, transfected BHK cells will express urokinase in addition to plasminogen. the pI of plasminogen and urokinase is 6.7 to 8.1 and 8.5. It follows that both plasminogen and urokinase are bound to an ion exchange column, while the co-expression produced by the plasminogen activator inhibitor appears in the void volume.

Although high yields of plasminogen are obtained in this way, it has been found that activation of plasminogen may occur to some extent during the affinity chromatography step. This is probably due to the fact that the plasminogen activator inhibitor is removed in the previous ion exchange column purification step. When plasminogen and plasminogen activator elute together from an ion exchange column (S-sepharose), even small amounts of plasminogen activator (urokinase) in the eluate can cause activation of plasminogen to plasmin. As a result, the plasminogen product contains a small amount of plasmin.

Surprisingly, the use of 6-AHA in the first ion exchange purification step resulted in selectively eluting lys-plasminogen without co-eluting urokinase. This avoids unintended activation of plasminogen to plasmin in the eluate by urokinase. This gives a purer product compared to elution by a salt gradient method.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Purification of lys-plasminogen

Lys-plasminogen is produced by a method analogous to that described in European Patent Application No. 319 944.

Urokinase (uk) secreted by BHK cells is inhibited by α-1-antitrypsin, which is obtained by coexpression with plasminogen. The presence of the activator could be demonstrated by exponential product development in chromatogenic analysis of plasmin. The presence of uk in the culture medium was demonstrated by the fibrin overflow technique [Granelli-Piperno and Reich: J. Exp. Copper. 148. 233 (1978). ].

The culture medium sample is divided into two equal fractions. Comparison of elution from the ion exchanger column, S-Sepharose, in the first step with either 6-AHA buffer or 0.5 M NaCl. Each e11ilate is further dialyzed and purified on lysine-sepharose.

Experiment 1

S-Sepharose, NaCl elution (Test No. 910723)

Equilibration buffer: 0.02M NaH ₂ PO ₄ , 0.04M NaCl, 0.02% Tween 30, 3 mg / l aprotinin, pH 5.0, wash buffer 1: equilibration buffer, wash buffer 2: equilibration buffer with 0, 15M NaCl, pH 5.5, elution buffer 3: equilibration buffer with 0.50M NaCl and without aprotinin, pH 5.5.

Aprotinin is added to each fraction after plasmin has been determined in the sample. A 750 ml culture medium is fed to a column (5 ml) at a flow rate of 125 ml / h. The elution profile is shown in Figure 1. The elution profile is somewhat steep. Lys-plasminogen is in the second maximum.

In Figure 1, the numbers below the baseline (1, 2 and 3) indicate when different buffers (1. equilibration buffer, 2. wash buffer (0.15M NaCl, pH 5.5) and 3. elution buffer (0, 5M NaCl, pH 5.5) The numbers above the baseline (22, ... 30) are the fraction numbers used in SDS PAGE (Figure 3).

Experiment 2

S-Sepharose, 6-AHA elution (Test No. 910724)

The same experiment as above was performed except that the column was eluted in two steps: The first elution was performed with an elution buffer containing 0.02 M NaH ₂ PO ₄ , 0.18 M NaCl, 0.02% Tween 80 and 3 mM 6 -AHA, pH 5.0. The second elution was performed as in Experiment 1.

The elution profile is shown in Figure 2. Lys-plasminogen is at the second maximum. The third maximum is different impurities.

In Figure 2, the numbers below the baseline (1, 2, 3, and 4) indicate when different buffers were added (1. equilibration buffer, 2. wash buffer (0.5M NaCl, pH 5.5), 3. elution buffer I (0.18M NaCl, 3mM 6-AHA, pH 5.5) and 4th Elution Buffer II (0.5M NaCl, pH 5.5) The numbers above the baseline (16 to 42) are the fraction numbers used in SDS PAGE (Figure 3).

Figure 3 shows the non-reduced SDS-PAGE fraction from the elution from both experiments. Very many high molecular weight complexes elute with aprotinin.

The belt numbers indicate the following:

1. molecular weight standard

2. Plasma lys-plasminogen (control)

3. culture medium used in test 910723

4. fractions C. 23 on giant. 1 5. fractions C. 24 on giant. 1 6. fractions C. 25 on giant. 1 7. fractions C. 26 on giant. 1 (attempt 1)

8. culture medium used in test 910724

9. fraction no. 16 in FIG. 2 10. fraction no. 22 in FIG 11. fraction no. 23 in FIG. 2 12. fraction no. 26 in FIG. 2 13 . fraction no. 30 in FIG. 2 14. fraction no. 42 in FIG. 2 15 Dec standard molecular weight

(Experiment 2)

It can be seen that the elution of 3mM 6-AHA (lanes 10-13) results in a pure product (lys-plasminogen) which does not contain high molecular weight complexes, while the elution of NaCl (lanes 4 to 7) results in a product contaminated by some impurities with high molecular weights.

To determine if the isolated lys-plasminogen contains less plasmin (due to urokinase activation in the ion exchange column eluate), further lysine-sepharose purification was performed. This purification inter alia removes aprotinin.

Both eluates are dialyzed against the equilibration buffer below and purified by lysine-sepharose affinity chromatography:

column size: 5 ml, flow rate: 50 ml / h, equilibration buffer: 0.1M Na ₂ HP0 _{4 /} 0.02% Tween 80, 3 mg / l apr., pH 7.4, wash buffer 1: equilibration buffer, Wash Buffer 2: equilibration buffer with 0.15M NaCl but without aprotinin.

Elution is performed with a gradient of 0.1 M Na ₂ HPO ₄ containing 0.02% Tween 80 and from 0 to 5 mM 6-AHA, 50 ml each. Figure 4 shows the elution profile of the dialyzed eluate from Experiment 12 (NaCl).

The numbers below the baseline (1, 2, 3 and 4) refer to the following steps:

1. sample application

2. Wash Buffer 1;

3. Wash Buffer 2 a

4. start of elution gradient.

The numbers above the top line (8, 10, 12) indicate the fraction numbers used in SDS-PAGE (Figure 6).

Figure 5 shows the elution profile of the dialyzed eluate from Run 2 (6-AHA).

The numbers below the baseline (1, 2, 3 and 4) refer to the following steps:

1. sample application

2. Wash Buffer 1;

3. Wash Buffer 2 a

4. start of elution gradient.

In some cases the saddle is visible in the picture. It is known from the literature that the two glycosylation forms of plasminogen have different affinities for lysine-sepharose.

Figure 6, which is SDS-PAGE of elution fractions from a lysine-sepharose column, shows that lys-plasminogen is a fairly pure product, considering that it has undergone only two purification steps.

The belt numbers indicate the following:

1. molecular weight standard

2. Plasma lys-plasminogen

3. the sample applied to Figure 4 (from Test No. 910723 without 6-AHA, Figure 1);

4. = 3,

Fraction No. 7 in Figure 4

Fraction No. 9 in Figure 4

Fraction No. 11 in Figure 4

8. Sample applied in Figure 5 (from Test No. 910724 with 6-AHA, Figure 2)

9. = 8

Fraction No. 7 in Figure 5

11th fraction No. 8 in FIG. 5

Fraction No. 9 in Figure 5

Fraction No. 10 in Figure 5

14. molecular weight standard

Figure 7 shows the results of plasmin analysis of purified lys-plasminogen purified according to the invention (A), ie eluting 6-AHA in the S-sepharose purification step and eluting with NaCl (B). The analyzes were performed using the chromogenic substrate S2251. Standard assay assay where p-NA does not contain S 2251 ^IR (HD-Val-Leu-Lys-pNA), by enzymatic treatment with plasmin, developing a yellow color measured at 405 nm. Prior to the assay, all samples are diluted to have the same protein concentration. It is clearly seen that plasmin is not detectable in the product that eluted from S-Sepharose 6-AHA (no color appeared). When elution is carried out with NaCl, the product contains plasmin, indicating that lys-plasmin-

Claims (7)

Patent Claims'! and ^L ^ 'I A method for purifying a kringl-containing protein which is capable of binding to ω-amino acids from a protein solution comprising such a kringl-containing protein, comprising the steps of: (a) the kringl-containing protein solution is applied to an ion exchange column under conditions such that the kringl-containing protein binds to the column material; (b) the protein containing kringl is eluted by the action of the ω-amino acid without substantially altering the pH and ionic strength; and c) isolating the kringl-containing protein from the eluate of step b) by further suitable purification steps. The method of purifying a kringl-containing protein according to claim 1, wherein the ω-amino acid is selected from the group consisting of 4-aminobutyric acid, 5-aminopentanoic acid, 6-aminohexanoic acid, 7-aminopentanoic acid, 8-aminoctanoic acid, lysine, arginine, and t-aminomethylcyclohexane-1-carboxylic acid. A method of purifying a kringl-containing protein according to claim 1 or 2, wherein the kringl-containing protein is t-PA or a t-PA analog. A method of purifying a kringl-containing protein according to claim 1 or 2, wherein the protein is plasminogen or a plasminogen derivative. 5. The method of claim 4, wherein the plasminogen derivative is lys-plasminogen. The method of purifying a kringl-containing protein according to any one of claims 1 to 5, wherein the ω-amino acid is 6-aminohexanoic acid (6-AHA). A process for purifying a kringl-containing protein according to any one of claims 1 to 6, characterized in that the eluate of step b) is subjected to affinity chromatography. · '