JPH01148185A - Purification of t-pa by reversed phase liquid chromatography - Google Patents

Abstract

PURPOSE:To easily obtain t-PA useful in pharmaceutical field, in high purity, by developing a t-PA-containing solution with reversed phase liquid chromatography using a specific mobile phase. CONSTITUTION:A solution (A) containing t-PA (tissue plasminogen activation factor) can be separated from an extracted fraction of human kidney, etc. An aqueous solution (a) having a pH controlled with trifluoroacetic acid, etc., is mixed with a solution (b) containing a highly volatile organic solvent to obtain a polar mobile phase (B) having pH 4.5-1. The mobile phase does not dissolve column carrier at the lower pH limit and elutes the t-PA at the upper pH limit. A filler having small particle size is packed in a column to obtain a high-performance reversed phase liquid chromatography. The solution A and a solution of the phase B is supplied to the component C to effect the development of the component C. The component adsorbed to the column is eluted and purified with NaCl, etc., to recover the t-PA having a molecular weight of about 65,000.

Description

DETAILED DESCRIPTION OF THE INVENTION (1) Industrial application field The present invention provides tissue plasminogen activator (1-P^)
Tissue extraction fractions, cultured cell extraction fractions, cultured cell culture supernatants, or their concentration fractions and crudely purified fractions containing t-PA are developed using reversed-phase liquid chromatography to extract t-PA from other cell components or culture medium components. t-PA.

(2) Conventional technology Technologies related to the purification of t-PA include column chromatography using a zinc chelate column or a concanavalin A column (Rijken, D, C, & Co.
11en, D., (1981), J. Biol.
Chew, -Dadan, 7035), Erythrina Trypsin Inhibitor Affinity Column Chromatography (JP-A-59-110625), Column Chromatography Using Ion Exchange Column 4- (Sueishi, et al, + (19B2) , Biochim,
Biophys, Acta Ju, 327), anti-t-
p^Antibody affinity column chromatography, lysine or arginine sepharose column chromatography (Einarsson, M, et al, +
(1985) + Biochis, Biophys
, Acta, l 1 ) and other liquid column chromatography methods are known.

Separate t-PA using various liquid column chromatography,
When purifying t-PA, various organic or inorganic substances are added to the eluent for the purpose of aiding the separation of t-PA, so it is necessary to separate t-PA and these additives in the next step.

For this purpose, it is usually necessary to perform another liquid column chromatography, which requires a complicated and long process to obtain a purified product.

(3) Problems to be solved by the invention T-
Purification of PA required a great deal of effort and time to separate t-PA from various organic or inorganic substances contained in the eluent used to elute t-PA from a column.

Therefore, if it is possible to separate and purify t-PA using reversed-phase liquid chromatography and a polar mobile phase containing an easily volatile solvent, it is possible to separate and purify t-PA using a polar mobile phase containing t-PA and a polar mobile phase in that step. This is useful because it can be easily separated from the solution by evaporation under reduced pressure.

However, $J! When attempting to separate t-p^, which has a relatively large molecular weight (M, approximately 11.TOKDa) and a complex three-dimensional structure, using reversed-phase liquid chromatography, it was found that due to its properties, Then t-
Unexpected interactions occur between PA and the active groups constituting the reversed phase column or the carriers of the active groups, making it impossible to separate with the expected sharp development pattern.

The present inventors solved the above-mentioned problem of non-specific adsorption in a t-PA reversed-phase column that contains an easily volatile organic solvent, and the hydrogen ion concentration is such that the column carrier does not dissolve at the lower limit and the upper limit t-
We solved the problem by using a polar mobile phase that satisfies the conditions for PA@t81111, and discovered that the resulting highly purified t-PA had no decrease in physiological activity, and completed the present invention. It has arrived.

Using the present invention, single heavy chain t-
It was found that PA and two-strand It-PA could be easily separated, and one-strand tit-PA (Rtj
Ken, D, C, et al, + (1982), J.
Biol, Chem, 25, 2920) is useful because it can be easily obtained.

The present invention also relates to so-called high performance liquid chromatography.

The use of graph (HPLC) is useful because it allows separation to be easily completed in a much shorter time than conventional column chromatography.

(4) Means for Solving the Problem The reversed phase liquid chromatography referred to in the present invention is composed of a nonpolar stationary phase and a polar mobile phase.

The active group (R) of the nonpolar stationary phase includes an alkyl group in which h is O to 20 (R(CHt)-CHs), a cyanoalkyl group (R(CHz)aCN), a phenylalkyl group, a diphenylalkyl group. Silica is usually used as a carrier for active groups, and a nonpolar stationary phase composed of both of them is packed in a column and used.

A C4 or CIl reverse phase column in which the active group is an alkyl group is commercially available, easily available, and achieves the purpose of the present invention.
1, C6 or any of the other non-polar active groups mentioned above can be used.

The polar mobile phase used in the present invention is a solution supplied by mixing a solution A containing water as a main component and a solution B containing a readily volatile organic solvent as a main component in an arbitrary ratio.

The hydrogen ion concentration of the polar mobile phase used in the present invention is set within a range where the lower limit does not dissolve the column carrier and the upper limit satisfies the conditions that t-p^ is eluted.

That is, the hydrogen ions in the polar mobile phase are set lower than around neutrality in order to avoid unexpected interactions between t-PA and the non-polar stationary phase. When separating t-PA using a reverse phase column under certain specific conditions, the hydrogen ion concentration of the polar mobile phase is set to be lower than 4.5. More preferably lower than 3.5.

Of course, if the hydrogen ion concentration in the polar mobile phase is too low,
The problem of dissolution of the nonpolar stationary phase carrier arises, and the possibility of denaturation of t-PA itself increases, so the hydrogen ion concentration must be such that these problems do not occur.

That is, when t-p^ is separated using a reversed phase column under certain specific conditions, the hydrogen ion concentration of the polar mobile phase is higher than 1.0.

The main component of solution B used for the polar mobile phase may be any hydrophilic and easily volatile organic solvent, but plasminol can be easily removed if acetonitrile, isopropyl alcohol, methanol, ethanol, n-propyl alcohol, or a mixture thereof is used. Efficient separation and purification can be performed without impairing the activity of the gene activator, and after separation, the hydrophilic and easily volatile organic solvent can be efficiently removed by evaporation under reduced pressure.

The main component of solution A used for the polar mobile phase is water, but P
Adjust and use R. Volatile vinegar, acid, etc. may be used for p adjustment, but it is preferable to use trifluoroacetic acid (TFA) or perchloric acid, which act as effective ion-pairing agents, as they improve the separation ability. 9H of solution
is about 1.7, so using this as a standard pn 1.9
When the amount is higher than that, it is preferable to prepare a 0.1xTFA solution by titrating it with, for example, trimethylamine. TFA may or may not be added to solution B.

The reversed-phase liquid chromatography of the present invention is a so-called high-performance liquid chromatograph (I PLC
;Old gh Performance 1iquid c
chromatography) can perform efficient separation in a short time, so it is recommended to use HPLC as necessary. Regarding HPLC, see (Hancock, @
, S., and Sparrow, J.? ,,(1984
)+HPLC Analysis of Biology
calCo*poundS+A Laborator
y Guide, Marcel [1ekker,
INC, P11361) has a detailed explanation.

Tissue plasminogen activator (t-P) as used in the present invention
A) is an enzyme that works in the fibrinolysis system, and when it works, it produces fibrrin-clot, which is a component of blood clots.
), it is useful as a new therapeutic agent for thrombosis.

t-PA is a glycoprotein with a molecular weight of 65,000 and s-
It is said that it takes a three-dimensional structure with two kringles due to the s-bond (Vehar, G.A., et al., (1
984), BiotechnoIogVt! +10
51) *t-PA is originally single-stranded, but becomes double-stranded after being degraded by plasmin (Wallen).
P, et al. + (1983) + Eur, J. Bio.
chem, 132:681). This two-terminal chain t-
When p^ is reduced with mercaptoethanol etc., the S-8 bond is cleaved, and 2 is separated by SO3-polyacrylamide electrophoresis.
Detected as a book band.

t-PA consists of a finger region, a kringle region, and a serine protease region following it; the serine protease region has an enzyme active center, and the former two regions have a fibrin-binding site and an inhibitor-binding site. If the region is missing < t-PA (Ka
gitani, H. et al. (1985), FBB
Examples of t-p^ without kringle (Japanese Patent Application Laid-Open No. 62-48378), polykringle (for example, 3 to 4 kringles), and t-PA (Japanese Patent Application Laid-open No. 62-104577) have been reported. These deletions, modifications, or mutations of t-PA are also included in t-p^ here as related substances.

A plasminogen activator other than t-PA, which has a molecular weight of 54 KDa and was called urokinase.
Urinary plasminogen activator (U-PA) or its related substances, enzymes other than plasminogen activator, growth factors such as lyphokines such as T- or β-interferon, colony stimulating factor or erythropoietin. Proteins with sugar chains, such as hormones, viral antigens such as HBsAg, or cancer-related antigens, have a common feature with plasminogen activators in that they contain sugar chains, and can be separated using the reversed-phase liquid column chromatography method of the present invention. It can be easily assumed that it can be purified.

A specific example of the present invention will be described in detail below, taking the separation and purification of human t-PA as an example.

In the method of the present invention, a mixture containing human t-p^ is developed using a reverse phase liquid column chromatography to separate and purify t-PA from other contaminants. Further, depending on how the separation conditions of reversed phase liquid chromatography are set, it is possible to separate subtypes of t-PA, ie, single-chain t-PA and double-end chain t-PA.

H) The starting materials for the separation and purification of t-PA include human kidney extract fraction (JP-A-59-80614), melanoma Bowes strain culture supernatant (Rijken, Co., Ltd., C, a).
nd Co11en, D., (1981), J.
, Biol, Ches, , j26.7035) or human normal cells (2n=46), recombinant mouse C127 cells into which human t-PA gene has been introduced, recombinant myeloma cells, recombinant Chinese cells, hamster cells, or Recombinant human cells (JP-A-62-126978
) culture supernatants etc., but are limited to these)
Any tissue extract fraction, cultured cell extract fraction, cultured cell culture supernatant, recombinant extract fraction, recombinant culture supernatant, or other mixture containing t-PA containing PA may be used.

h) The above-mentioned mixture containing t-PA is usually h) t-
Since the content of PA is low, an anti-t-PA antibody column, lysine or arginine sepharose column (Ei
Narsson, M. et al. (1985)
Biochim, Biophys.

Acta 83 1-10), erythrina trypsin inhibitor column (JP-A-59-110625), zinc chelate column or concanavalin A column (R
ijken, D, C, & Co11sn, 01 (19
81), J. Biol, Chem, 256.7035
), ion exchange column chromatography (Sue
ishi et al. (1982) Jioch
im, Biophys, Aeta shi 1i327~33
It is preferable to concentrate human t-PA using a method such as 6) and to purify it to some extent at the same time because the effects of the present invention can be better obtained.

The H1-t-PA fraction containing a small amount of impurities obtained through partial purification is dissolved in a solution under the initial conditions of the polar mobile phase that constitutes reversed-phase liquid chromatography, and applied to a reversed-phase column to increase the concentration. t-PA with other mixtures,
In other words, it is separated from cell components, culture medium components, and other additives to obtain high-purity t-PA, or if necessary, one 1 it-P can be obtained by strictly setting development conditions.
Separate A and the double chain t-p^.

The polar mobile phase is a solution in which a solution A containing water as a main component is mixed with a solution B containing an easily volatile organic solvent in a gradient or stepwise manner.

h) Separation of t-PA from other substances, and 1-terminal chain t-P
The separation of A and the two tl'Jt-PAs is achieved by the use of a polar mobile phase with increasing organic solvent concentration.

When separating and purifying t-p^ using a C4 reverse phase column, the hydrogen ion concentration of the polar mobile phase should be 1 or more and 4.5 or less,
Preferably, it is adjusted to 1 or more and 3.5 or less, and in that case, about 0.1% of trifluoroacetic acid (
It is preferable to add TFA) because it increases the resolution.

Human t-p^ is, for example, pH 1 containing 0.1% TFA,
9. Solution A at or around pH 2.5 and solution B in acetonitrile or isopropyl alcohol.
It is separated as a specific peak on a C4 reverse phase column using a gradient elution method.

When the separated human t-p^ was redeveloped and analyzed using the above method using a C4 reverse phase column, no or only a few peaks derived from other contaminating proteins were observed, and when separated using the above method, the purity was It is calculated to be over 99%.

Separation of the 1st chain t-PA and the 2nd chain t-PA can be performed using, for example, C4
Using a reverse phase column, p) containing TFA at a concentration of around 0.1%, from 35% to 37.5% using solution A of a polar mobile phase around 12.5 and solution B which is acetonitrile. Alternatively, it can be done by using a linear gradient method in the vicinity.

Of course, the above separation example is just one example, and any reversed-phase column can be used in principle, and as long as the hydrogen ion concentration conditions are met, small amounts of other additives can be added to solution A. An ion forming agent other than TF^ may be included, and for solution B, an easily volatile and hydrophilic organic solvent other than acetonitrile or isopropyl alcohol may be used.

An important point from another aspect of the present invention is that human t-PA that is isolated and recovered by evaporation of the solvent has its physiological activity.

The human L-PA separated and purified by the above method is TF
It is in the form of an aqueous solution containing A and acetonitrile, and this aqueous solution is lyophilized to remove substances other than the t-PA group by evaporation, and then redissolved in an appropriate solution.

The redissolved t-PA was subjected to 5O5-polyacrylic electrophoresis (Laemmli, O., (1970), Natu
re, 227 680) using fibrin-agar plates. Zymograph method (Granel 1
i-Piperno+^, and Re1ch+[i,
, (1978), J.I! xp, Med, J4
When its activity was investigated using t-PA, 223), it was found that fibrin was present at the same molecular weight as authentic t-PA.
Clot lytic activity was observed.

That is, according to the method of the present invention, human t-PA or single-chain t-PA can be isolated while retaining its physiological activity.

It can be separated and purified.

Next, the present invention will be explained in more detail with reference to Examples.
This does not limit the scope of the present invention.

By using the method of the present invention, highly pure t-p^ can be purified in a short time without impairing physiological activity,
Furthermore, single heavy chain t-PA and second terminal chain t-p^ can also be separated and purified. Since highly purified t-p^ or single-chain t-PA is expected to be used as a medicine, it is also industrially useful in the field of separating and purifying these medicines.

Example Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example I Method of Rijken and Co11en (J, Biol, C
hes, (1981).

Melanoma Bows (2567035)
es) strain was cultured, and Dowdle's method (Unexamined Japanese Patent Publication No. 59-11
0625) A partially purified product of t-PA was obtained.

That is, Tween 8 was added to the culture supernatant obtained by culturing the melanoma Bowes strain and using a serum-free medium containing aprotinin.
0 to 0.1%, NaCl to a final concentration of 0.4M, and the pH was adjusted with acetic acid (pH 5,
5 to 6.0). This solution was applied to an erythrina trypsin inhibitor (ETI) cellulose column at a flow rate of 4.
5m A! I applied it with /h. After washing the column with more than 6 times its volume of phosphate buffer containing 0.4 M NaCl and 0.1 χ Tween 80, the adsorbed proteins on the ET1 column were washed with 1.6 M potassium thiocyanate and 0.1 χ Tween 80.
Elution was performed with saline containing 4M NaCl. The protein concentration of each obtained portion is ^3. . In addition, the plasminogen-dependent fibrinolytic activity was examined using a plasminogen-containing fibrin plate, and the fraction of the protein peak with t-p^ activity was collected, and the plasminogen-dependent fibrinolytic activity was determined using a plasminogen-containing fibrin plate. Dialysis was performed to obtain a partially purified t-PA.

The obtained t-PA partially purified product (1-region t-p^, 2-region t-p^ mixture) by the ETI column was subjected to SO3-polyacrylamide gel electrophoresis (Laesmli, U.
K, + (1970) + Nature43J,
680) revealed that some contaminating proteins were present.

Approximately 2 melanoma t-p^ partially purified products obtained with ETI column
00 μg was lyophilized and diluted with 0.1% trifluoroacetic acid (T
FA), an aqueous solution containing 0.07% trimethylamine (TMA) and 15% acetonitrile (pH 2,5)
and apply it to a C4 reverse phase column (Synchropack
RP-4; (manufactured by 5ynchro-) was applied at a flow rate of 1 mj! Solution A (containing 0.1 χ TF^, 0.07 χ TIIA) constituting the polar mobile phase using a Gilson HPLC system with /sin and a column temperature of 34 °C.
l 2.5 aqueous solution) and solution B (acetonitrile)
Separation was performed using a linear gradient from 15% to 50% acetonitrile over 35 minutes.

When separated using a linear gradient using the above method, 280n
Besides many small peaks indicating the absorption of w, one peak appeared at the lithine silane time of about 26 minutes (RTζ26).

A percentage of the main peak was collected and freeze-dried, and 100 μl of the dried product was added to 0.11'! It was dissolved in acetic acid.

A sample dissolved in 0.1M acetic acid was separated by 5OS-polyacrylamide gel electrophoresis without reduction treatment, and then separated by silver staining or zymography (Granel
1i-Piperno, ^, and Re1ch,
E., (1978), J.

Exp, Med,” arc, 223) was analyzed. As a result of silver staining, a major doublet band at a molecular weight of about 70 Da and a minor band considered to be a dimer at a molecular weight of about 140 KDa were observed, and no other bands derived from contaminating proteins were observed. Fibrinolytic activity was detected at the same molecular weight position as the silver-stained protein band using zymograph, and this reversed-phase column chromatography separation demonstrated that melanoma t-PA can be separated while maintaining its physiological activity. The specific activity of isolated t-PA was ascertained by measuring the clot lysis time (Gaffney
, P. J., and A. D., Curtis+ (198
5), Thro@bosis and Haemost
asis, and 134) about 4 X1G'lU/m
g protein.

160 μg of the t-p^ dry product obtained by the above linear gradient method was dissolved in an aqueous solution (pH 2.5) containing 0.1χ TFA, 0.07χ TM^ and 15χ acetonitrile, and the same was dissolved in the reverse phase again using the above linear gradient method. Expand in column A□. When the peaks of t-PA were investigated, a single peak appeared around 26 minutes of IIT, and other peaks derived from contaminating proteins were not observed or were slight, and from calculation of the peak area, the purity of t-PA was over 99%. Met.

When aprotinin contained in the culture medium of melanoma cells, a small amount of bovine serum-derived protein, or ETI contained in the ETI column are developed on a reversed-phase column using the linear gradient method described above, the RT for ETI is approximately 24 minutes, and the RT for ETI is approximately 24 minutes for ETI. Approximately 28 minutes for major proteins derived from serum, and approximately 8 minutes for aprotinin, which does not overlap with the approximately 26 minutes for human t-PA, and ETI, bovine serum white protein, aprotinin, and human t-PA in equal amounts. It was confirmed that when a mixed sample was separated, each protein could be separated based on the difference in RT.

In order to investigate the influence of the hydrogen ion concentration of the polar mobile phase on reversed phase column chromatography, the polar mobile phase solution A was
．． Different concentrations of TMA were added to 1.chi. TPA and 15.chi. acetonitrile to prepare various hydrogen ion concentrations, and the separation of partially purified t-PA was carried out as described below without changing other conditions.

When the TMA concentration is 0.1%, the pH of solution A is 1.9, and when separation is performed using the linear gradient method described above at this hydrogen ion concentration, the separation is as sharp as that at pH 2.5 described above. Human t-PA could be isolated.

If the TMA concentration is 0.42%, the pH of solution A is 3.5.
When separation was performed using the above-mentioned linear gradient method at this hydrogen ion concentration, the t-PA fraction was obtained as a peak that was slightly less sharp than that at pH 2.5.

When the TMA concentration was further reduced and the pH of solution A was increased to 4.5 or higher, separation was performed using the linear gradient method described above, the t-PA peak lacked sharpness and the peak area also decreased. When the inside of the reverse phase column was washed with 100% acetonitrile, t-p^ remaining in the column due to non-specific adsorption was eluted.

In the above-mentioned linear gradient method, if isopropyl alcohol is used instead of acetonitrile in solution B that constitutes the polar mobile phase, the RT of t-PA will be fast (p) of 1.9 or 2.5. However, by changing the gradient conditions to a linear gradient of 25% to 35% isopropyl alcohol (35 minutes), good separation from other contaminating proteins was obtained, and a sharp peak of t-PA was obtained. It was confirmed by zymography that the isolated t-PA has fibrinolytic activity.

Example 2 Method of Rijken and Colen (J, Biol, C
hem, (1981).

Melanoma Bows (2567035)
es) strain was cultured and the method of Einarsson et al. (B
iochim+Biophys, ^cta, (198
5), a partially purified product of t-PA was obtained in 83.1).

That is, Tween 8 was added to the culture supernatant obtained by culturing the melanoma Bowes strain and using a serum-free medium containing aprotinin.
Add 0 to 0.1%, p.

was adjusted to 7.3, and the culture supernatant was applied to an anti-t-PA antibody-Sepharose column equilibrated with 0.1M sodium phosphate buffer containing 0.01% Tween 80.

Thereafter, the column was treated with 0.25 h potassium thiocyanate, 0.25 h at least 6 times the column volume. The next adsorbed protein was washed with 0.1M phosphate buffer containing 1% Tween 80.
．． 0.1M sodium phosphate buffer containing 0M potassium thiocyanate and 0.01% Tween 80 pH 7.3
It was eluted with The protein concentration was determined by measuring the absorption of 28hs+, and the plasminogen-dependent fibrinolytic activity was determined using a plasminogen-containing fibrin plate. Fractions obtained by fractionating protein peaks corresponding to fibrinolytic activity were collected.

This fraction was dialyzed against O, 1M acetic acid to obtain a partially purified product.

The t-PA partially purified product obtained by the anti-t-p^ antibody column was subjected to SO3-polyacrylamide gel electrophoresis (La
ew+*l L U, (1970) + Na t
As a result of analysis using ureI+680), contaminating protein was detected.

Approximately 20 melanoma t-PA partially purified products obtained using an antibody column
Freeze-dry 0μg, 0.1! ) Lifluoroacetic acid (TP
A), dissolved in an aqueous solution (pH 2,5) containing 0.07% trimethylamine (TMA) and 15% acetonitrile, and loaded onto a C4 reverse phase column (Synchropack RP).
-4; Synchrom) was applied to a polar mobile phase (containing 0.1χTFA, 0.07χTM^) using a Gilson HPLC system at a carding speed of 11ffi/11n and a column temperature of 34°C. pH 2,5 aqueous solution) and solution B (acetonitrile)
Separation was performed using a linear gradient from 15% to 50% acetonitrile over 35 minutes.

When separated using a linear gradient using the above method, 280n
In addition to a small peak of poly(m) indicating absorption of m, a main peak appeared at a retention time of about 26 minutes (RT-26).

Fractions constituting the main peak were collected and freeze-dried, and the resulting dried product was dissolved in 100 μl of O, 1M acetic acid.

0, 1? Samples dissolved in I acetic acid were separated by S[lS-polyacrylamide gel electrophoresis without reduction treatment, and then silver-stained or zymograph method (Granel I
t-Piperno+A, and Re1ch, E...
(1978)+J,t! xp, Med1.223). As a result of silver staining, a main doublet band at a molecular weight of 70 KDa and a minor band considered to be a dimer at a molecular weight of 140[1a] were observed, and no other bands derived from contaminating proteins were observed. Fibrinolytic activity was detected at the same molecular weight position as the silver-stained protein band using zymograph, and this reversed-phase column chromatography separation demonstrated that melanoma t-PA can be separated while maintaining its physiological activity. The specific activity of isolated t-PA was determined by measuring the t9 resolution time (Gaffney+P, J, and A, D, Curt
is+ (1985), Thros+bosis and
Haewsostasls, J3.134) was determined to be approximately 4 x 10'rtl/mg protein.

160 μg of the t-p^ dried product obtained by the above linear gradient method was added to an aqueous solution (pi(2,5)
A. When the peaks of t-PA were examined, a single peak appeared at around 26 minutes RT, and other peaks derived from contaminating proteins were not observed or were slight, and from calculation of the peak area, the purity of t-PA was over 99%. Met.

When aprotinin contained in the culture medium of melanoma cells, a small amount of bovine serum-derived protein, or IgG contained in an antibody column are developed on a reversed-phase column using the linear gradient method described above, the RT for IgG is approximately 30 minutes, and the RT for bovine serum is approximately 30 minutes. It takes approximately 28 minutes for major serum-derived proteins, and approximately 8 minutes for aprotinin, which does not overlap with the approximately 26 minutes for human t-PA. It was confirmed that each protein could be separated by the difference in resolution by separating a sample containing equal amounts of the two proteins.

Implementation 913 The method of Rijken and Co11en et al. (J, Biol.
Melanoma Bowe S strain was cultured according to Chem, + (1981), 2567035), and partially purified t-PA was obtained using a concanavalin A column according to their method.

That is, Tween 8 was added to the culture supernatant obtained by culturing the melanoma Bowes strain and using a serum-free medium containing aprotinin.
0 to 0.11, adjust the pH to 7.2,
The culture supernatant was prepared in 1. OM NaCl and 0
．． 0.01% containing Tween 80. Concanavalin A (Co) equilibrated with OIM phosphate buffer pi 7.5
nA) Applied to Sepharose column. The column was then washed with at least 6 times its volume of equilibration buffer.
Next, 0,0 containing 0.4M α-D-methylmannoside and 2M potassium thiocyanate, 0.01% Tween 80
α-D-methylmannoside 0 to 0 in 1M phosphate buffer
．． A linear gradient of 4M and potassium thiocyanate from 0 to 2M was applied to elute the adsorbed protein.

Protein concentration was determined by measuring absorption at 280 nm, and plasminogen-dependent fibrinolytic activity was determined using a plasminogen-containing fibrin plate. Fractions containing the protein peak corresponding to the fibrinolytic activity thus obtained were collected. This hundredth is 0.1M
Dialysis was performed against acetic acid to obtain a partially purified t-PA.

The obtained t-PA partially purified product by Con A column (
5DS-
Polyacrylamide gel electrophoresis (Laemmli,
tl,)1. , (1970), Nature, Ji
, 680), some contaminating proteins were observed.

Approximately 200 ag of partially purified melanoma t-PA obtained using a ConA column was lyophilized and treated with 0.1% trifluoroacetic acid (TFA) and 0.07% trimethylamine (TMA).
) and an aqueous solution containing 15% acetonitrile (pH 2
, 5) and applied to a C4 reverse phase column (Synchrona
ck RP-4 (manufactured by Synchrom) at a flow rate of 1 ml/sin and a column temperature of 34°C (manufactured by Gilson). Solution A (0,1χTFA, 0.07χTM) constitutes the polar mobile phase using a 1PLc system.
An aqueous solution containing 8 (pH 2.5) and solution B (acetonitrile) were separated over 35 minutes using a linear gradient of acetonitrile from 15χ to 50χ.

When separated using a linear gradient using the above method, 280n
Besides many small peaks indicating the absorption of m, a main peak appeared at a retention time of about 26 minutes (RT-26).

Both components constituting the main peak were collected and freeze-dried, and the resulting dried product was dissolved in 100 μl of 0.1)'l acetic acid.

A sample dissolved in 0.1M acetic acid was directly subjected to S without reduction treatment.
After separation by O3-polyacrylamide gel electrophoresis,
The zymograph method (Granelli-Pi) is a silver staining method.
perno+ A, and Re1ch+ E, +
(1978) + J, Exp, Med, 14fi+2
23).

As a result of silver staining, a major doublet band with a molecular weight of 70 KDa and a minor band with a molecular weight of 140 KDa thought to be a dimer were observed, and no other bands derived from contaminating proteins were observed. Fibrinolytic activity was detected at the same molecular weight position as the silver-stained protein band using zymograph, and this reversed-phase column chromatography separation demonstrated that melanoma t-PA can be separated while maintaining its physiological activity. It was confirmed.

The specific activity of isolated t-PA was determined by measuring the clot lysis time (Gaffney+P, J, and A, D, C
urtis, (1985).

Thrombosis and Hacvostasi
s 53 +134) Approximately 4 XIO'lU/m
g protein.

160 μg of the dried t-PA obtained by the above linear gradient method was added to an aqueous solution (pH 2.5) containing 0.1χTFA, 0.07χTMA, and 15χ acetonitrile.
Dissolve it in A and develop it again on a reversed phase column using the linear gradient method described above. . When I checked the peak of , RT was 26.
A single peak appeared around 10 minutes, and other peaks derived from contaminating proteins were not observed or were slight, and the purity of t-pa was 99% or more based on calculation of the peak area.

When aprotinin contained in the culture medium of melanoma cells and a small amount of protein derived from bovine serum are developed on a reversed phase column using the linear gradient method described above, the RT
It takes approximately 8 minutes for aprotinin and approximately 28 minutes for the main protein derived from bovine serum, which does not overlap with the approximately 26 minutes for human t-PA. It was confirmed that each protein could be separated by the difference in RT when a sample was mixed in quantity.

Example 4 The method of Rijken and Colen et al. (J, Biol.
Melanoma Bowes strain was cultured according to Chem., (1981), 2567035);
Using their method, a partially purified t-PA was obtained using a zinc chelate column.

That is, melanoma Bowes strain was cultured, and Tween 80 was added to the culture supernatant using a serum-free medium containing aprotinin.
was added to give o, oiχ and adjusted to pH 7.5,
The culture supernatant was applied to a zinc chelate agarose column equilibrated with a 0.02 M l-lith hydrochloric acid buffer containing LM NaCl and 0.01 χ Tween 80, pH 7.5.

After this, the column was IM NaCl and I) containing 0.01χ Tween 80! (More than 6 times the column volume was washed using 0.02M Tris-HCl buffer in 7.5 as a washing solution.Next, imidazole was added to the above washing solution and a linear graph of imidazole from 0 to 0.05M was applied to the column. The adsorbed proteins were eluted by applying gel.

Protein concentration was determined by measuring absorption at 2B0 nm, and plasminogen-dependent fibrinolytic activity was determined using a plasminogen-containing fibrin plate. Fractions containing the protein peak corresponding to the fibrinolytic activity thus obtained were collected. Both parts are 0.1M
Dialysis was performed against acetic acid to obtain a partially purified t-PA.

The obtained t-PA partially purified product (1 zero chain t-PA, 2 terminal chain t-PA mixture) by the zinc chelate column was purified by 5OS.
-Polyacrylamide gel electrophoresis (LaeIII1
1, IJ, (1970), Nature,
As a result of analysis using 227.680), it was found that there was some contaminating protein present.

Approximately 200 tIg of partially purified melanoma t-PA obtained using a zinc chelate column was lyophilized and treated with 0.1% trifluoroacetic acid (TMA) and 0.07χ trimethylamine (
TMA) and an aqueous solution containing 15χ acetonitrile (
pH 2, 5) and applied to a C4 reverse phase column (Synchr
OPACK RP-4i (manufactured by SVnchrom) at a flow rate of Is 1 /win.

Solution A (0,1χ↑PA,
aqueous solution of p)l 2.5 containing 0.07χTMA) and solution B (acetonitrile) in 15%
Separation was performed as a linear gradient from to 50% over 35 minutes.

When separated using a linear gradient using the above method, 280n
Besides many small peaks indicating the absorption of m, a main peak appeared at a latency time of about 26 minutes (RTζ26).

Both components constituting the main peak were collected and freeze-dried, and the resulting dried product was dissolved in 100#ffi of 0.1M acetic acid.

A sample dissolved in 0.1M acetic acid was separated by 5IllS-polyacrylamide gel electrophoresis without reduction treatment, and then separated by silver staining or zymography (Granel
li-Piperno+ A, and Re1
ch, E-+(197B)+J, Exp-Med,
148223). As a result of silver staining, a main doublet band with a molecular weight of 70 KDa and a minor band considered to be a dimer with a molecular weight of 140 KDa were observed, and no other bands derived from contaminating proteins were observed. Fibrin-dissolving activity was detected at the same molecular weight position as the silver-stained protein band using a zymograph, and this separation using reversed-phase column chromatography revealed that melanoma t-
It was confirmed that PA was separated while maintaining its physiological activity.

The specific activity of isolated t-PA was determined by measuring the clot lysis time (Gaffney, P, J, and^, D, C
urtis+ (1985) +τhro+*bosi
s and Haemostasis, 53134)
Approximately 4 x 10'lLI/mg protein
Met.

160 μg of the dried t-PA obtained by the above linear gradient method was added to an aqueous solution (pH 2.5) containing 0.1% TFA, 0.07% TMA and 15% acetonitrile.
Dissolved in Ao and developed again on a reversed phase column using the linear gradient method described above. When the peaks of t-PA were examined, a single peak appeared at around 26 minutes RT, and other peaks derived from contaminating proteins were not observed or were slight, and from calculation of the peak area, the purity of t-PA was over 99%. Met.

When aprotinin contained in the culture solution of melanoma cells and a small amount of protein derived from bovine serum are developed on a reversed phase column using the linear gradient method described above, the R
T is approximately 8 minutes for aprotinin and approximately 28 minutes for a major protein derived from bovine serum, which does not overlap with approximately 26 minutes for human t-PA, and that It was confirmed that when samples mixed in equal amounts were separated, each protein could be separated based on the difference in RT.

Example 5 Culture supernatant of normal human cells (2n = 46) was separated using a Concanavalin A (Con A) Sepharose column in the same manner as in Example 3 to obtain a partially purified product of t-PA.

That is, human normal cell MTC017 strain (Japanese Patent Application Laid-open No. 1986-
126978) and culture supernatant obtained using a medium containing aprotinin and a small amount of fetal bovine serum.
A partially purified t-PA was obtained using the A column.

The obtained t-PA partially purified product by Con A column (
Single chain t-PA, double end chain t-PA mixture) was subjected to 5OS-polyacrylamide gel electrophoresis (Laemmlk, υ
J., (1970), Nature, u, 68
As a result of analysis with 0), some contaminating proteins were present.

0 μg of the normal cell t-PA partially purified product obtained using the Con A column was freeze-dried, and 0.1χ trifluoroacetic acid (
TF^), 0.07% trimethylamine (TMA)
and an aqueous solution containing 15% acetonitrile (pif 2
．． 5) and applied to a C4 reverse phase column (Synchrona
ck RP-4; manufactured by 5ynchro+s) at a flow rate of 1+ll/win and a column temperature of 34"C.
Solution A (0.1% TF^, 0.07% TMA) constituting the polar mobile phase was prepared using a Gilson HPLC system.
(aqueous solution containing pH 2.5) and Solution B (acetonitrile) were separated over 35 minutes as a linear gradient from 15% to 50% acetonitrile9.

Separate with a linear gradient using the above method and 28o
Besides many small peaks indicating n- absorption, a main peak appeared at about 26 min (RT-26). Fractions constituting the main peak were collected and freeze-dried, and the resulting dried product was dissolved in 100μ2 of 0.1M acetic acid.

After separating the sample dissolved in 0.1M acetic acid by 5DS-polyacrylamide gel electrophoresis without reduction treatment, it was separated by silver staining or by zymography method (Granel 1
i-Piperno+^, and Re1ch,! ,,
(197B), J, Exp, Med, 148.22
3) was analyzed. 1! As a result of staining, the molecular weight is approximately 70K.
The main band of doublet is Da, and the molecular weight is about 140K.
A minor band thought to be a dimer was observed in Da,
No other bands derived from contaminating proteins were observed. Fibrinolytic activity is detected at the same molecular weight position as the silver-stained protein band using a zymograph, and normal cells t-p^ can be separated while maintaining their physiological activity by separation using main phase column chromatography. was confirmed 0
The specific activity of isolated t-PA was determined by measuring the clot lysis time (Gaffney, P, J, and^, D, C
urtis, (1985), Thro+*bosis
and Haemostasis, 53.134) was determined to be approximately 4X 10'lLI/mg protein.

160 μg of the t-p^ dried product obtained by the above linear gradient method was added to 0. A! ,. When the peaks of t-PA were investigated, a single peak appeared at around 26 minutes RT, and other peaks derived from contaminating proteins were not observed or were slight, and from the peak area calculation, the purity of t-PA was 99%.
That was it.

When aprotinin contained in a normal cell culture solution and a small amount of protein derived from bovine serum are developed using a reversed phase column using the linear gradient method described above, the RT for aprotinin is approximately 8 minutes, and for the main protein derived from bovine serum, the RT is approximately 8 minutes. It is approximately 28 minutes, which does not overlap with the approximately 26 minutes of human t-PA, and when a sample containing equal amounts of aprotinin, bovine serum-derived protein, and human t-PA is separated, each protein is separated by RT. It was confirmed that separation was possible based on the difference.

Example 6 h) The culture supernatant of Chinese hamster ovary cells containing the t-FA gene was treated with anti-t in the same manner as in Example 2.
-pa antibody column to obtain a partially purified product of t-PA.

That is, recombinant Chinese hamster ovary cells (JP-A No. 62-126978) were cultured, and the culture supernatant obtained using a medium containing aprotinin and a small amount of fetal bovine serum was subjected to t-PA using a Can A column. A partially purified product was obtained.

The t-PA partially purified product (1-end chain t-PA, 2-end chain t-PA mixture) obtained using the antibody column was subjected to 5DS-polyacrylamide gel electrophoresis (Laessli, υ, (1970), Nature+J As a result of analysis using Nail, 680), some contaminating proteins were present.

Partially purified recombinant t-PA obtained using an antibody column
freeze-dry ag, 0.1! ) Lifluoroacetic acid (TFA
), 0.07χ trimethylamine (TMA) and 15% acetonitrile in an aqueous solution (pH 2,5) and loaded onto a C4 reverse phase column (Synchropack R
Solution A (0.1% ↑F^, 0.1% ↑F^, 0.1% ↑F^, 0.5% ↑F^, 0.1% ↑F^, 0.1% ↑F^, 0.1% ↑F^, 0.5% ↑F^, 0.5% ↑F^, 0.1% ↑F^, 0.5% was applied to a polar mobile phase using a Gilson HPLC system at a flow rate of 1mff1/mis and a column temperature of 34°C. p containing 07%TM^
Aqueous solution of H2.5) and solution B (acetonitrile) were separated as a linear gradient from 15% to 50% acetonitrile over 35 minutes.

When separated by linear gradient using the above method, 280+
Besides many small peaks indicating absorption of wa+, a main peak appeared at about 26 min (RT-26). Both components constituting the main peak were collected and freeze-dried, and the resulting dried product was dissolved in 100 μl of 0.1M acetic acid.

Samples dissolved in 0 and 1M acetic acid were separated by 5DS-polyacrylamide gel electrophoresis without reduction treatment, and then separated by silver staining or zymography (Granelli).
-Piperno, A. and Re1ch, E.
(1978), J. Exp.

The analysis was carried out in Med, "■, 223). 1! As a result of staining, a major doublet band with a molecular weight of about 70 KDa and a minor band considered to be a dimer with a molecular weight of 140 KDa were observed, and no other bands derived from contaminating proteins were observed. Fibrinolytic activity was detected at the same molecular weight position as the silver-stained protein band using zymograph, and recombinant t-PA was separated while maintaining its physiological activity through separation using this reversed-phase column chromatography. This was confirmed.

The specific activity of isolated t-PA was determined by measuring the riqt dissolution time (Gaffney, P, J, and^, D, C
urtis, (1985LT Thrombosis a
nd 1Iae+5ostasis+ J3,134
) is about 4 XIO'lU/++g protei
It was n.

160 μg of the dried t-PA obtained by the above linear gradient method was added to an aqueous solution (pil 2.
5) and develop it again on a reverse phase column using the linear gradient method described above. When the peaks of t-PA were examined, a single peak appeared at around 26 minutes RT, and other peaks derived from contaminating proteins were not observed or were slight, and from calculation of the peak area, the purity of t-PA was over 99%. Met.

Aprotinin contained in the recombinant culture medium, a small amount of bovine serum-derived protein, or Ig contained in the antibody column
When each G is developed using a reversed-phase column using the linear gradient method described above, each column will last approximately 30 minutes with IgG.
It takes about 28 minutes for the main protein derived from bovine serum, and about 8 minutes for aprotinin, which does not overlap with the approximately 26 minutes for human t-PA, and aprotinin, a protein derived from bovine serum,
It was confirmed that when a sample containing equal amounts of IgG and human t-PA was mixed, each protein could be separated based on the difference in RT.

Example 7 The culture supernatant of mouse C127 cells containing the human t-PA gene was separated using an ETI Sepharose column in the same manner as in Example 1 to obtain a partially purified t-PA.

That is, recombinant mouse C127 cells (Japanese Patent Application Laid-Open No. 1983-
126978) and obtained ETI from the culture supernatant obtained using a medium containing aprotinin and a small amount of fetal bovine serum.
A partially purified t-PA product was obtained using a column.

The obtained t-p^ partially purified product (1 t-p^, double-stranded t-PA mixture) by the ETI column was subjected to SO3-submerged acrylamide gel electrophoresis (Lae+u+1e+υ,
(1970), Nature, mL680) showed that some contaminating proteins were present.

0 μg of the recombinant t-p^ partially purified product obtained using the ETI column was freeze-dried, and 0.1% trifluoroacetic acid (TF
A), aqueous solution containing 0.07% trimethylamine (TMA) and 15% acetonitrile (pH 2,5)
C1 reverse phase column (Synchropack
RP-4: Applied to 5ynchro- Co., Ltd.) at a flow rate of 1 sj! Solution A (pH 2.5 aqueous solution containing 0.1% TFA, O,Q 7% TMA) and Solution B (acetonitrile) were used to form polar mobile phases using a Gilson HPLC system at a column temperature of 34°C.
Separation was performed using a linear gradient from 15% to 50% acetonitrile over 35 minutes.

When separated using a linear gradient using the above method, 280n
Besides many small peaks indicative of the absorption of Toshi, a main peak appeared at a retention time of about 26 minutes (RT-26).

Both components constituting the main peak were collected and freeze-dried, and the resulting dried product was dissolved in 100 μl of 0.1M acetic acid.

A sample dissolved in 0.1M acetic acid was separated by 5DS-polyacrylamide gel electrophoresis without reduction treatment, followed by silver staining or zymography (Granelli method).
-Piperno, ^, and Re1ch+E, +
(197B) +J, Exp, Med,” ■, 223
) was analyzed. As a result of 11 staining, the molecular weight was 70KD.
The main band of the doublet is shown in a, and the molecular weight is 140KD.
A minor band considered to be a dimer was observed in a, and no other bands derived from contaminating proteins were observed. Fibrin-dissolving activity was detected at the same molecular weight position as the silver-stained protein band using a zymograph, and recombinant t-PA could be separated while maintaining its physiological activity by separation using zero-reversed phase column chromatography. The specific activity of isolated t-PA was confirmed by measuring the clot lysis time (
Gaffney, P., J., and^, D., Curtis.
, (1985) + Thr.

mbosis and ) Iaesostasis+
, 51+134) Approximately 4XLO' 10/mg
It was protein.

160 μg of the dried t-PA obtained by the above linear gradient method was added to an aqueous solution (pH 2.5) containing 0.1% TFA, 0.07% TM^ and 15% acetonitrile.
A1. . When I checked the peak of , RT was 26.
A single peak appeared around 10 minutes, and other peaks derived from contaminating proteins were not observed or were slight, and the purity of t-PA was 99% or more based on calculation of the peak area.

When aprotinin contained in the culture medium of recombinant cells, a small amount of bovine serum-derived protein, or ETI contained in the ET1 column are developed on a reversed-phase column using the linear gradient method described above, the RT for aprotinin is approximately 8 minutes. , approximately 28 minutes for major proteins derived from bovine serum, and approximately 24 minutes for ETI, which does not overlap with approximately 26 minutes for human t-PA, and aprotinin, ETI,
It was confirmed that when a sample containing equal amounts of bovine serum-derived protein and human t-PA was separated, each protein could be separated based on the difference in RT.

Example 8 Method of Rijken and Colen (J, Biol, C
hem, (1981).

■Melanoma Bow (Bow 7035)
es) strain was cultured, and Dowdle's method (Unexamined Japanese Patent Publication No. 59-11
0625) to obtain a t-p^ partially purified product. That is, melanoma Bowes strain was cultured, and a t-p^ partially purified product was obtained from the culture supernatant obtained using a serum-free medium containing aprotinin using an ET1 column.

The obtained t-PA partially purified product (1-terminal t-p^, double-strand t-PA mixture) by the ET1 column was subjected to 5OS-polyacrylamide gel electrophoresis (Laemmli, Ll
, (1970) +Nature, 221.
680) showed that some contaminating proteins were present.

The freeze-dried partially purified t-PA was mixed with 0.1% trifluoroacetic acid (TF^) and 0.07% triethylamine (T
MA ) and a solution containing 35% acetonitrile (pH
2,5) and applied to a C4 reverse phase column (Synchronp
ack RP-4 (manufactured by SynChrom), and the acetonitrile concentration was applied to a linear gradient method from 35% to 37.5% over 30 minutes using a Gilson HPLC system at a flow rate of 1+wl/+min and a column temperature of 34°C. It eluted.

By this method, one main peak was obtained in the range of 12 to 15 minutes of lithine silane time, and another main peak was obtained in the range of 15 to 20 minutes.

These two fractions were subjected to SO3-polyacrylamide electrophoresis with or without reduction with mercaptoethanol, and examined by silver staining. Non-reduced molecular weight 70KD
a, Bands were detected at about 30 KDa and about 40 KDa upon reduction. Furthermore, the fraction eluted in the range of 15 to 20 minutes had a weight of about 70 KDa even after reduction, and no bands of 30 KDa and 40 KDa were detected.

From this result, the t-
It was confirmed that p^ is one terminal chain t-p^, and the t-PA eluted in the range of 15 minutes to 20 minutes is a double chain t-p^.

Two of these single-stranded t-PA units $1 t-PA Ha5
Fibrinolytic activity was detected as a result of zymography analysis after 05-ho17 acrylamide electrophoresis.

Regarding the separation conditions in the above-mentioned one-piece 1t-p^ and two-piece 11t-PA reverse phase column chromatography, the column temperature or pl+ was varied and the effect thereof was investigated.

When the column temperature was set at approximately 16°C under the above separation conditions, and when it was set at 40°C, the first chain t-p^ and double chain t -PA
The separation was not excellent.

When the pH was raised to 3.5 by increasing the concentration of TMA contained in the polar mobile phase under the above separation conditions, single-chain t-
The separation between PA and two $1t-PA is slightly inferior, and TM^
When the concentration of t-PA was further increased and pi was raised to 4.5, the separation of 1-bottle $Jjt-P^ and 2-bottle t-PA was even slightly worse, and nonspecific adsorption of t-PA in the column was observed. Admitted.

When isopropyl alcohol is used instead of acetonitrile under the above separation conditions, single-chain and double-chain t-PA
The separation was slightly poorer.

Patent applicant: Mitsui Toatsu Chemical Co., Ltd.

Claims (1)

[Claims] 1) A solution containing t-PA is mixed with a polar mobile phase that contains an easily volatile organic solvent and has a hydrogen ion concentration within a range where the lower limit does not dissolve the column carrier and the upper limit satisfies the conditions that elute t-PA. 1. A method for purifying t-PA, which is characterized by developing the t-PA using reverse phase liquid chromatography.