New! View global litigation for patent families

US3293236A - Process for isolating alpha1-antitrypsin - Google Patents

Process for isolating alpha1-antitrypsin Download PDF

Info

Publication number
US3293236A
US3293236A US37542464A US3293236A US 3293236 A US3293236 A US 3293236A US 37542464 A US37542464 A US 37542464A US 3293236 A US3293236 A US 3293236A
Authority
US
Grant status
Grant
Patent type
Prior art keywords
antitrypsin
solution
serum
buffer
process
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Lifetime
Application number
Inventor
Hermann E Schultze
Heimburger Norbert
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Behringwerke AG
Original Assignee
Behringwerke AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Grant date

Links

Images

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81Protease inhibitors
    • C07K14/8107Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121Serpins
    • C07K14/8125Alpha-1-antitrypsin
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01JCHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS, COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J47/00Ion-exchange processes in general; Apparatus therefor
    • B01J47/014Ion-exchange processes in general; Apparatus therefor in which the adsorbent properties of the ion-exchanger are involved, e.g. recovery of proteins or other high-molecular compounds
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/827Proteins from mammals or birds
    • Y10S530/829Blood
    • Y10S530/83Plasma; serum

Description

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.

We claim:

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.

Claims (1)

1. A PROCESS FOR ISOLATING A1-ANTITRYSPIN FROM A SOLUTION COMPRISING HUMAN OR ANIMAL BODY FLUIDS, WHICH PROCESS CONSISTS SOLELY OF BUFFERING 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 BUFFERED SOLUTION WITH AN EXCHANGER CONTAINING CARBOXYMETHYL GROUPS TO ADSORB DISSOLVED SERUM SUBSTANCES OTHER THAN A1-ANTITRYSPIN THEREFROM AND THEN RECOVERING A1-ANTITRYSPIN FROM THE SOLUTION CONTACTED WITH SAID EXCHANGER.
US3293236A 1963-06-12 1964-06-11 Process for isolating alpha1-antitrypsin Expired - Lifetime US3293236A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
DE1963B0072263 DE1189674B (en) 1963-06-12 1963-06-12 A process for the isolation of alpha-antitrypsin

Publications (1)

Publication Number Publication Date
US3293236A true US3293236A (en) 1966-12-20

Family

ID=6977373

Family Applications (1)

Application Number Title Priority Date Filing Date
US3293236A Expired - Lifetime US3293236A (en) 1963-06-12 1964-06-11 Process for isolating alpha1-antitrypsin

Country Status (5)

Country Link
US (1) US3293236A (en)
BE (1) BE649238A (en)
DE (1) DE1189674B (en)
GB (1) GB1062434A (en)
NL (1) NL6406622A (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0035204A2 (en) * 1980-03-05 1981-09-09 Miles Inc. Pasteurized therapeutically active protein compositions
EP0698615A1 (en) * 1994-08-24 1996-02-28 Bayer Corporation Purification of alpha-1 proteinase inhibitor using novel chromatographic separation conditions
WO1997009350A1 (en) * 1995-09-07 1997-03-13 Ppl Therapeutics (Scotland) Ltd. Purification of alpha-1 proteinase inhibitor
US6093804A (en) * 1998-09-24 2000-07-25 American National Red Cross Method for purification of alpha-1 proteinase inhibitor
US6462180B1 (en) 1999-11-24 2002-10-08 Bayer Corporation Method of preparing α-1 proteinase inhibitor
EP2295126A1 (en) 2002-12-31 2011-03-16 CSL Behring LLC Method for purification of alpha-1-antitrypsin

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3271827D1 (en) * 1981-05-01 1986-07-31 Medical Research Inst Of San F Method for isolating alpha-1-antitrypsin

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3002823A (en) * 1958-04-16 1961-10-03 Pharmacia Ab Process of separating materials having different molecular weights and dimensions
US3042667A (en) * 1959-03-10 1962-07-03 Pharmacia Ab Process for producing dextran derivatives and products resulting therefrom

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3002823A (en) * 1958-04-16 1961-10-03 Pharmacia Ab Process of separating materials having different molecular weights and dimensions
US3042667A (en) * 1959-03-10 1962-07-03 Pharmacia Ab Process for producing dextran derivatives and products resulting therefrom

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0035204A2 (en) * 1980-03-05 1981-09-09 Miles Inc. Pasteurized therapeutically active protein compositions
EP0035204A3 (en) * 1980-03-05 1982-11-10 Miles Laboratories, Inc. Pasteurized therapeutically active protein compositions
DK156698B (en) * 1980-03-05 1989-09-25 Miles Inc Method for the pasteurization of a material containing a thermally sensitive, therapeutically active protein selected from alpha-l-antitrypsin, antithrombin-III, prekallikrein, antihemophilic factor (faktorviii) and fibronectin
EP0698615A1 (en) * 1994-08-24 1996-02-28 Bayer Corporation Purification of alpha-1 proteinase inhibitor using novel chromatographic separation conditions
US5610285A (en) * 1994-08-24 1997-03-11 Bayer Corporation Purification of α-1 proteinase inhibitor using novel chromatographic separation conditions
WO1997009350A1 (en) * 1995-09-07 1997-03-13 Ppl Therapeutics (Scotland) Ltd. Purification of alpha-1 proteinase inhibitor
US6525176B1 (en) 1995-09-07 2003-02-25 Ppl (Holdings) Limited Purification of alpha-1 proteinase inhibitor
US6194553B1 (en) 1995-09-07 2001-02-27 Ppl Therapeutics (Scotland) Limited Purification of alpha-1 proteinase inhibitor
EP1736483A3 (en) * 1995-09-07 2008-07-30 Pharming Intellectual Property BV Purification of alpha-1 proteinase inhibitor
US6093804A (en) * 1998-09-24 2000-07-25 American National Red Cross Method for purification of alpha-1 proteinase inhibitor
US6462180B1 (en) 1999-11-24 2002-10-08 Bayer Corporation Method of preparing α-1 proteinase inhibitor
EP2295126A1 (en) 2002-12-31 2011-03-16 CSL Behring LLC Method for purification of alpha-1-antitrypsin
EP3006462A1 (en) 2002-12-31 2016-04-13 CSL Behring LLC Method for purification of alpha-1-antitrypsin

Also Published As

Publication number Publication date Type
DE1189674B (en) 1965-03-25 application
BE649238A (en) 1964-12-14 grant
GB1062434A (en) 1967-03-22 application
NL6406622A (en) 1964-12-14 application

Similar Documents

Publication Publication Date Title
Osserman et al. Serum and urinary lysozyme (muramidase) in monocytic and monomyelocytic leukemia
US3719655A (en) Process for the crystallization of the ammonium and alkali metal salts in insulin
James et al. Studies on a unique diagnostic serum globulin in papular mucinosis (lichen myxedematosus)
Frommhagen et al. The role of aggregated γ-globulins in the anticomplementary activity of human and animal sera
Gilbert et al. The chemistry of xanthine oxidase. 9. An improved method of preparing the bovine milk enzyme
Tishkoff et al. Preparation of Highly Purified Prothrombin Complex I. CRYSTALIZATION, BIOLOGICAL ACTIVITY, AND MOLECULAR PROPERTIES
US4269605A (en) Method and kit for separation of glycoproteins
Miletich et al. The synthesis of sulfated dextran beads for isolation of human plasma coagulation factors II, IX, and X
US3931399A (en) Process for isolating a fibrin-stabilizing factor
Laurell Purification and properties of different haptoglobins
Inman et al. Coupling of large haptens to proteins and cell surfaces: Preparation of stable, optimally sensitized erhythrocytes for hapten-specific, hemolytic plaque assays
USRE32011E (en) Ultrapurification of factor VIII using monoclonal antibodies
US4568488A (en) Reverse immunoaffinity chromatography purification method
Curzon et al. The purification of human caeruloplasmin
US4396762A (en) Heparinase derived anticoagulants
Worwood et al. The purification and properties of ferritin from human serum.
US4719108A (en) Chymopapain composition and method for its use
US3842061A (en) Method for isolation of antithrombin from animal tissue materials by adsorption on sulfated carbohydrate gel
Heidelberger et al. THE SPECIFIC POLYSACCHARIDES OF TYPES I, II, AND III PNEUMOCOCCUS: A REVISION OF METHODS AND DATA
Lotan et al. The purification, composition, and specificity of the anti-T lectin from peanut (Arachis hypogaea).
US4340589A (en) Antithrombin preparation and process for the production thereof
US4027010A (en) Antistaphylococcous human immunoglobulin and method of preparing same
US4421896A (en) Method of coupling a protein to a polymer particle containing hydrazide groups in a polymer latex and the products formed therefrom
US3869437A (en) Mono-, di, and N{HD A1{B , N{HU B1{B , N{HU B29{B -tri-acylated insulin
US5986062A (en) Recombinant human serum albumin, process for producing the same and pharmaceutical preparation containing the same