FR2590274A1 - Process for the industrial preparation of a superoxide dismutase extract - Google Patents

Abstract

This process for preparing a superoxide dismutase extract from a lysate of red blood cells or another animal cell extract comprises the steps consisting of: 1.treating, at about 4 DEG C, the lysate or extract by rapidly adding not less than 0.7 volumes of a chloroform/ethanol or methylene chloride/ethanol mixture in proportions of 2 to 7:5 by volume in order to precipitate in a lump most of the protein substances other than SOD; 2.after allowing to stand, treating the supernatant obtained by adding 200 to 400 g/l of disodium or dipotassium phosphate; 3.allowing to stand until a brownish ring separating the two liquid phases is obtained; and 4.treating the upper liquid phase, cooled to about 4 DEG C, with 0.6 to 1 volume of acetone, cooled to the same temperature, in order to precipitate the SOD. Application especially in pharmacy.

Description

Process for the industrial preparation of a superoxide dismutase extract
The invention relates to a process for the industrial preparation of superoxide dismutase (SOD) from animal cells.

 More specifically, the subject of the invention is a simplified, rapid and economically advantageous process for the industrial production of superoxide dismutase from animal cells, in particular erythrocytes, and a new protein concentrate, rich in superoxide dismutase .

O2 + H202
I1 a été démontré que grâce à cette propriété, la superoxyde dismutase qui est connue en pharmacie sous le nom d'orgotéine, peut être efficacement utilisée dans le traitement de différentes maladies inflammatoires ainsi que de diverses maladies dues à la production de radicaux oxygène libres dans l'organisme ou à la surface de la peau.We know that superoxide dismutase is an enzyme that catalyzes the dismutation of free radical oxide superoxides according to the reaction 20 + 2H +

O2 + H202
It has been shown that thanks to this property, superoxide dismutase which is known in pharmacies as orgotein, can be effectively used in the treatment of various inflammatory diseases as well as various diseases due to the production of free oxygen radicals in the body or on the surface of the skin.

 For the past fifteen years, a great deal of research has been carried out with the aim of developing a process making it possible to obtain a preparation of superoxide dismutase of high purity, suitable for use without harmful side effects, in pharmaceutical compositions, in particular injectable solutions.

 The sources of superoxide dismutase are very varied. It can for example be extracted from certain strains of bacteria. However, it seems particularly advantageous to extract it from animal cells, in particular cells from certain mammals, in particular liver and heart cells and especially from erythrocytes from cattle, sheep, pigs and humans.

 The techniques proposed so far for extracting superoxide dismutase from animal cells, in particular erythrocytes, lead, at least for some of them, to a product of a purity satisfactory for pharmaceutical use, but are difficult to use industrially. , due to their complexity and / or their high cost price.

 Thus Joe M. McCord and Irwin Fridovich g J.

Biol. Chem. 244, 6049-6055 (1969) j describe the extraction of SOD, on a laboratory scale, from beef blood. The red blood cells separated from the plasma by centrifugation are washed with physiological saline and lysed with deionized water. The lysed cells are then treated as follows.

 Hemoglobin is precipitated according to the conventional method of Tsuchihashi L iochem. Z, lq9, 65 (1923) 2, using a mixture of chloroform and ethanol (0.15 and 0.5 volumes respectively). The supernatant is then separated from the precipitate by centrifugation and then it is treated with K2HPO4, which causes a separation in two phases. The enzyme is found in the upper phase which represents approximately 40% of the total. The method provides for a second centrifugation to clarify the liquid which contains practically all of the enzyme. The solution is then cooled to 4 ° C. and the SOD precipitated with 0.75 volume of cold acetone. After centrifugation, the precipitate is dissolved in distilled water and dialyzed against 2.5 mM potassium phosphate at pH 7. , 4 to then be applied to a chromatography column containing DE-32 cellulose. The column is balanced with the buffer used for dialysis and eluted with a potassium phosphate gradient whose molarity varies between 2.5 and 200mM. A blue-green protein (SOD) is eluted in a single peak and thus separated from other contaminating proteins. The enzyme obtained by this technique is very pure. However, this process cannot be used on an industrial scale, for the reasons indicated below.

 The mixture obtained after precipitation of the hemoglobin becomes very thick when it is stirred for 15 minutes. Even if we add, as the authors suggest, 0.1 vol. of water, the problem of centrifugation is not resolved.

Indeed, the plates of laminar flow centrifuges are quickly clogged. The centrifuges with pots or drums must be continuously emptied of a very hard to unhook pellet, which represents approximately 30% of the total volume. Large machines such as garbage collectors or clarifiers (manufactured by Kraus
Maffei) are unusable because they give off too much solvent vapor.

 The centrifugation necessary to clarify the liquid phase containing the SOD, obtained after treatment with K2HPo4, also complicates the implementation of this process.

 Finally, the final purification using a buffer concentration gradient requires complicated installations and significant monitoring, with the intervention of qualified personnel.

 The applicants have set themselves the aim of remedying, at least in part, the drawbacks set out above and of developing a simplified process, advantageous from an economic point of view, making it possible to obtain SOD on an industrial scale. from animal cells.

 In a first step in their research to simplify the McCord and Fridovich process, the applicants established that, under very specific conditions which they determined, it was possible to precipitate the bulk of most of the protein substances other than SOD, especially hemoglobin, from a lysate of red blood cells or another extract of animal cells, without the need for centrifugation.

 These very precise conditions consist in treating the lysate or the extract, at refrigerator temperature (approximately 4 ° C.), by rapid addition of at least 0.7 volume, preferably 0.8 volume, of a mixture chloroform / ethanol or methylene chloride / ethanol in the proportions of 2 to 7: 5 by volume. Under these conditions, substances such as hemoglobin precipitate as a block in the form of a hard "cake" which remains at the bottom of the container. Lighter particles still settle out by standing for a few hours, for example overnight.

 The supernatant obtained lends itself without further modification to the next phase of separation, namely salting out with a salt. This release is carried out by adding 200 to 400 g / l, preferably 300 g / l, of disodium or dipotassium phosphate.

 In a second stage of their research, the applicants established that at this stage, according to the invention and contrary to what happened according to the prior art, it is not necessary to centrifuge to clarify the liquid obtained by the treatment release. It suffices to wait for a brownish ring containing the major part of the impurities to form (rest for approximately 2 hours) between the two liquid phases which separate. Some impurities also settle on the bottom of the containers.

 The upper phase contains SOD in aqueous ethanol. It is cooled to about 4-C and treated with 0.6 to 1, preferably 0.75 volume of acetone cooled to around the same temperature to precipitate the SOD.

 In a third stage of their research, the applicants established that it was possible to extract the SOD with high purity from the crude extract obtained by dissolving in the water the precipitate obtained during the treatment with acetone , by simple chromatography possibly preceded by dialysis, without having to use a concentration gradient, and without it being necessary to concentrate or lyophilize beforehand the crude extract. This considerable simplification of the processing of the crude extract is made possible on the one hand thanks to the particular qualities of this raw extract, improved compared to those of the extract of the prior art thanks to the modifications made according to the invention to its preparation process and on the other hand, thanks to a particular choice both of the chromatography column and of the characteristics of the buffer used to balance the column and to elute it.

This particular choice consists in using an ion exchange column of low ionic strength of the type
DEAE (that is to say containing diethylaminoethyl groups), such as those which are marketed by the company Pharmacia, in particular under the trade name DEAE-Sephacel and to work at a pH value of 7.3 to 8, 3, preferably 7.8, the conductivity of the balancing buffer being substantially equal to the conductivity of the crude extract and the conductivity of the elution buffer being substantially double that of the balancing buffer.

The subject of the invention is therefore a process for the industrial preparation of an extract of superoxide dismutase (SOD) from a lysate of red blood cells or of another extract of animal cells, according to which the cells are rid of the major part of the protein substances other than the SoD by precipitation by a solvent, the SOD is released from its solution by means of a mineral salt then is precipitated by acetone, characterized in that it essentially comprises the stages consisting of 8:
1) treat, at refrigerator temperature (approximately 4 ° C.), the lysate or the extract by rapid addition of at least 0.7 volume, preferably 0.8 volume, of a chloroform / ethanol mixture or methylene chloride / ethanol in the proportions of 2 to 7: 5, preferably 3 :: 5, in volumes to precipitate in bulk the major part of the protein substances other than SOn
2) after standing, treat the supernatant obtained under 1) by adding 200 to 400 g / l, preferably 300 g / l, of disodium or dipotassium phosphate
3) let stand until a brownish ring separating the two liquid phases met and
4) treating the upper liquid phase cooled to about 4 ° C with 0.6 to 1, preferably 0.75 volume of acetone cooled to the same temperature to precipitate the SOD.

 The SOD extract thus obtained is not yet sufficiently purified to be used in all types of pharmaceutical preparations, in particular injectables. However, it represents a new industrial product which can be stored in frozen aqueous solution or lyophilized and be used in particular in pharmaceutical preparations for external use. As indicated above, this product already has a purity such that it leads after a simple treatment, explained above, to a preparation of SOD having a high purity.

 The invention also relates to this new industrial product, namely a crude preparation of superoxide dismutase extracted from animal cells, in particular from erythrocytes, in the form of a greenish solution in water, frozen or in the lyophilized state, characterized in that it has a specific activity in the range from 2000 to 2500 units per mg of protein (riboflavin-nitroblue tetrazolium method) and, according to polyacrylamide electrophoresis, contains neither albumin nor catalase and essentially gives a main band followed by 'a small band.

 The invention further relates to a process for obtaining a preparation of superoxide dismutase suitable for pharmaceutical use, characterized in that it essentially consists in chromatography, possibly after reduction of its mineral salt content, the crude preparation of superoxide dismutase described above, on a low ionic strength ion exchange column of the DEAE-Sephacel type, at a pH value of 7.3 to 8.3, preferably 7.8, the conductivity of the buffer balancing being substantially equal to the conductivity of the crude extract and the conductivity of the elution buffer being substantially double that of the balancing buffer.

The invention finally relates to a process for the industrial preparation of an extract of superoxide dismutase
(SOD) suitable for pharmaceutical use from a lysate of red blood cells or other extract of animal cells, according to which the cells are freed from most of the protein substances other than SOD by precipitation by a solvent , the SOD is released from its solution by means of a mineral salt and then is precipitated by acetone, characterized in that it essentially comprises the steps consisting in
1) process, at refrigerator temperature
(approximately 4 * C) the lysate or the extract by rapid addition of at least 0.7 volume, preferably 0.8 volume, of a chloroform / ethanol or methylene chloride / ethanol mixture in the proportions of 2 at 7: 5, preferably 3 :: 5, in volumes to bulk precipitate most of the peptide substances other than SOD
2) after standing, treat the supernatant obtained under 1) by adding 200 to 400 g / l, preferably 300 g / l, of disodium or dipotassium phosphate
3) let stand until a brownish ring separating the two liquid phases is obtained
4) treating the upper liquid phase cooled to about 4-C with 0.6 to 1, preferably 0.75 volume of acetone cooled to the same temperature to precipitate the SOD; and
5) chromatograph, possibly after reduction of its mineral salt content, the SOD thus precipitated, on an ion exchange column of low ionic strength of the DEAE-Sephacel type, at a pH value of 7.3 to 8.3, preferably 7.8, the conductivity of the balancing buffer being substantially equal to the conductivity of the crude extract and the conductivity of the elution buffer being substantially double that of the balancing buffer.

 According to the invention, use is preferably made, as animal cells, of red blood cells, in particular red blood cells originating from ox blood, because of the great availability and the low cost of this product.

 The invention will be better understood with the aid of the detailed description which follows of a nonlimiting example of implementation of the invention.

Example
Oxygen blood is used as the starting material.

1. Hemolysis of beef sana
400 l of ox blood drawn from the carotid artery of 40 animals are collected in citrate buffer to prevent coagulation. The red blood cells are separated from the plasma by continuous centrifugation at low speed and collected directly in an equal volume of physiological sodium chloride solution at 0.9 t. in large plastic containers. This operation is repeated twice to remove all traces of plasma, but in the last step the cells are added to an equal volume of deionized water to cause their lysis. After mixing, the lysis is allowed to take place for about 2 hours 4 * C (fridge temperature).

2. Elimination of hemoglobin by treatment with chloroform / ethanol melanae
The cell lysate is briefly agitated, for example by hand, before the rapid addition of 0.8 volumes of 3: 5 mixture by volumes of chloroform / ethanol.

 Under these conditions, a precipitate immediately forms which sediments at the bottom of the container in the form of a hard "cake", without the need to centrifuge.

This "cake" is rejected. After two hours, the translucent supernatant contains only traces of a flocculent suspension and can be used as it is for further treatment.

 This supernatant can be stored for several hours at room temperature without disadvantage for the enzyme.

3. Purification of the enzyme By relaraaae and Acetone Precipitation
The purification of the supernatant is continued by salting out using 300 g / l of K2HPO4, in a large separation column, at room temperature. Two layers of liquids form in about two hours. The upper layer contains the enzyme in water-ethanol solution, the lower layer is a concentrated salt solution. The two layers are separated by a brownish ring containing a large part of the impurities, other impurities possibly deposit at the base of the lower layer.

 The lower phase and all the impurities are rejected. The upper phase containing the enzyme is cooled to about 4C and treated, in a separation column, with 0.75 volume of acetone cooled with ice.

 A gray-white precipitate sediments by gravity and is collected after one hour in the form of a suspension. The residual solvent (approximately 20 l in the case of the treatment of 400 l of blood) is removed by centrifugation at low speed (4000 rpm, 10 minutes, 4 ° C.). The pellet which contains the "crude" enzyme is dissolved in 4 l of deionized water. The total protein concentration is then around 4 g / l.

 4. Final purification by adsorption on a DEAE-Senhacel column (trade name of the company Pharmacia) and elution in one step.

The potassium phosphate content of the crude enzyme solution is lowered, so that it is less than 10 mM (which corresponds to a conductivity less than 1.8 mSiemens at 20-C), by ultrafiltration against water using a type filter
PTGC 00005 Pellicon 210K marketed by the company
Millipore (retaining molecules with a molar mass greater than 10,000 daltons).

 Aliquots of one liter of the crude enzyme solution are applied to 5 x 13 cm DEAE-Sephacel columns equilibrated with 10 mM potassium phosphate buffer, pH 7.8 and 4-C. A brownish ring about 0.5 cm high remains adsorbed at the top of the column and the upper third of it has the blue-green color characteristic of SOD.

 The enzyme is eluted with 20 mM phosphate buffer at pH 7.8. Elution of the enzyme begins after the flow of an "empty" volume of 240 ml; the fractions corresponding to the concentration peak are combined in 200 ml of elution buffer which contain approximately 80% of the purified enzyme. The remaining 20 can be eluted from the column in 500 ml of the same buffer and, if desired, rechromatographed on another column of the same type.

Results
The "raw" SOD preparation obtained by acetone precipitation is a greenish solution having a specific activity in the range from 2000 to 2500 units / dosing riboflavin-nitrobleu tetrazolium (NBT) / per mg of protein.

 According to polyacrylamide electrophoresis, this preparation contains neither albumin nor catalase and gives, in this analysis, a main band followed by a minor band, as well as weak traces of impurities.

 Analysis by high performance liquid chromatography (HPLC) on a column of Aquapore AX-300 type, sold by the company Kontrvn, in 20 mM Tris-HCl phase, pH 7.5, with a NaCl gradient from 0 to 0, 15 M in 30 min, at a flow rate of 1.0 Q ml / min, at 280 nm, shows a main peak accompanied by two minor peaks.

 The purified enzyme is blue-green and has a specific activity in the range from 2600 to 3200 units (riboflavin-NLT dosage) per mg of protein.

 Contrary to what is observed with the raw material, the polyamide gel eiectrophoresis reveals only the main band and a second minor band.

 Analysis by HPLC shows a single peak.

 The enzyme has a characteristic UV absorption spectrum with a shoulder at 258 nm and a broad maximum at 280 nm in visible light.

Efficiency of the method according to the invention
This process provides 35 to 40 mg of crude enzyme, 20 to 25% of which is lost during purification.

It therefore provides 26 to 32 mg of purified enzyme per liter of blood.

 The McCord-Fridovich process, which can only be carried out on a laboratory scale, provides approximately 30 mg of purified enzyme.

 The yields of the two methods relative to the quantities of blood used are therefore quite comparable.

 Furthermore, the McCord-Fridovich process provides 90,000 units of SOD for 30 mg of protein, or 3,000 units of SOD for i mg, which is also quite comparable to the specific activity of 2,600 to 3,200 units of SOD. purified, obtained according to the invention.

 In conclusion, the method according to the invention makes it possible, thanks to all the simplifications which it introduces, to obtain a high quality SOD, comparable to that obtained by the closest prior art complex method, and this with an equivalent yield.

Claims (5)

 1. Process for the industrial preparation of an extract of superoxide dismutase (SOD) from a lysate of red blood cells or another extract of animal cells, according to which the cells are rid of most of the protein substances other that the SOD by precipitation with a solvent, the SOD is released from its solution by means of a mineral salt and then is precipitated by acetone, characterized in that it essentially comprises the steps consisting in  1) treat, at refrigerator temperature (about 4 ° C.), the lysate or the extract by rapid addition of at least 0.7 volume, preferably 0.8 volume, of a chloroform / ethanol or chloride mixture of methylene / ethanol in the proportions of 2 to 7: 5, preferably 3 :: 5, in volumes to precipitate in bulk the major part of the protein substances other than SOD  2) after standing, treat the supernatant obtained under 1) by adding 200 to 400 g / l, preferably 300 g / l, of disodium or dipotassium phosphate  3) let stand until a brownish ring separating the two liquid phases is obtained  4) treating the upper liquid phase cooled to about 4-C with 0.6 to 1, preferably 0.75 volume of acetone cooled to the same temperature to precipitate the SOD.  2. Raw preparation of superoxide dismutase extracted from animal cells, in particular erythrocytes, in the form of a greenish solution in water, frozen or in the lyophilized state, characterized in that it has a specific activity in the range from 2000 to 2500 units per mg of protein, riboflavin-nitroblue tetrazolium method) and, according to polyacrylamide electrophoresis, contains neither albumin nor catalase and gives essentially a main band followed by a band of low importance.  3. Method for obtaining a preparation of superoxide dismutase suitable for pharmaceutical use, characterized in that it essentially consists in chromatography, optionally after reduction of its mineral salt content, the crude preparation of superoxide dismutase described below above, on an ion exchange column of low ionic strength of the DEAE type Sephacel, at a pH value of 7.3 to 8.3, preferably 7.8, the conductivity of the balancing buffer being substantially equal to the conductivity of the crude extract and the conductivity of the elution buffer being substantially double that of the balancing buffer.  4. Process for the industrial preparation of a superoxide dismutase (SOD) extract suitable for pharmaceutical use from a lysate of red blood cells or another extract of animal cells, according to which the cells are rid of the most of the protein substances other than SOD by precipitation with a solvent, SOD is released from its solution by means of a mineral salt and then is precipitated by acetone, characterized in that it essentially comprises the steps consisting in  1) treat, at refrigerator temperature (around 4 ° C.) the lysate or the extract by rapid addition of at least 0.7 volume, preferably 0.8 volume, of a chloroform / ethanol or methylene chloride mixture / ethanol in the proportions of 2 to 7: 5, preferably 3 :: 5, in volumes to precipitate in bulk the majority of the peptide substances other than SOD  2) after standing, treat the supernatant obtained under 1) by adding 200 to 400 g / l, preferably 300 g, of disodium or dipotassium phosphate  3) let stand until a brownish ring separating the two liquid phases is obtained  4) treating the upper liquid phase cooled to about 4-C with 0.6 to i, preferably 0.75 volume of acetone cooled to the same temperature to precipitate the SOD; and  5) chromatograph, possibly after reduction of its mineral salt content, the SOD thus precipitated, on an ion exchange column of low ionic strength of the DEAE-Sephacel type, at a pH value of 7.3 to 8.3, preferably 7.8, the conductivity of the balancing buffer being substantially equal to the conductivity of the crude extract and the conductivity of the elution buffer being substantially double that of the balancing buffer.  5. Method according to one of claims 1,3 or 4, characterized in that the animal cells are red blood cells, in particular beef blood.