Process for purifying a plasminogen activator.
The present invention relates to a purification process, and in particular to a process for purifying human tissue plasminogen activator (tPA). It is envisaged that the process will be of particular, but not exclusive, application in the purification of tPA produced by recombinant DNA technology. tPA is an enzyme (and therefore a protein) which is found in the blood and various tissues of the body. It is involved in the processes in the body whereby an intact blood circulation system is maintained. In particular, tPA is involved in the regulation of blood clot formation.
A blood clot is formed by laying down a mass of fibrin which forms the basis of the clot.
However, the fibrin has associated with it an enzyme precursor, plasminogen. Plasminogen can be activated to produce the enzyme plasmin which acts on the fibrin in the blood clot to cause dissolution of the clot (thrombolysis) .
There are three commonly available enzymes which can act on plasminogen to activate it to plasmin. The first is streptokinase, which is an enzyme derived from various streptococcus strains. Streptokinase has been used as a thrombolytic agent, but has the disadvantages that it activates plasminogen whether it is circulating or bound in a clot and that it is antigenic. The first disadvantage can lead to uncontrolled bleeding in parts of the body remote from the targetted clot, and the second disadvantage can lead to adverse immunological reactions in the body which can lead
fccc as. reduction in the efficiency of the action of the streptokinase or to undesirable clinical symptoms.
The second plasminogen activator is generally known as urokinase since it was originally isolated from human urine. More recently, it has been isolated from tissue culture systems or has been produced by recombinant DNA technology. Since urokinase is of human origin, it is not antigenic. - However, it has the disadvantage that it activates both circulating and bound plasminogen, leading to the problem set out above.
The third plasminogen activator is tPA which is also non-antigenic in humans. Moreover tPA is very specifically bound to fibrin and is therefore able to act almost exclusively on plasminogen bound into a blood clot. Thus, tPA has the advantage that it is a highly specific thrombolytic agent. It has already been used clinically for the treatment of thrombi in patients who have received a kidney transplant (see Weimar et al. , The Lancet, 7th November, 1981, pages 1018 and 1019)
Until the end of the 1970 's, tPA could only be isolated in small quantities from human tissues, such as human uterine tissue. Thus, although its use would very clearly have been advantageous, it had not been possible to produce it. in commercially useful quantities.
In 1981 it was discovered that a particular melanoma cell line (known as the Bowes melanoma cell line) secreted large amounts of tPA. (see Rijken and Collen, J. Biol. Che ., 7035-7041, 1981). It was thus possible to produce large quantities of tPA by culturing the cell line and harvesting the supernatant.
More recently, it has been proposed that tPA should be produced by use of recombinant DNA technology. For instance, Pennica et al. disclose the cloning and expression of the tPA gene in E. Coli (see Pennica, D. et al. , Nature, 301, 214-221, 1983) and GB-A-2 119 804 (Genentech) discloses the cloning and expression of the tPA gene in both prokaryotic and eukaryotic cell lines. Thus tPA should now be available in commercially viable quantities. However, in order to be able to meet the requirements of various national regulatory authorities to enable tPA to be used clinically, it will be necessary to purify the tPA to substantially complete purity. Thus, for tPA produced by melanoma cell culture, it will be necessary to separate it from the components of the culture medium and such other human proteins as are normally produced by the melanoma cells. Similarly, for tPA produced by recombinant DNA technology, it will be necessary to separate it from the components of the culture medium and such other proteins as are normally produced by the host cell used for tPA expression. It is known that tPA as it is normally produced, for instance by the melanoma cell line or by recombinant DNA technology, is a one chain protein. However, it is subsequently cleaved into a two chain, disulphide-linked protein. This cleavage can occur spontaneously during handling of the tPA following its production.
There is now some evidence from clinical tests in humans that the one chain form is more efficacious in clearing blockages in arteries (see Garabedian, H.D. et al. , JACC, , 3, 599-607, 1987). It is preferred therefore that the tPA as purified should be in the one chain form so that, after
purification, it can be used in the one chain form. Both one chain and two chain molecules can each be found in two variants which differ in molecular weight by about 3,000 Daltons. This difference is probably due to different degrees of glycosylation at one (Asn-184) of four potential glycosylation recognition sites in the molecule.
Moreover, both one chain forms of tPA contain N-terminal sequence variants, (see Jornvall, H. et al., FEBS Letters, 156, 1, 47-50, 1983). In the material purified from Bowes cell supernatants, there is an approximately 50:50 mixture of the following two N-terminal sequences
H2N-G-A-R-S-Y-Q-V-I H2N-S-Y-Q-V-I-
A number of purification procedures are already known. However, these are either too complex, too low in yield or too small scale to enable them to be used in a commercial production process. Moreover some of them are known to promote the cleavage of the tPA into its two chain form. For instance, it is known that gel electrophoresis can be used as an efficient, one step purification process, but preparative gel electrophoresis is time consuming, of low yield and very expensive.
It has been proposed in DE-A-3 222 08.4 (Bayer) that tPA should be purified by affinity chromatography using an anti-tPA monoclonal antibody bound to a chromatography medium such as Sepharose 4B. However, no details of any equilibration or elution buffers are given. It is envisaged that, for instance, supernatant from a melanoma cell line
culture could be treated by such a process to produce purified tPA. It is indicated that the yield of tPA is as much as 90%, but no indication of the purity of the tPA, nor of the ratio of one to two-chain types, is given.
It has been found that using affinity chromatography, even under the optimum conditions specified below in relation to the present invention, does not produce sufficiently pure tPA in a good enough yield to enable a commercial process to be set up.
A problem which is common to all tPA purification procedures is that tPA is generally highly adherent to vessels, in particular glass vessels, in which it is stored or through which it .is passed. There can therefore be a considerable loss of material during processing. Moreover, it is generally difficult to maintain a high concentration of pure tPA in solution. At a concentration of about 1 mg/ml, the tPA tends to precipitate out. This makes it less useful in the therapeutic field, where a high concentration solution is desirable.
It has been proposed that tPA could be kept in solution by the addition of auxiliary agents, such as IM ammonium bicarbonate, lysine, arginine, other amino acids or other amines. These are also considered to be active in maintaining the stability of the tPA. However, these proposals have the disadvantage that the auxiliaries may not be acceptable in a therapeutic context. It would therefore be desirable to produce a high concentration, stable tPA solution without needing to use any possibly unacceptable auxiliaries.
According to the present invention, there is provided a process for the purification of tPA comprising carrying out a) an ion exchange chromatography step on a highly anionic chromatography medium and b) an affinity chromatography step on a mother solution containing tPA.
Preferably, all the purification steps are carried out at as low a pH as possible without adversely affecting the tPA or the properties of the chromatography media used. Generally the pH should not fall below 4. It has surprisingly been found that at such low pHs, the tPA is both storage stable and able to stay in solution at concentrations well above lmg/ l.
Advantageously, at least the later purification steps, and preferably the whole purification procedure, is carried out in the presence of about 0.01% of a detergent, preferably a non-ionic detergent. A particularly suitable detergent is Tween R 80, which is known to be therapeutically acceptable. Such detergents appear to reduce the amount of tPA adhering to the purification or storage vessels. Preferably, the process comprises: applying the mother solution containing tPA to a highly anionic chromatography medium equilibrated with a buffer at 25 mS ionic strength or lower and at a pH between 4 and 7; eluting the tPA from the highly anionic chromatography medium with a high ionic strength buffer; applying the eluate from the highly anionic chromatography medium to an affinity chromatography
medium, comprising an anti-tPA antibody covalently bound to a chromatography material, equilibrated with a buffer which does not substantially interfere with the binding of the tPA to the antibody; and eluting the tPA from the affinity chromatography medium with a buffer which disrupts the binding of the antibody to the tPA.
The mother solution containing the tPA may be the supernatant from a culture of tPA secreting cells, such as Bowes melanoma cells. Alternatively the mother solution may be the supernatant from a culture of a eukaryotic cell line which has been transformed by recombinant DNA technology so that it is able to express and secrete tPA. The mother solution, if necessary, may be clarified to remove any cells αr cell debris.
The clarification may be carried out, for instance, by centrifugatiori or filtration. However, the clarification should not be carried out by any process, such as ultrafiltration, which could adversely affect the yield of tPA. Ultrafiltration in particular has been found to reduce significantly the yield of tPA.
Preferably, the mother solution is clarified, where necessary, by filtration, for instance using a hydrophilic resin-impregnated glass-fibre filter, such as a Balston LP-200-50-80 cartridge filter (which is a polypropylene and borosilicate glass-fibre depth filter having a 0.22 urn nominal pore size) .
Advantageously, the solution in which the cells are cultured to produce the mother solution contains a serine protease inhibitor, such as aprotinin, to inhibit any enzymatic cleavage of the
tPA. If such an inhibitor is used, it will be necessary to maintain the pH in the mother solution and in at least the initial purification steps above 4 so that the inhibitor remains active. It has been found that a preferred level of aprotinin for obtaining one chain tPA is from 10 to 100 KlU/ml of mother solution.
It is also highly advantageous to process the mother solution as soon as possible after it has been produced. As is shown below in the Example, swift processing of the mother solution leads to an unexpectedly high yield of homogeneous tPA.
Advantageously, the highly anionic chromatography medium (HACM) is of the rigid type so that fast flow rates without compaction of the medium can be achieved. Rigid HACMs are well known in the art.
The anionic groups on the HACM may be any of those known in the art, such as carbox late, sulphate, sulphonate, sulphopropyl or phosphate groups. Preferably, the anionic groups are sulphonate groups.
A particularly suitable HACM is S-Sepharose Fast Flow (sold by Pharmacia Ltd.), which is a rigid medium comprising a highly cross-linked beaded agarose matrix having attached thereto -CH2~Sθ3 a (sulphonate) groups.
Advantageously, the HACM is washed with equilibration buffer following application of the (clarified) mother solution.
The pH of the HACM equilibration buffer is preferably between 4 and 7 and is preferably about 6 or lower. It has been found that if the pH is too
low, undesired proteins become attached to the medium, whereas if the pH is too high, the t?A does not become attached to the medium in sufficient quantity. The ionic strength of the HACM equilibration buffer should be adjusted to suit the pH used. For high pHs, low ionic strengths should be used. For instance, at pH 6.2 an ionic strength not above 10 mS is suitable. At a pH of 4, the ionic strength could rise to about 25mS.
Preferably, the buffer used to equilibrate the HACM comprises 50 mM acetate, 0.01% Tween 80 adjusted to pH 6.0.
However, it will be readily appreciated by the skilled person that other buffering agents, such as phosp.hate or citrate buffers or mixtures thereof, could be used and that ionic strength adjusting agents, such as sodium or potassium chloride, could be included in the HACM equilibration buffer so long as the pH of the buffer and its ionic strength are as specified above.
Preferably, the high ionic strength buffer used to elute the tPA from the HACM comprises 50 mM * acetate, 0.01% Tween 80 and 500 mM sodium chloride adjusted to pH 6.0.
It will again be readily appreciated, as with the equilibration buffer, that other buffering agents and ionic strength adjusting agents could be used. Preferably, these agents are chosen to be compatible with the agents used in the equilibration buffer. Preferably, the ionic strength of the HACM elution buffer is not less than that of a solution containing 50 mM acetate and 200 mM, preferably 250 mM, sodium chloride adjusted to pH 6.0, and is advantageously the same as or greater than that of
the preferred elution buffer. However, it should be ensured that the ionic strength of the buffer is not so high as to affect adversely the tPA.
The affinity chromatography medium (ACM) preferably comprises a monoclonal anti-tPA antibody, for instance of the type disclosed in DE-A-3 222 084, covalently bound to a suitable chromatography material. However, the antibody may comprise a polyclonal anti-tPA antibody. Preparation of ACMs is well known in the art and any one of these known methods may be used. Conveniently the anti-tPA antibody is coupled to the chromatography material by use of cyanogen bromide activation, for instance as described in Affinity Chromatography, Principles and Methods, pages 11 to 18, by Pharmacia Ltd. (and available from them).
Chromatography materials suitable for the preparation of ACMs are well known in the art and a suitable one can readily be selected by those skilled in the art. Preferably, the chromatography material is rigid, for the reasons set out above in relation to the HACM.
A particularly suitable chromatography material for producing the ACM is Sepharose CL4B (sold by Pharmacia Ltd.) which is a semi-rigid material comprising a cross-linked agarose polymer.
Advantageously, the ACM is washed with equilibration buffer after the application thereto of the HACM eluate. The pH and ionic strength of the ACM equilibration buffer should be adjusted to facilitate the binding of the tPA to the antibody, and in particular the ionic strength should not be so great or so low as to disturb such binding.
Preferably, the pH of the ACM equilibration buffer is between 5 and 10, and is advantageously between 5 and 7 and most preferably is about 5.5. The ACM equilibration buffer is preferably of high ionic strength, for instance, equivalent to or greater than that of a solution containing lOOmM sodium chloride, since in some cases this reduces the incidence of non-specific binding of undesired protein to the ACM. Preferably, the ACM equilibration buffer contains 100 mM acetate, 200mM sodium chloride and 0.01% Tween 80 adjusted to pH 5.5.
As with the HACM buffers, it will be apparent to the skilled person that other buffering agents and ionic strength adjusting agents could be used in 'the ACM equilibration buffer.
The ACM elution buffer should be able readily to disrupt the binding of the tPA to the antibody without adversely affecting either. There are a number of types of buffer which can disrupt the binding between an antibody on an ACM and its substrate, and any of these types of buffers can be used. For instance, the buffer may have an extremely high or an extremely low pH or ionic strength. Alternatively, the buffer may contain a chaotropic salt, such as magnesium chloride, potassium thiocyanate or potassium iodide, or a protein denaturing agent, such as urea or guanidine. In a further alternative, the buffer may contain a disruptive solvent, such as propanol or ethylene glycol.
Preferably, the ACM elution buffer is a low pH buffer, for instance containing 50 mM acetate, 0.01% Tween 80 and 200 mM sodium chloride adjusted
to pH 3.5. Surprisingly, the use of such low pHs does not present any problems as regards the stability of the tPA. Alternatively, the ACM elution buffer contains a chaotropic salt, and ma'y, for instance, contain 5M magnesium chloride, and 20 mM Tris adjus-ted to pH 6.2 with hydrochloric acid„
As with the ACM equilibration buffer, variations in the buffering agents and ionic strength adjusting agents may be made in the ACM elution buffer.
Preferably, the ACM eluate is buffer-exchanged using a gel filtration chromatography medium having a low molecular weight exclusion limit. A particularly suitable such medium is Sephadex G-25 (sold by Pharmacia Ltd). Advantageously, the gel filtration chromatography medium is equilibrated with 50mM acetate, 0.01% Tween 80 adjusted to pH 4.0.
Advantageously, the eluate from the gel filtration step is concentrated, preferably using a membrane concentration system, such as a Millipore Minitan system.
In carrying out all the above process steps, it is necessary to ensure that all possible precautions are taken for the exclusion of protease contamination from the chromatography media and equipment.
The buffer exchange step may be used to put the tPA into a buffer suitable for intravenous administration, suitable for freezing, or suitable for freeze drying.
Since the present process can be carried out speedily and without substantially degrading the tPA, it can be carried out at room temperature. This is advantageous over prior art processes, which are usually carried out at 4°C, since it avoids problems of increased solution viscosities and increased sterilizing times.
Using the process of the present invention it is possible to purify tPA on a commercial scale to substantially complete purity with little cleavage of the tPA into its two chain form, in a simple two step purification process. This clearly provides a distinct advantage over the previously known processes. Moreover, the process of the present invention provides substantially pure, substantially only one chain tPA having substantially only one type of N-terminal sequence. Such tPA provides a second aspect of the present invention. A further aspect of the invention is the provision of a stable, concentrated tPA solution comprising the tPA described above in a low pH buffer, which preferably contains a detergent.
The present invention is now described by way of illustration only with reference to the accompanying drawings, in which:
Figure 1 shows a series of sodium dodecyl sulphate (SDS) 7 to 15% polyacrylamide gradient gels used to monitor the progress of the purification wherein: lane 1 shows molecular weight markers; lane 2 shows mother solution; lane 3 shows eluate from the HACM; lanes 4 and 5 show eluates from the ACM; lanes 6 to 9 show eluates from a buffer exchange column; and lanes 10 to 13 show eluates from a second buffer exchange column; and
Figure 2 shows a series of SDS 7 to 15% polyacrylamide gels used to show the purity of the product, wherein: lanes 1 and 5 are molecular weight markers, and lanes 2, 3 and 4 are the eluate from a buffer exchange column at loadings of 6, 12 and 18 micrograms respectively.
In all cases, the samples were reduced and alkylated before application to the gels. In the gels of Figure 1, the loading was 15 micrograms of protein per lane.
For ease of processing, the ACM purification step was carried out in two stages and the buffer •exchange was carried out in four stages, as shown in lanes 6 to 9. The buffer exchanged material was then concentrated in four stages on a Millipore Minitan system, as shown in lanes. 10 to 13.
During the purification procedure described below, the operation of the chromatography stages was monitored, in conventional fashion, by observing the absorbance of the eluates at 280 nm. Elution was continued until the peak indicating protein elution had been fully eluted. The activity of the tPA was measured at all stages by the fibrin agar assay as described by Cederholm-Williams, S.A., Houlbrook, S., Porter, N.W. , Marshall, J.M. and Chissic, H. in "Characterisation of Plasminogen Activators secreted by human Malignant Cells in Treatment of Metastasis: Problems and Prospects", 1985, Eds. Hellman, K. and Eccles, S., Taylor Francis, London. Protein content was estimated using conventional protein assays.
EXAMPLE
A rat cell line which is able to express and secrete tPA was produced by recombinant DNA technology in a manner similar to that described in GB-B-2 119 804. This was cultured in a standard culture medium containing 50 KlU/ml aprotinin in a fermenter to produce a mother solution containing tPA and aprotinin.
In all the following procedures, all equipment and chromatography media were carefully treated to ensure there was no protease contamination.
200 1 of the mother solution containing impure tPA from the fermenter was collected and immediately clarified by filtration through a Balston LP-200 filter diluted to less than 25 mS ionic strength and adjusted to pH 6.0 with hydrochloric acid. The clarified mother solution contained gram quantities of tPA.
An 8 litre column of S-Sepharose Fast Flow was equilibrated with a buffer containing 50 mM acetate, 0.01% Tween 80 adjusted to pH 6.0. The clarified mother solution was immediately applied to the equilibrated column, which was then washed with equilibration medium until all unbound proteins had been eluted (as judged by the absorbance of eluted buffer at 280 n ) .
After washing, the column was eluted using a buffer containing 50 mM acetate, 0.01% Tween 80 and 500 mM sodium chloride adjusted to pH 6.0 until elution of the tPA was complete.
The eluate contained 87% of the applied tPA which was about 30% pure and which had a specific activity of 30 x 103 units of tPA activity per milligram of protein (U/mg).
An affinity chromatography column was prepared by coupling 3g of a monoclonal antibody, MAC 10 (supplied by the Culture Products Division of Celltech Limited), to 1 litre of cyanogen bromide-activated Sepharose CL4B. Monoclonal antibody MAC 10 is specific for tPA. The affinity chromatography column was equilibrated with a buffer containing 100 mM acetate, 200 M sodium chloride, 0.01% Tween 80 adjusted to pH 5.5. The eluate from the S-Sepharose column was immediately applied to the affinity chromatography column, which was then washed with the affinity chromatography equilibration buffer until no more protein was eluted. The tPA was then eluted from the column by use of a buffer containing 50 mM acetate, 0.01% Tween 80 200mM sodium chloride adjusted to pH 3.5 acid until all the protein had been eluted.
The affinity chromatography eluate contained 86% of the applied tPA, which was substantially pure and which had a specific activity of greater than 100 x 103 ϋ/mg.
The affinity chromatography eluate was applied to a 2.2 1 Sephadex G25-M gel filtration column equilibrated with 50mM acetate, 0.01% Tween 80 adjusted to pH 4.0. The tPA was eluted in the buffer free of the components of the ACM elution buffer, which were retained on the column. The collected buffer contained 88% of the applied tPA which was substantially pure and which had a specific activity of 110 x 103 U/mg. The tPA was further concentrated by application to a Millipore Minitan system.
It can thus be seen that the process of the present invention produced a quantity of the tPA which is substantially pure in a simple operation carried out at room temperature. The overall yield of the process was 67% The process can readily be operated on a scale sufficient to produce commercially viable quantities of tPA.
The progress of the purification can be seen from the SDS polyacrylamide gel electrophoresis (SDS-PAGΞ) results shown in Figure 1 of the accompanying drawings. Figure 1 shows a 7.5% - 15% gradient SDS-PAGE (reduced) in which the lanes are as follows:
I molecular weight marker 2 tissue culture 15 ug protein loaded in
3 S-Sepharose F.F. eluate . each case
4 Immunopurification 1 eluate
5 Immunopurification 2 eluate
6 G25 1 7 G25 2 The two immunopurification
8 G25 3 eluates were buffer-exchanged
9 G25 4 on a G25 column (4 mins G25 1
10 G25 cone. 1 to 4) , then concentrated on a
II G25 cone. 2 Minitan system (4 cycles G25 12 G25 cone. 3 cone. 1 to 4)
13 G25 cone. 4
It will be seen from this, and especially lanes 6 to 13, that the tPA produced by the process is mainly in the one chain form with only minor amounts of the two chain form. This is further confirmed by the gels shown in Figure 2 of the accompanying drawings, which show that, even with a highly overloaded gel, lane 4, only very minor amounts of the two chain form are visible.
A sample of one chain tPA was tested in an in vivo assay using beagle dogs at a level of 0.05 to 0.4 mg/kg body weight and found to be fully potent. Moreover, it was found that this solution, containing the tPA at a concentration in excess of Img/ml in a low pH buffer containing a detergent, did not form precipitates, was storage stable and did not enable the tPA to adhere to the walls of the container.
It has been shown by amino acid analysis, enzyme activity, fibrin binding activity and N-terminal analysis that the product of the process is tPA substantially uncontaminated by other material.
In particular, N-terminal analysis of the product of this process showed that 94% of it had the N-terminal sequence. H2-G-A-R-S-Y-Q-V-I (that is, the full amino acid sequence), the remaining 6% having the N-terminal sequence.
As a control, the same fermentation as described above was carried out and the product tPA was separated according to a conventional procedure at conventionally used pHs and ionic strengths. No special precautions were taken to carry out the purification as fast as possible. The product from this control run consisted essentially of the one chain form of tPA. However, it had a variety of species having different N-terminal sequences. The following distribution was observed:
NH2-G-A-R-S-Y-Q-V-I 58 %
NH2-S-Y-Q-V-I 12%
NH2-Y-Q-V-I 11%
It can thus be seen that the process of the present invention results in a highly homogeneous, highly pure, single chain tPA which cannot otherwise be produced by any process known to the present applicants. It will be appreciated that the present process has been described above purely by way of example only and that other variations and modifications of detail can be made without departing from the scope of the invention as defined in the appended claims.