US 3293236 A
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United States Patent 3,293,236 PROCESS FOR ISOLATllN G a -ANTITRYPSHN Hermann E. Schultze and Norbert Heimhurger, Marbach,
near Mar-burg (Lahn), Germany, assignors to Behringwerke Aktiengesellschaft, Mai-burg (Lahn), Germany, a corporation of Germany No Drawing. Filed lune 11, 1964, Ser. No. 375,424 Claims priority, application Germany, June 12, 1963,
4 Claims. (Cl. 260-112) The present invention relates to a process for isolating o -antitrypsin, in particular from hum-an or animal body fluids, with exchangers which contain carboxymethyl groups.
As known in the art, the electrophoresis of serum globulins in an alkaline medium produces three principal fractions, the most mobile of which is identified as aglobulin and is in turn comprised of two further mate-rials identified as oqand ri -globulin respectively. The 4x globulin fraction contains a -antitrypsinz u antitrypsin is found in the or -globulin.
Attempts have been made to isolate u -antitrypsin by means of DEAE-cellulose columns [1. Biol. Chem. 235, 56 (1960)] or Dowex columns [1. Biol. Chem. 236, 2672 (1961)]. However, these processes have the disadvantage of being feasible only when using fractions that have been pre-purified in several stages. Furthermore, the yields of these processes are very loW, so that a large scale production of a -antitrypsin is uneconomical. In addition, these multistage processes involve heavy losses of activity which are due to the lability of the ca -antitrypsin. In many cases, the preparations obtained by the above-mentioned processes must be purified once :again.
Now, we have found that u -antitrypsin can be isolated in a simple manner by adjusting an a -antitrypsin-containing solution to a pH-value in the range from 5.0 to 5.5 with a buffer salt molarity of 0.005 to 0.05, treating the said solution with an exchanger containing carboxymethyl groups, separating the a -antitrypsin-containing solution from the exchanger, if desired or required removing in known manner the buffer salt from the solution obtained and isolating a -antitrypsin in known manner.
As d -antitrypsin-containing solutions, human or animal body fluids, in particular human serum, animal sera and ascitic fluids which have been adjusted to a pH-value in the range of 5.5 and 5 .0 and comprise a butter salt molarity of 0.005 to 0.05 can 'be used according to the invention. The adjustment can be made, for example, by dialyzing the fluid against a 0.005 to 0.05 molar, preferably 0.01 molar, bufier solution having a pH-value ranging from 5.5 to 5 .0, preferably 5.3, by diluting 'with a buffer solution having a pH-value ranging from 5 .0 to 5.5, preferably 5.3, to a buffer salt molarity of 0.005 to 0.05, by using a cationic exchanger, or by removing undesired low molecular Weight substances using the process described in French Patent 1,300,388, with the addition of a 0.005 to 0.05 molar, preferably 0.01 molar, buffer solution having a pH-value in the range from 5.0 to 5.5, preferably 5.3. Suitable buffer solutions are, for example, phosphate buffer or acetate bulfer, but preferably an about 0.01 molar citrate buffer having a pH-value of 5.3 is used. The adjustment of the solutions to the desired pH-value and to the desired molarity is advantageously carried out at temperatures below room temperature, preferably at temperatures in the range of '0 and C., and dialysis is carried out with stirring. During dialysis it has proved advantageous to maintain a volume ratio of a -antitrypsin solution to buffer solution of 1:25 to 1:200, preferably 1:50 to 1:100. Advantageously, dialysis is carried out for about 4 to 6 hours.
Instead of the above-mentioned native sera, those so amounts into rabbits, which develop lutions containing a -antitrypsin which are obtained by the fractionation of native sera by known methods using ammonium sulfate or alcohol can also be used after having carried out the above-described steps for adjusting the desired pH-value and the desired molarity. In addition, the active substance may also be isolated by the process of the present invention from impure oq-antitrypsin solutions prepared according to the processes described earlier herein.
The process of the present invention is advantageously carried out by treating the aqueous a -antitrypsin-containing solutions which have been dialyzed and adjusted to a pH of 5.0 to 5.5 and a buffer salt molarity of 0.005 to 0.05 as described above with an exchanger containing carboxymethyl groups by chromatographing the solution in a column charged with the exchanger. The latter is advantageously Wetted with buffer solution, for example with the buffer used for dialysis. Alternatively, the socalled batch process may be used, in which the solution is brought into mechanical contact with the adsorbing agent, preferably with stirring. According to the invention, all exchangers may be used, provided they contain carboxymethyl groups. Preferably, carboxyrnethylpolysaccharides, for example, carboxymethyl cellulose, are used. Cross-linked dextrans which are etherified with carboxymethyl groups, obtainable by the process described in US. Patent 3,042,667, proved particularly advantageous. The accompanying substances are bound by the adsorbing agent, whereas the a aant-itrypsin is not adsorbed. In the process using a column, the purified a -antitrypsin solution runs off at the bottom of the column and is collected there. It is advantageous to wash the column with a buffer solution having the same composition as that used for dialysis. In the batch process, the aqueous phase is separated in known manner, for example by filtration or centrifugation. The buffer salts can then be eliminated from the solution in known manner, for example by dialysis. For isolating the u -antitrypsin, the dialyzed solution is suitably lyophilized.
The process of the present invention offers the advantage that it can be carried out rapidly and under mild, but above all under sterile, conditions and that it permits the isolation of a -antitrypsin in good yields even from crude serum. The biological activity is fully maintained in the process of the present invention. A further particular advantage of the present process is that when using serum as starting material, the other biologically valuable serum proteins such as gamma globulin, albumin and others can be eluted from the exchanger and recovered.
The product obtained by the process is immuno-eleo trophoretically uniform against anti-human-serums from rabbits.
The a -antitrypsin obtained according to the present in vention binds trypsin, plasmin or chymotrypsin and there by inhibits the activity of trypsin, pl asmin or chymotrypsin. By reason of this property, a -antitrypsin is therapeutically important in cases of pancreatitis and burns, since it is able to inhibit the enzymatic activity of trypsin. (Plasmin is present in the blood and plays a role in blood coagulation.)
The product of the present invention serves for the preparation of antisera which are of interest for diagnostic purposes. It is, namely, possible to diagnose by means of the antisera the a antitrypsin concentration in sera, particularly a reduction or increase of the a -antitrypsin content in the serum of patients.
In order to prepare antiserum for the detection or proof of u -antitrypsin, a -antitrypsin, dissolved in a 0.85% sodium chloride solution, is injected in increasing antibodies against the injected material. These antibodies are contained in the serum recovered from the blood of the rabbits. When a sample of this antibodies-containing rabbit serum is mixed-witha sample of human ordinary serum, or when rabbit serum andhuman ordinary serun are allowedto 'diifusehga'iiist'one another in agar, the a -antitrypsin, which normally is present in' the blood in a certain concentration, of the human serum reacts with the antibodies contained in the rabbit serum. A precipitate forms in the mixture of both sera, which precipitate appears, for example, in the test tube in the form of flakes or may be made visible in the agar in the form of a white bent line.
If a human serum having an a -antitrypsin concentration increased or reduced due to pathological reasons, is reacted with a sample of rabbit serum containing antibodies, a precipitate is also formed. The quantity of precipitate, however, is higher or lower with respect to the precipitate formed with human normal serum.
An alteration of the a -antitrypsin concentration has been proved hitherto in the cases of the following diseases:
Morbus haemolyticus meanatorum Rheumatic diseases Liver diseases The indicated method represents the most specific test for the detection of a antitrypsin and is an important support for the diagnosis of the above-mentioned diseases.
The same test also proved suitable for the diagnosis of pulmonary emphysema, because in this disease the ca antitrypsin content is reduced, though this reduction is genetically dependent.
The following examples illustrate the invention, but they are not intended to limit it.
Example 1 A column for chromatography, having an internal diameter of 2.3 cm. and a length of 40 cm., is charged up to a height of 30 cm. with a cross-linked dextran etheritied with carboxymethyl groups (CM-Sephadex C-50), hereinafter designated as CMD, equilibrated with a 0.01 molar sodium citrate butter having a pH-value of 5.25 and is then charged with 12 ml. of human serum which had been dialyzed against 1200 ml. of the equilibration bufier for 5 hours at C. with stirring. The column is then washed with 100 ml. of a 0.01 molar sodium citrate buffer having a pH-value of 5.25. The a -antitrypsin-containing solution is subsequently dialyzed against water at a pH-value of 7 and finally freeze-dried. The yield from 12 ml. of human serum amounts to 38 mg. of pure a -antitrypsin.
Example 2 Into a column (2.3 x 50 cm.) charged with CMD and equilibrated as described in Example 1, there are introduced 20 ml. of fresh human serum which had been dialyzed against 2 liters of 0.01 molar sodium cit-rate buffer having a pH-value of 5.3, for hours at 0 C. and while stirring. The yield of immuno-electrophoretically uniform a antitrypsin is 66 mg.
A column (34x23 cm.) filled with CMD-andequilibrated as described in Example 1 is charged with 10 ml. of a 3.6% serum protein solution obtained by fractionation with ammonium sulfate from human serum and which had been dialyzed against 500 ml. of a 0.01 molar sodium citrate buffer having a pH of 5.3. The operation is carried out as described in Example 1. The yield of oi -antitrypsin is 45 mg.
' Example 4 g. of CMD impregnated with a 0.01 molar sodium citrate buflfer having a pH-value of 5.3 are added to 15 ml. of human serum previously di-alyzed at 0 C. with stirring against 1500 ml. of 0.01 molar sodium citrate buffer; the mixture is stirred for 20 minutes and the Ol antitrypsin containing solution is recovered by centrifugation. The s-oltuion is then dialyzred and freeze-dried as described in Example 1. The yield is 33 mg. of u -antitrypsin.
1. A process for isolating a -antitrypsin from a solution comprising human or animal body fluids, which process consists solely of 'bufiering said body fluids at a pH between 5.0 and 5.5 with a buffer salt to give a solution containing buffer salt at a molarity between 0.005 and 0.05, contacting the bufiered solution with an exchanger containing carboxymethyl groups to adsorb dissolved serum substances other than a -antitrypsin therefrom and then recovering a -antitrypsin from the solution contacted with said exchanger.
2. A process as in claim 1 wherein said exchanger is a polysaccharide containing carboxymeth'yl groups.
3. A process as in claim 1 wherein said exchanger is carboxymethyl cellulose.
, 4. A process as in claim 1 wherein said exchanger is cross-linked dextran etherified with carboxymethyl groups.
References Cited by the Examiner UNITED STATES PATENTS 10/1961 Flodin et a1. -66 X 7/1962 Flodin et al. 260209 OTHER REFERENCES A. LOUIS MONACELL, Primary Examiner.
L. M. SHAPIRO. Assistant Examiner.