CA1236015A - Process for the preparation of the c1 inactivator and its use - Google Patents

Abstract

Divisional ABSTRACT OF THE DISCLOSURE

The invention relates to a process for preparing a hepatitis-safe medicament of C1 inactivator by heating a solution of the C1 inactivator in the presence of a stabilizer until the solution has lost its infectiousness caused by the hepatitis B virus.

Description

S

82/B 011 = Ma 411 This Application is a Divisional of Canadian Patent Application Serial Number 433,647, Filed July 29, 1983.

The invention relates to the preparation of a pro-tein called the Cl inactivator and its use as a medicament.
The Cl inactivator, called after its property of inactivating Cl esterase in the complement system, addi-tionally also "controls" important enzymes for the clotting of blood, especially in the contact phase, that is to say prekallikrein and factors XI and XII, as well as plasmin. On the basis of this specificity, the Cl inactivator has a particular physiological function. In general, it can be said that it is consumed when blood comes into contact with surfaces (for example in a heart-lung machine), as a result of neutralization of the enzymes thereby formed, as well as in disease patterns which lead to activation of the coagulation cascade, for example immunocomplexes, such as occur in connection with chronic, chiefly rheumatic diseases.
Cl inactivator is the medicament of choice for hereditary angioedema.
There are a number of processes for the preparation of Cl inactivator from human plasma. Besides multi-stage processes, for example the method of Haupt, Heimburger et al:
Beitrag zur Isolierung und Charakterisierung des Cl-Inaktivators aus Humanplasma (Contri~ution on the isolation and characterization of Cl inactivator from hu~an plasma);
Eur. J. Biochem. _, 254-261 (1970), affinity chromatography ~2~~
~ 3~
is also used (Reboul et alO: A simplified procedure for the purification of C1 ;nactivator fro~ humar, plasma;
FE8S Letters 79, 45 (1977))~ Such processes have certain deficienc;es: they are still not simple enough, wasteful S and t;me-consuming. rven ;on exchanger chroma~ography, if neeessary combined wi~h gel filtration and aff;n;ty chromatography, has not led to the desired success. The - process of E.F. Vogelaar et ~l~, Vox. Sang. 2h: 118-127 ~1~74), by wh;ch C1 inactivator can be prepared on a large scale for clin;cal use, does not futf;l the present r~u;rements made of such a productO
It ~as therefor~ the object to prepare C1 inact;-vator by a process which ;s readily reproducible and ~eads to a high yield of a h;ghly pure product with a good therapeutic tolerance.
It has now been found, surprisingly~ that C1 inac-tivator ;s a relatively hydroph;lic prote;n and the pro-te;ns wh;ch usually accompany C1 ;nactivator and reduce its specific acti~ity tconcomitant proteins) have aff;n;ties for hydroph~b;c groups, in particular aromatic compounds, and that they are thus adsorbed from the C1 inactivator ~lth the aid of such groups fixed to a v1rtually water-~nsoluble carr;er and can in th;s manner be separated from the inactivator. Preferred aromatic compounds are phenyl compounds. Suitable carri~rs are the mater;als which are known per se, such as those used for hydrophobic chroma-tography with various active groups~ Crosslinked agarose containing aromatic groups which may be bonded via a spacer is preferably used.

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Carr;er compounds hav;ng the following structure are thus su;table for ;solat;ng the C1 ;nact;vator:
A-B-Aro., in which A represents ~he high-molecular, virtually ~ater-S insoluble carrier, for example crosslinked agarose, pre-ferably SEPHAROSE(R), and 3 ;s an alipha~ic bonding member of the aro~atic radical, preferably a phenyl group, bonded to this carrier. Pre-ferred bonding members, also called spacers, are NH
-O-CH2-CH-CH2-O- and -O-C-N-CH2-CH2-.
OH H
A suitable commercial product of the formula AB-Aro is PHENYL-SEPHAROSE~R) CL-4B. Preferred products are pro-ducts ~h;ch are similar to th;s~ such as those ~hich can be obtained, for example~ by reacting a crosslinked agarose, for example SEPHAROSEtR) 4B, uith cyanogen bromide and then with an aromatic amine, for example phenylethyLamineO
The adsorption of the concomitant proteins of C1 lnactivator on~o such hydrophob;c carriers and their removaL
from the ;nactivator ;s a step which is essential to the

2~ invention in the process for the preparation of a pure product and ~hich can, if appropriate, be preceded or followed by process steps which are known per se.
Human plasma is the preferred startin~ materia~
for isolating C1 inactivator; however, ~he process step according to the invention can also be used on other aqueous soLutions conta;ning C1 ;nactivator and concomitant proteins.

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The comb;nat;on of ~he process step according to the in~ent;on and a non-hydrophob;c adsorbent, preferably an ion exchanger~ espeGially ~ith diethylaminoethyl groups, is advantageous. Ion exchangers containin~ QAE
groups also lead ~o ~ concen~ra~ion of ~he C1 inactivator, as do mineral adsorbents, such as~ for example, calcium phosphate Precip;tat;on processes ~ith neutral salts9 such as, for example, w;th ammon;um suLfate~ are also kno~n and su;table as purif;cation steps for C1 ;nact;vator.
It is essential that therapeut;c use of C1 ;nacti-vator presents no danger to the patient. The exclusion of transm;ss;on of hepat;tis ;s based on the assumption that prote;n solut;ons kept at about ~0C for several hours can no longer transmit hepat1t;s a even ;f these soLutions contained ;nfectious hepatitis 3 v;rus before be;ng ~armed.
In a further development of the present invention, there ~as thus the object of providing the product obtained accord;ng to the invent;on ;n a hepat;t;s-free form.
Solutions conta;ning C1 ;nactivator can be kept at about 2~ ~C for several hours ~ith virtually no loss in acti-vity ;f these solut;ons conta;n compounds which stabilize the activity of C1 inact;vator. Such compounds are am;no acids, sugars and/sugar alcohols, and mixtures thereofO
A solut;on conta;ning C1 inactivator can be heated at 60C
w;th a m;xture of 2 moles/l;ter of glyclne and 60X t~eight/
volume) of sucrose for 10 hours without los5 in activi~y~
Generally, am;no ac;ds are used in a concentration of 1-3 moles/l;ter, monosacchar;des and ol;gosaccharides are used in a concentration of 20-60% (we;ght/volume) and ~6f~

sugaroalcohols are used in a concentrat;on of up to 75~
(weight/volume). For the ~arm;ng operat;on, the solution containing C1 inactivator and stab;lizer is brought to a pH
value between 5.5 and 8.5, preferably between 6.5 and 7.5.
Apart from glycine, the amino acid preferably used, the follo~ing amino acids are also suitable for the stabil;za-tion: L-aspart;c acid, L-~rine, L-valine, L-~ys;ne, L-threonine, L-tyrosine~ L-phenylalanine, L-leucine, L-alanine, L-methionine, L-proline~ L-hydroxrproline, L-arginine ~ or ~-alanine, glutamine and ~-~ or ~-am;nobutyric acid; besides sucrose, th~ following sugars are suitable: arabinose, glucose, galactose~ fructose, ribose, mannose, rhamnose, maltose and raffinoseO and the follo~;ng sugar-alcohols are suitable: erythritol, rib;tol~
sorbitol and mannitol.
As a resu~t of above stabilizing substances, a solution containing C1 inactiva~or can be ~ar~ed at 30 to 100C for 1 minute to 48 hours, preferably at 60C for 10 hour~, in vie~ of the necessity of avoiding trans-~;ssion of hepatitis.
The ;nvention thus furthermore relates to a process,~hich comprises heating a C1 inactivaeor-containing solu-tion in the presence of stab;lizers until the solu~ion has lost its infectiousness caused by a content of hepatitis a v;rus.
Appropriately prepurified material uhich has been kept, ~here relevant, at about 60C for several hours, contains C1 inact;vator and concomitant proteins and has a purity of about 23 2S units of C1 inactivator/mg ~spe-cific activity) is trea~ed, according to the invent;on, ;naqueous solution ~ith an adsorben~ A-~-Aro containing hydrophob;c substituents, for example P~ENYL-SEPHAROSE(R).
The concomitant prote;ns of ~he C1 inactiva~or are adsorbed at a ~eakly acid, neutral to ~eakly alkaline pH value~
preferably at pH 6 to pH 9O The conductivity of th~
solution ;s advantageously 60-120 ms~ The adsorpt;on step ~th the hydrophobic carrier material ;s advantageously com bined ~i~h a prec;pitation st~p for concen~ration, for ~hich the C1 inactivator is precipitated from the solution.
Th~ precipitated C1 inactivator is then taken up in an aqueous solut1On contaln~ng the prec;p~an~ in a concen-tration at ~hich the C1 ;nactivator does not precip;tate.
If, for exampLe, the C1 inact;vator is precipitated ~;th a neutral salt, for example ammonium sulfate, the precipitate can be taken up in an aqueous solution of a neu~ral salt in a concentration at ~hich the C1 inactivator remains in solution, for example an ammon;un suLfate con-centration of 7-14%, directly after the ~recip;tation~ ay using such a solut10n, bonding of the conventional proteins accompanying C1 ;nactivator as an impurity to the hydro-phob;c carr;er is achieved and highly pure C1 inac~ivator can be separated off fro~ the adsorbent, ~h;ch retains the impurities.
Protein-stabilizing substances ~hich are kno~n per se, for example an amino acid, such as glycine, are added to C1 inactivator for therapeutic use. The produc~, ~hich is finally purified by hydrophobic chromatography, i 5 sterilized by filtration, brought to the desired concentra-~3~

tion effective for therapy, filled into containers and, if desired, lyophil;2ed. T-he amino acid added stabilizes the Cl ;nactivator during freeze-drying~
A preferred proc~ss is carried outO for example~
i as in the follo~ing general descr;pt10n, the base substance o~ C1 ;nact;vator, human plasma, b~ng used here as the starting mater;al:
~ u~an plasma ~hich contains, for example, citrate and is free fro~ cryoglobulins and prothrombin factors ;s treated ~ith an anion exchanger and the eluate contain1ng C~ inactivator ;s fractionated uith neutral salts. The concomitant prote1ns of C1 ~nactivator are adsorbed from ~03t of the oth~r plasma proteins ~ith a hydrophobic adsorbent. A good yield of highly pure C1 inactivator from which concomitant proteins have been removed can thus be isolated in a single step by means of hydrophobic chromatography. C1 inactivator passes out of a hydro~
phobic column, for example PHENYLSEPHAROSEtR) in a salt containing solution of appropriate COnGentratiOn~ whilst most of the concomitant proteins, especially ceruloplasmin, ~hich is as a rule highly concentrated in this fraction~
are retained.
If desired, the C1 ;nactivator in the precipita-tion res;due can be heated at 60C for 10 hours~ after dissolving ;n distilLed ~ater and adding a sugar, for example sucrose, to the extent of 60X (weighttvolume) ~ith-out substantial loss in activity. After the sucrose has been removed by reprecipitation wi~h ammonium sulfate from dilute solution, a good yield of highly pure C1 inactivator ., ~23~

g from which cancomitant proteins have been removed can then be isolated immedia~ely, also using the hydrophobic chroma-tography technique and ~n a single step.
The ~xample ~hieh follo~s illustrates the invention:
S ~:
Deep-froz~n ci~rated plasma which has been freed from cryoglobulins and from ~h1ch factor VIII, Cig and human fibrinogen haYe bcen isolated, ~as adsorbeJ wi~h DEAE-S~P~ADEX~R) accord;ng to ~ 30 43 857.4, P 30 45 1537 ~nd P 31 01 752.S in order to obtain pro~hrombin concen-trateO After the DEAE-SE~H~DEX~R) had been s~parated off, 10 9 of QAE-SEPHADEX(R) per l~t2r of pLasma ~ere added to the supernatant liquor and the suspension ~as stirred at 12~C for ~0 minutes~ the ~AE-adsorbent ~as 1~ then separated off and ~ashed with 0.15 mol (sic) NaCl.
1.0 mol tsic)NacL~ pH8.09 and 0.0025 mole/liter of EDTA, in a ~olume of 0.45 liter of buffer per 10 liters o~ plasma, was used for the elution. The QAE eluate is a deep blue solut~on which chiefly conta;ns, in addition to cerulop~asmin, C1 inactivator and factor V$I. Th2 eluate ~as fractionated with liqu;d ammonium sulfate at 20C.
60X saturation ~as achieved by adding 1,500 ml of saturated ammon~um sulfate solution/liter of eluate.
The 60X ammon;um suLfate residue in ~hich C1 inac-tivator ~as concentrated ~as then subjected ~o hydrophobicchromatography on PHENYL-SEPHAROSE~R)~ For this~ the ~
precipitate ~as taken up in distilled ~ater to g;ve a solu-tion corrosponding to an optical density at 280 nm of ~- 55, the ammonium sulfate concentration w3s adjusted to 7% and ~23~

the pH value was adjusted eo 7.2 to 7~6. After clari-fication and ster;~ization by filtrat;on, 1.3 l;ters of this solution ~ere separated over a gel bed w;th PHENYL-SE~HAROSE(R) in the form of a column 31 cm high and 12 cm ~;de. The first ~ater-clear runnings contained C1 inacti-vator, ~ell-separated fro~ ~he ceruloplasmin pass;ng through the column as a blue band. The fraction contain-1ng C1 ln~ctiva~or ~as concentra~ed by adding solid ammonium sulfate: 340 9 of ammon;um su~fate per li~r of the frac-10 t~on running through ~ere added at 6Co ~he precipi~ateuas centrifuged off and dissol~ed in distilled water. The c~arificat;on and steriLization by filtration ~ere follo~ed by dialys;s against a 1.5X strength glyc~ne buffer. The protein content of C1 inactivator in a portion of the solu-tion ~as determined ~ith the aid of ~he rad;o immuno dif-fus~on technique, and the activity was determined by ~he m~thod of Levy and Lepow ~roc. Soc.~xp.aiol.Med. 101, 6û8 t1959)), using N-acetyl L-tyrosine e~hyl es~er as the substrate. The activity was given in C1 inactivator units, 2û 1 U thereby being def;ned as the amount which inhibited 10 U of C1 esterase. On a~erage, products containing about 40 U of C1 inactivator/mg of C1 inactivator protein were obtained in a yield of 20X, based on the starting plasma. The resulting product ~as abou~ 90Z pure and pyrogen-free and could be used in animal experiments with-out side effects and in the therapy of angioedema~
If desired, it ~as possible to dissolve the 60%
ammonium sulfate precipitation residue ;n dis~;lled water, to add 6û% weight/volume of sucrose, to bring ~he pH to ~23~0~5 7 - 7.5 and to heat the mixture at 60C for 10 hours.
After cooling, ~he solution ~as dilut~d 1:5 ~ith distil~ed water and pr~cipitated aga;n by add;ng saturated ammon;um sulfate solut;on up to 60% saturation in order to remove S th~ sucrose. This procedure ~as follo~d by hydrophobic chromatography 9

Claims (4)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the preparation of a hepatitis-safe medicament containing C1 inactivator, which comprises heating a C1 inactivator-containing solution in the presence of a stabilizer selected from an amino acid, a sugar, a sugar-alcohol or a mixture thereof, until the solution has lost its infectiousness caused by a content of hepatitis s virus.

2. The process of claim 1, wherein the stabilizer is glycine.

3. The process of claim 1, wherein the stabilizer is sucrose.

4. The process of claim 1, wherein the stabilizer is an oligosaccharide.