CZ20031277A3 - Process for preparing latent antithrombin III - Google Patents

Abstract

A process for the preparation of latent antithrombin III is provided. The process comprises incubation of a solution of native antithrombin III in the presence of sulfate ions and a buffer selected from Good's zwitterionic buffers.

Description

PROCEDURE FOR PREPARING LATENT ANTITROMBIN III

PREPARATION PROCESS

Technical field

The present invention relates to a process for the preparation of latent antithrombin III.

BACKGROUND OF THE INVENTION

Antithrombin III (AT) is a plasma glycoprotein with a total molecular weight of 58.1 kDa (Lebing et al., Vox Sang. 67, 117-124, 1994), which inhibits serine proteases in the coagulation cascade and therefore plays a major role in regulation blood clotting. Antithrombin III is an inhibitor of Factors IXa, Xa, XI and XIa as well as thrombin. Thus, AT regulates clot formation at various stages of the coagulation cascade. A small decrease in AT content in the blood is associated with an increased risk of thromboembolism. AT concentrates are used in the prevention and treatment of thromboembolic disorders in patients with acquired or hereditary antithrombin deficiency. In addition, AT has been reported to function in many other processes in the human body, for example during angiogenesis and response to inflammatory stimuli. The function of AT in these physiological processes is not fully investigated.

. A particular form of antithrombin III, which was first characterized by Wardell et al. (Biochemistry 36, 1313313142, 1997), is known as the latent form (L-AT). L-AT and a selectively cleaved elastase variant have been shown to have potent anti-angiogenic activity and also suppress tumor growth in mice injected subcutaneously with a human neuroblastoma cell line (O'Reilly et al., Science 285, 1926-1928, 1999, and WO 00/20026). For these reasons, L-AT must be considered as a potential human anti-cancer drug.-Φ φ · · · · · · · · · prot prot prot Clinical verification of this potential drug has yet to be performed.

Purification by AT affinity chromatography was performed using purified heparin as a tightly bound ligand as known in the art. MillerAndersson et al (Thrombosis Research 5, 439-452, 1974) demonstrated the use of heparin-Sepharose for purification of human AT. This chromatographic system is also useful for separating AT and L-AT, where the reduced affinity of heparin for L-AT compared to AT makes it possible to distinguish these two components as described in Chang and Harper (Thrombosis and Haemostasis 77, 323-328, 1997). Hydrophobic chromatography was used to separate native and latent forms of AT (Karlsson, G & Winge, S. (2001) Protein Expr. Purif. 21: 149-155).

Induction of the latent form of AT was previously performed as described by Wardell et al. (supra) who obtained 50-60% L-AT by incubating AT in 0.25 M citrate, 10 mM Tris / HCl, pH 7.4, for 15 h at 60 ° C.

After incubation of native antithrombin III at 60 ° C in medium or buffer alone, polymerized protein aggregates were often formed. The presence of these aggregates damages the high yield of latent antithrombin III and should be avoided wherever possible.

It is therefore an object of the present invention to provide a process for preparing latent antithrombin III, L-AT from a solution of native antithrombin III, AT, wherein such a process provides a higher yield of the desired product than the prior art known in the art.

It is another object of the invention to provide a process for preparing L-AT from AT, wherein formation of aggregates of AT polymers is minimal.

It is a further object of the invention to provide a process for converting AT to L-AT, wherein commercially available AT-L-ATs are used. Reagents and buffer solutions, which are carried out in vitro.

Yet another object of the invention is to provide a process for preparing L-AT from AT, which is readily scalable for industrial production of L-AT.

Description of the invention

The foregoing and other objects of the invention comply with the process defined by the claims. Therefore, a method is provided which comprises incubating a solution of native antithrombin III in the presence of sulfate ions and buffers selected from Good zwitterionic buffers. Surprisingly, it has been found that these incubation conditions, which avoid possible aggregation problems, allow recovery of L-AT from the process in yields that are substantially higher than those obtained by methods of the prior art (particularly the citrate conditions of Wardella et al.).

Descriptions for Figures

Figure 1: Heparin affinity chromatography of antithrombin using a 0-2 M sodium chloride gradient (5-60 min). The amount of protein injected was 100 pg for sample A-B and 150 pg for sample C-D. All samples were incubated at 60 ° C for 16 h except reference AT sample A, which was not heat treated (sample 7 in the example). Sample B (sample 6 in the example) was incubated according to Wardell, i.e. in 0.5 M citrate. Sample C (Sample 2 in the example) was incubated in 5 mM HEPES, pH 7.4 with 0.9 M ammonium sulfate and Sample D (Sample 1 in the example) was incubated in 5 mM HEPES, pH 7.4 with 0.8 M ammonium sulfate. Integration of the low affinity heparin-binding peak eluted at 22 min gave 44% of the total integration area of sample B, 71% of the total integration area of sample C and 89% of the total integration area of sample D.

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mM HEPES, O, 8 M sulfate ammonium, pH 7, 4 mM HEPES, O, 9 M sulfate ammonium, pH 7, 4 mM HEPES, 1, 1 M sulfate ammonium, pH 7, 4 mM HEPES, 1 , 4 M sulfate ammonium, pH 7 , 4 mM HEPES, 2 , 0 M sulfate ammonium, pH 7 , 4

Native AT was eluted at 39 min.

Figure 2: Native electrophoresis of antithrombin samples using 12.5% polyacrylamide in a homogeneous gel. The sample amount was 0.5 µg protein / lane and the gels were stained with silver at the end of the split. All samples, except lane 7, were incubated at 60 ° C for 16 h.

Track 1)

Runway 2)

Dráha 3)

Dráha 4)

Track 5)

Lane 6) 10 mmol Tris / HCl, 0.5 M trisodium citrate, pH 7.4 (according to Wardell et al. 1997)

Lane 7) reference AT sample, no heat treatment Lane 8) 25 mM sodium phosphate, 100 mM sodium chloride, pH 7.4

Lane 9) 25 mM HEPES, 0.8 M ammonium sulfate, pH 7.4

Lane 10) 5 mM HEPES, 0.5 M ammonium sulfate, pH 7.4 Lane 11) 5 mM HEPES, 2.0 M ammonium sulfate, pH 7.4 Lane 12) 5 mM HEPES, 0.8 M ammonium sulfate, pH 7.0 All numbers refer to the sample numbers given in the example below.

Detailed description of the invention

The invention provides a description for the preparation of latent antithrombin III (referred to as L-AT), starting from a solution of antithrombin III in its native form (referred to as AT). AT can be isolated from blood plasma by heparin-Sepharose chromatography as described. According to the invention, AT is then incubated in the presence of sulfate ions and buffers. The temperature and duration of the incubation can be readily determined by one of ordinary skill in the art, but it has been found that normal pasteurization conditions, such as a temperature of about 60 ° C for about 60 ° C, for approximately 9 9999. · · · · · · ····· · · ·

9 9 9 9 · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

The sulfate ions are preferably provided in the form of a sulfate salt. Here, the use of alkaline earth sulfate, alkali metal sulfate or ammonium sulfate is preferred. Especially preferred is the use of ammonium sulfate. A suitable concentration of sulfate ions in the process of the invention is in the range of from 0.5 to 2.0 M, more preferably from 0.7 to 1 M, and most preferably the concentration is between 0.8 and 0.9 M.

Another component in the incubation mixture is a buffer selected from Good zwitterionic buffers (Good et al., Biochemistry 5, 467-477, 1966). The choice of use of one of the shown buffers can be determined without conducting the experiment, bearing in mind that the buffer should meet most or all of the following requirements: it should have a pK _{a of} between about 6 and about 9, maximum solubility in water and minimal solubility in other solvents, creating a minimum of salt effects, being stable under the experimental conditions used and not absorbing light in the visible or ultraviolet spectral range (and thus not interfering with spectrophotometric measurements). Good zwitterion buffers, including buffers such as HEPES, MES, and PIPES, typically exhibit desirable characteristics. The use of HEPES is particularly preferred in the process of the invention. The widespread Tris buffer is unsuitable for the purposes of the invention. Preferred buffer concentrations are somewhat dependent on the selected buffer, but are generally in the range of from 1 to 25 mM, more preferably from 2.5 to 10 mM, and most preferably from 4 to 6 mM.

As indicated above, the pH of the incubation reaction should lie between pH 6 and pH 9, preferably between pH 7 and pH 8, and most preferably between pH 7.4 and pH 7.6.

Following the incubation of AT under the conditions shown above, it is 99 · · je · · · · · · · · · · · ·

The separation of the L-AT thus obtained from the remaining AT was carried out by means of affinity chromatography on heparin. LAT, which exhibits a lower binding affinity for heparin than AT, elutes significantly faster and allows easy separation of the two forms of antithrombin III.

The preparation of the L-AT thus obtained is preferably subjected to a treatment for inactivation or removal of pathogens, in particular in the form of viruses and prions. This can be done at any stage of the process using one of several methods of inactivation or removal known in the art, or a combination of such methods. Examples of such methods include chemical inactivation, thermal inactivation, light inactivation, microwave inactivation and removal by nanometric filtering. Cross-flow filtration with a high salt content, as described in WO96 / 00237, is particularly preferred, alone or in combination with other processes. Removal and inactivation of pathogens may be performed when the antithrombin III molecules are in the native state, prior to conversion to L-AT.

The invention is further illustrated by the following non-limiting example.

EXAMPLE

A laboratory AT sample of> 95% purity was obtained from

Plasma Products ,. Pharmacia, Stockholm, Sweden. This sample was prepared according to known methods (MillerAndersson et al., Supra) and used to induce the latent form of antithrombin.

Preparation of L-AT

The laboratory AT sample was transferred to the following solutions:

Sample 1) 5 mM HEPES, 0.8 M ammonium sulfate, pH 7.4 ·· · ♦ ··

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7 * • • • ·· · • · · • «♦ · »» · • ·· • 9 999 9 9 9 9 9 99 99 Sample 2) 5 mM HEPES, 0.9 M sulfate ammonium, pH ^{Ί '4} Sample 3) 5 mM HEPES, 1.1 M sulfate ammonium, pH Sample 4) 5 mM HEPES, 1.4 . M sulfate ammonium, pH 1.4 Sample 5) 5 mM HEPES, 2.0 M sulfate ammonium, pH 7.4 Sample 6) 10 mmol Tris / HCl, 0,5 M citrate sodium triple, pH ! 7, 4 (according to Wardell and kol.1997) Sample 7-8) 25 mM phosphate 100 mM chloride sodium pH 7.4 Sample 9) 25 mh 1 HEPES, 0, i 3 M 1 ammonium sulfate pH 7.4 Sample 10) 5 mM HEPES, 0.5 M sulfate ammonium, pH 7.4 Sample 11) 5 mM HEPES, 2.0 M sulfate ammonium, pH 7.4 Sample 12) 5 mM HEPES, 0.8 M sulfate ammonium, pH 7.0 Sample 13) 5 mM HEPES, 0.8 M sulfate ammonium, pH 7.8

All buffers listed above were adjusted to the desired pH at room temperature; 1 M HCl was used to adjust Sample 6, while 1 M sodium hydroxide was used to adjust the pH of all other samples.

AT at a final concentration of 6 mg / ml was incubated in solutions (Samples 1-13) in glass tubes for 16 h at 60 ° C (except for Sample 1, which was kept in a refrigerator at about 8 ° C) and transferred to a solution containing 50 ° C. mM Tris / HCl, 50 mM sodium chloride, pH 7.4, using small gel filtration columns (NAP-5 Amersham Pharmacia Biotech, Uppsala, Sweden).

The formation of L-AT in the samples was analyzed by affinity chromatography on heparin and the presence of aggregates was analyzed by native electrophoresis.

Affinity chromatography on heparin

This method was performed according to Chang and Harper (supra). An HPLC equipped with a TSK Heparin® column (Tosohaas, Stuttgart, Germany, 7.5 ID x 75 mm, 10 µm, 1000 Å) was used. 20 mM Tris / HCl buffer, pH 7.4 (Buffer A) and 2 M * were used as elution buffers.

· Chloride in 20 mM Tris / HCl buffer, pH 7.4 (buffer B). Linear gradient (0-5 min 0% B, 5-60 min 0-100% B, 60-90 min 0% B) was used. The flow rate was 0.4 ml / min and detection was performed by measuring absorbance at 280 nm.

Native polyacrylamide gel electrophoresis

Electrophoresis was performed using a 12.5% polyacrylamide homogeneous Phast ® gel (Amersham Pharmacia Biotech, Uppsala, Sweden) following the recommended parameters. Each lane was loaded with 0.5 μρ of protein at 1 μΐ. Diamino-silver staining was performed according to the Pharmacia & Upjohn Handbook (Phast System ™, Technical Note No 2, Two-Dimensional Electrophoresis with PhastGel ™ separation media, Pharmacia LKB Biotechnology AB, Uppsala, Sweden) except that a slightly stronger one was used. fixative solution containing 50% ethanol, 10% acetic acid and 40% water.

Antithrombin activity

Sample 2 (incubation in 0.9 M ammonium sulfate) was analyzed for biological AT activity with thrombin chromogenic peptide substrate (S-2238) (Chromogenix, Molndal, Sweden), according to Handeland et al. (Scand J. Haematol. 31: 427-436,1983). The test solution consisted of thrombin, heparin, chromogenic substrate and sample, and after incubation the response was recorded as a change in absorbance at 405 nm.

Results

Heparin affinity chromatography eluted native AT at 39 min (approximately 0.9 M sodium chloride) and the major latent peak eluted at 22 min. (Approximately 0.3 M sodium chloride) (Figure 1A-1B) (Figure 1A-1B) ). Integration of a low heparin-binding peak showed a yield of 44% (Figure 1B) for a sample prepared according to the Wardell method (Sample 6), while incubation in 0.9 M ammonium sulfate (sample 2) and 0.8 M ammonium sulfate ( Sample 1) provided 71% for Sample 2 and 89% for Sample 1 of the total integrated area (Figure 1C-1D). Table 1 shows that the percentage of L-AT formed decreases at increased ammonium sulfate / HEPES concentration or at higher pH.

Native AT electrophoresis incubated at 60 ° C in phosphate / NaCl (Sample 8) gave strong aggregation formation and only a minor portion of the protein remained in monomeric form (Figure 2, lane 8). AT incubated according to Wardell (Figure 2, lane 6), as well as unincubated AT (Figure 2, lane 7), did not form any aggregates. Incubation in 0.5 M ammonium sulfate (Sample 10) induced strong aggregation (Figure 2, lane 10), while 0.8 M (Sample 1) gave only a small fraction of the aggregates (Figure 2, lane 1). Ammonium sulphate at a concentration of 0.9 - 2.0 M (Samples 2-5) did not lead to the formation of visible aggregates (Figure 2, lanes 2-5). Many aggregates were observed at pH 7.0 (Figure 2, lane 12), while at pH 7.8 only a small amount of aggregates were formed (data not shown).

The antithrombin activity assay in Sample 2 (with 0.9 M ammonium sulfate) showed that 34% of the original specific activity remained; this should be compared with a 29% gain of AT binding with high affinity for heparin after analyzes of the same sample by affinity chromatography (Table 1).

· · 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

Table 1: Heparin affinity chromatography. L-AT formation in various sample buffers after 16 h incubation at 60 ° C.

Incubation solution Sample ¹ % AT with low affinity heparin 10 mmol Tris / HCl, 0.5 M citrate, pH 7.4 6 . . ★ 44 (Wardell) 5 mM Hepes, 0.5 M ammonium sulfate, pH 7.4 10 99 5 mM Hepes, 0.8 M ammonium sulfate, pH 7.4 1 89 5 mM Hepes, 0.9 M ammonium sulfate, pH 7.4 2 71 * 5 mM Hepes, 1.1 M ammonium sulfate, pH 7.4 3 56 * 5 mM Hepes, 1.4 M ammonium sulfate, pH 7.4 4 49 * 5 mM Hepes, 2.0 M ammonium sulfate, pH 7.4 5 48 * 25 mM Hepes, 0.8 M ammonium sulfate, pH 7.4 9 ⁷⁰ 5 mM Hepes, 0.8 M ammonium sulfate, pH 7.0 12 99 5 mM Hepes, 0.8 M ammonium sulfate, pH 7.8 13 65

¹ By example

Native electrophoresis analysis did not show aggregates (see Figure 2)

Experimental conclusions

By incubating AT in 5 mM HEPES, pH 7.4, containing 0.8 M ammonium sulfate at 60 ° C for 16h, approximately 85-90% AT was converted to the latent form and incubating AT in 5 mM HEPES, pH 7.4 containing 0.9 M ammonium sulfate at 60 ° C for 16h was converted to the latent form AT of approximately 70-75%. Native electrophoresis showed a small proportion of aggregates in 0.8 M ammonium sulfate, and no aggregates were observed at 0.9 M. By purification, such small amounts of aggregate can be easily removed.

Gel filtration or similar techniques.

Optimal concentration to convert AT to L-AT using sulfate. ammonium is 0.8-0.9 M. Good conversion results will also be obtained between 0.7 and 1 M, and certain results between 0.5 and 2.0 M. For L-AT formation, the procedure using 0, 5-2.0 M ammonium sulfate, preferably 0.8-0.9 M, and up to 25 mM HEPES, preferably no more than 10 mM, at a pH of about 7.4 found to give the most satisfactory results. The percentage of L-AT formed will decrease with a higher concentration of ammonium sulfate / HEPES or at a higher pH. In addition, it is necessary not to use too low a concentration of ammonium sulfate or too low a pH value to prevent the formation of aggregates.

Claims (11)

PATENT CLAIMS A process for preparing latent antithrombin III, comprising incubating a solution of native antithrombin III in the presence of sulfate ions and a buffer selected from Good zwitterionic buffers. The process of claim 1, wherein said sulfate ions are provided as a salt selected from ammonium sulfate, alkali metal sulfates and alkaline earth sulfates. The process of claim 2, wherein said sulfate is ammonium sulfate. The method of any one of claims 1-3, wherein the concentration of said sulfate ions is in the range of 0.5 to 2.0 M. 5. Procedure according to claim 4, where concentration sulphate ion is in range from 0.7 up to 1 M • 6. Procedure according to claim 5, where concentration sulphate ion is in range from 0.8 do 0,9 M. The process of any one of claims 1-6, wherein said buffer comprises a HEPES buffer. The method of any one of claims 1-7, wherein the concentration of said buffer is in the range of 1 to 25 mM. The method of claim 8, wherein the buffer concentration is in the range of 2.5 to 10 mM. The method according to claim 9, wherein the concentration of the buffer is "A". In the range of 4 to 6 mM. 11. Procedure according to any from claims 1-10, where the pH is ranging from 6 to 9. 12. Procedure according to claim 11, where pH Yippee in the range of 7 to 8. 13. Procedure according to claim 12, where pH Yippee ranging from 7.4 to 7.6. The method of any one of claims 1-13, further comprising treating inactivation or removal of pathogens, particularly viruses or prions. The method of claim 1, wherein the native antithrombin III has been adapted to inactivate or remove pathogens, particularly viruses and prions. The method of claim 14 or 15, wherein said treatment comprises one or a combination of methods selected from chemical inactivation, thermal inactivation, light inactivation, microwave inactivation and removal by nanometric filtering.