AU660410B2 - Decontaminated biological material - Google Patents

Abstract

The invention concerns decontaminated biological material, in particular liquids, arising from treatment with acid followed by treatment with alkali. The progress of the treatment can be monitored and/or the end point determined by determining the residual LDH (lactate dehydrogenase) activity. The invention also concerns a process for decontaminating biological material, in particular biological liquids, in which the pH value is lowered by the addition of acid and, after a given length of time, raised again by the addition of alkali. The progress of the treatment can be monitored and/or the end point determined by determining the (residual) LDH activity.

Description

OPT IATE J-1/11/91 APPLN ID 75640O 91 PLT AOJP DATE 19/12/91 PCT NUMBER PflT/DEq1/0O3fl7

INTERINATIO

INTERNATIO,,,.-- f.i itiur' i-Cavi uIrnl I t'ta rA., I LIN I WrIWI'Nb (FtL (51) Internationale Patentlassifikation 5 (11) Internationale Wsiiffcntlichungsnummer: WO 91/16082 A61L 2/00, 2/18 Al (43) Wnernationales Verbffenilichungsdatum: 31. Oktober 199 1 (31.10.91) (21) Internationales Aktenzeichen: PCT/DE91/00307 (81) Bestimmungsstaaten: AT (europaisches Patent), AU, B13, BE (europilisches Patent), BF (OAPI Patent), BG, BJ (22) Internationales Anmeldedatum: 13. April 1991 (13.04.9 1) (OAPI Patent), BR, CA, CF (OAPI Patent), CG (OAPI Patent), CH (europliisches Patent), CM (OAPI 'Patent), DEU (erpiisches Patent), DK (europfiisches Patent), ES Pioiritiitsdzten: (europliisches Patent), Fl, FR (europiisches Patent), GA P 40 12 323.5 18. April 1990 (18.04.90) DE (QAPI Patent), GB (europllisches Patent), OR (europfli- P 40 14 063.6 9. Mai 1990 (09,05.90) D E sches Patent), HU, IT (europfiisches Patent), JP, KP, KR, LK, LU (europliisches Patent), MC, MG, ML (QA- PI Patent), MR (OAPI Patent), M' NL (europtiisches (71)(72) Anmelder und Erfinder: BOHN, Burghard [DE/DE]; Patent), NO, PL, RO, SD, SE (eurog ,ches Patent), SN Karisruher Stra&e 6, D-6900 Heidelberg (QAPI Patent), SU, TD (QAPI Patent), TG (OAPI Patent), US, (74) Anwalt: RATZEL, Gerhard; Seckenheimerstrage 36a, D- 6800 Mannheim 1 (DE).

Veriiffentlicht Mit internationalen, Reclierchenbericht.

(54) Title: DECONTAMINATED BIOLOGICAL MATERIAL (54)13ezeichnung: DEKONTAMINIERTES BIOLOGISCHES MATERIAL (57) Abstract The invention concerns decontaminated biological material, in particular liquids, arisirig from treatment with acid followed by treatment with alkali. The progress of the treatment can be monitored and/or the end point determined by determining the residual LDH (lactate dehydrogenase) activity. The invention also concerns a process for decontaminating biological material, in particular biological liquids, in which the pH value is lowered bv the addition of acid and, after a given length of time, raised again by the addition of alkali. The progress of the treatment can te monitored and/or the end point determined by determining the (residual) LDH activity.

(57) Zusanimeiifassung Die Erfindung betrifft dekontaminiertes biologisches Material, insbesondere Flflssigkeit, das erhilltlich ist dutch Behandlung mit Sflure gefolgt von Laugenbehandlung; dabei kanti der Behandlungszustand und/oder der Behand~ungsabschlug durch die Bestimmung der Restaktivitilt von LDH (Lactat-Dehydrogenase) verfolgt bzw. bestimmt werden. Die Erfiridung betrifft ferner ein Verfahren zur Dekontamination von biolisgischemn Material, insbesondere biologischer Fltlssigkeiten, bei dem tier pH- Wert durch Sflurezugabe abgesenkt und nach einer definierten Zeitspanne wieder durch Laugenzugabe angehoben wird; dabei kann der Behandlungsverlauf und/oder der Behandlungsabschlug dutch Bestimmung der (Rest-) Aktivitiit von LDH (Lactat- Dehydrogenase) verfolgt bzw, bestimmt werden.

-1- DECONTAMINATED BIOLOGICAL MATERIAL The invention concerns decontaminated biological material, especially liquids, and a method for the decontamination of biological material, especially biological liquids. In numerous fields of research and development as well as in industrial and professional fields serum, especially calf serum and fetal calf serum is used. This serum is employed in cell cultures for cultivating human and animal cells.

Furthermore it is used during the preparation of proteins and similar pharmaceutical products, which are prepared by means of cell cultures of fused cells of human or animal origin, and where necessary, from insects also.

The term biological material or biological liquid, respectively, means material or liquid, respectively, derived from biological origin.

The quality of such serum depends on its ingredients; for example insulin and transferrin are of special importance.

Serum, especially calf serum and fetal calf serum are sensitive to heat especially with respect to important biological components.

In consequence it is not possible, for example, to obtain a decontam-ination of these biological materials by rise in temperature, by making the serum hot, or by similar processes respectively.

Such decontamination is necessary for inactivating viruses in particular, such as, for example, foot and mouth virus; retroviruses, for instance HIV virus or related viruses, as well as other infectious agents.

For inactivation it does not suffice for instance to perform a so called "pasteurization" at 600C. Also, it is not possible to perform a biological decontamination in a satisfactory manner by heating over this pasteurizing temperature, for example by heating at 65 0 C for two periods of half an hour. These denaturising conditions causes changes of biological activity, based for instance on a depolymerization of aggregates and a denaturation of proteins.

Concerning the methods in the prior art, of particular i 39 note the biological decontamination by X-ray radiation, ,WDNf -2especially the inactivation of viruses. In this connection the reference "Canadian Journal of comparative Medicine", 45:397-399 (1981) is to mention.

The radiation of biological material for destruction of viruses is, in the case where high doses are required, injurious to the quality of the biological material.

Also the chemical agents employed for biological decontamination, especially for inactivation of viruses, for instance Ethylenoxide, cause in some cases an insufficient decontamination on the one hand and on the other hand an impairment of the biological material.

An impairment of the biological material means for instance an impairment of the promotion of cell growth by reason of the described changes of components.

Consequently, the methods in the prior art for biological decontamination of biological material, especially biological liquids, have the disadvantages of insufficient efficiency and/or of impairment of the quality of the biological material or its components, respectively.

Therefore it is an object of the present invention to provide a method for biological decontamination and to provide decontaminated biological material, whereby the biological decontamination, especially the inactivation of viruses, is obtained in an effective and lasting manner.

Concerning decontaminated biological material, especially liquids, in accordance to the invention this object is solved in that it is obtained from treatment with acid following by treatment with alkali, in the enclosed -laims this way of solution is specified, as %ell as numerous preferred embodiments of this way of solution.

It turns out that the inactivation of viruses, for instance of acid-labile retroviruses, is sharply increased.

Thereby, as shown by cell culture cultivation experiments, the biological components of the seruin will not be impaired, Consequently, a serum according to the invention or treated according to the invention, in particular calf serum and fetal calf serum respectively, has a sharply increased efficiency with use in research and industry.

In particular it is a positive feature of the present

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invention that the ionic strength of the serum according to the invention or treated according to the invention, respectively, only changes insignificantly and in consequence only small changes of osmolarity appear, for instance in the range of 20 to 30 m Osmol. In the method according to the invention or the product according to the invention respectively, the buffer capacity is relative quickly broken through and a lowering of the pH value is obtained, whereby with the parameters according to the invention the inactivation of viruses for example, and in general the biological decontamination is obtained.

The present invention concerns a method for decontaminating biological material including adding acid to the biological material to decrease the pH to a value of 3.5 to 6.5 and after a given length of time, adding alkali to the biological material to increase the pH of the biological material. Preferably the pH of the biological material is lowered to a value of 4.5 to more preferably 4.9. In a further preferred embodiment the pH is kept low for 2 to 5 hours, more preferably to 4 hours and most preferably 3 hours.

The mineral acid used to lower the pH of the 25 biological material is preferably a mineral acid of low :e normality, for example 1 N hydrochloric acid.

:The biological material is preferably raised to a physiologically compatible value on the addition of an alkali, more preferably the pH is raised to a value of S. to thermolabile at lowered pH value remain intact.

39 3-

A

•r With method according to the invention or in the product according to the invention all viruses as well as retroviruses are effectively able to be inactivated or inactivated, respectively.

For lifting the pH's to a desirable, in particular a physiological value of about 7 to 7.5 a base of low normality, especially 1 N calcium hydroxide is used.

For lowering the pH's other suitable acids with an appropriate dissociation constant may be used. Also other bases such as potassium oxide lye or organic bases may be employed.

The selection of the suitable bases or acid respectively, depends in case on the specific purpose of the serum with regard to cell culture, composition of the media, ionic content and suchlike.

A suitable selection for the particular case can be made by the expert on the basis of his general specialist knowledge without anything further.

By the method according to the invention it is also 9 s* 39- -4possible to employ sera or fetal calf serum, respectively, deriving from regions with high potential biological load of contamination, in cell culture as well as in the fields of industry and pharmacy and for research.

In the following an embodiment of the invention is pointed out.

One litre of fetal calf serum is lowered to a pH value of 4.9 with one normal of sterile HC1 and maintained at this value for two hours at +4C (cool room).

Subsequently the pH is raised to a value of pH 7.3 by the addition of sterile 1 N NaOH.

The material obtained in that way is used for cultivation of adherent cells.

Thereby 1x10 cells were seeded in a cell culture dish with 20 ml liquid culture volume and 10% serum content. The number of cells obtained is 3.2x10 after 2 days, 2.3x10 after days and 4.8x10 after 7 days.

The cells show a healthy appearance; cytopathic or virus-specific effects are not observable.

Performed experiments or tests respectively for the presence and absence or inactivation, respectively, were performed with foot and mouth virus and retrovirus and show complete inactivation or removal of viruses.

In many cases during special applications it is desirable to observe the time slope of the efficiency of the acid/alkali treatment, for instance to select the period which is as short as possible. Besides that, it is imperative to test whether an introduced material had been treated according to the invention, of particular relevance being laws of infection regulation.

It is a further object of the present invention to provide a characteristic or a measuring method, respectively, which allows a monitoring or verification of a decontaminating treatment. For decontaminated biological material this object is solved in accordance with the above mentioned designation, in that the status of the treatment and/or the end of the treatment is monitored or determined respectively by determining the (residual) LDH (Lactat-Dehydrogenase) activity.

Furthermore a method is claimed for decontamination of biological material, in particular biological liquids, in w t I';f.

*i accordance with the above mentioned class which is characterized in that the progress of the treatment and/or the end point of the treatment is monitored or determined respectively by determining the (residual) LDH (Lactate-Dehydrogenase) activity.

In a preferred embodiment, the LDH activity of the biological material is decreased by the acid/alkali treatment by the factor 4 to 15, more preferably the LDH activity is decreased by the factor 8 to 12.

0 0 0 0 0*0 0 0* 0000 0 0 0 A? 39 (3 v 4A in that the progress of the treatment end point of the treatment is r determined respectively by t r C3Zi rqa 1 TiDH (1t a t Yoqa a ad Furthermore the present invention concerns also a method for verifying the status of treatment or the performance of a treatment respectively, by using a key enzyme. The LDH (Lactate-Dehydrogenase) is an enzyme, which is ubiquitously present in calf serum and bovine serum and is existent .for instance in calf serum in a concentration of about lQ4%'i and in bovine serum in a concentration of about 100 to 5004 I. For the desired efficiency of sera, in particular for cell cultivation and cell growth, LDH is of no account. Consequently its decay is not relevant for the quality of the serum or the sera respectively. On the contrary, since potential side effects of LDH enzymatic activity are reduced, the biological material or the method, respectively, according to the invention, provide an improvement of quality of the sera or the biological material, respectively.

Additionally in some cases a decay of pyrogens occurs surprisingly, providing a further advantage of the present invention.

In practice it is recommendable to determine the decay of LDH activity either within the bounds of measurement of LDH activity before and after a treatment or within the bounds of measurement of biological materials and baseline pattern belonging to. Furthermore, for every biological material, a specific limit or threshold value is offered, for example, less than exactly 401Bt for calf serum is the specified measuring limit or limiting value.

Embodiments of the present invention are following: Example 1 Fetal calf serum, treated three hours at 4 0 C and at a pH value of 4.9 (followed by subsequent raising of the pH value) was evaluated with respect to LDH activity before and after the treatment. The L ctivity amounted to 100 AU74 before the treatment and 8.3jVP after the treatment.

3A2 ASimultaneously, the content of pyrogen dropped from 1.2 -6ng/ml before to 0.3 ng/ after ng/ml before to 0.3 ng/% after the treatment.

Example 2 Bovine serum was treated analogously to example 1 and the content of LDH was evaluated before and after the treatment, respectively. The value amounted to 3 5 0 4@A before and 251.M after the treatment.

Example 3 Bovine serum was treated accordin to example 1. Before treatment the LDH value amounted to 3004M and after treatment to 16.6 5 L c.r.

3 WD

Claims (6)

1. Decontaminated biological material, obtained from treatment with acid followed by treatment with alkali, namely by treatment with acid of low normality for lowering the pH to the value of 4.5 to 5.5 for 2.5 to 4 hours and subsequent raising of the pH to the value of to 7.5 by treatment with alkali.

2. Decontaminated biological material, arising from treatment with acid followed by treatment with alkali, according to claim 1, wherein the status of the treatment and/or the end of the treatment is monitored or determined respectively by determining the (residual) LDH (Lactate-Dehydrogenase) activity.

3. Decontaminated biological material according to claim 2, wherein the LDH activity is decreased by acid/alkali treatment by the factor 4 to

4. Decontaminated biological materiaI according to claim 3 wherein the LDH activity is decreased by acid/alkali treatment by the factor 8 to 12. 25 5. Decontaminated biological material obtained by treatment with acid of low normality for lowering the pH to the value of 4.5 to 5.5 for 2.5 to 4 hours and subsequent raising of the pH to the value of 7.0 to "according to any one of claims 2 to 4, wherein the decay 30 of LDH activity is determined by measuring the activity i before and after the acid/alkali treatment.

6. Decontamined biological material according to any one of claims 1-5 wherein the decontaminated biological 0 35 material is a liquid. .b o 7. Decontaminated biological material according to

39. 7- claim 6 whe;ein the liquid decontaminated biological material is sera. 8. Method for decontaminating biological material, including adding acid to the biological material to decrease the pH to a value of 3.5 to 6.5 and after a given length of time adding alkali to the biological material to increase the pH of the biological material. 9. Method according to claim 8 wherein the pH is decreased to a value of 4.5 to Method according to claim 8 wherein the pH is decreased to a value of 4.9, 11. Method according to any one of claims 8-10 wherein the decontaminated biological material is a liquid. 12. Method according to claim 11 wherein the liquid decontaminated biological material is sera. 13. Method according to any one of claims 8-12, wherein a mineral and/or organic acid is used. 25 14. Method according to claim 13, wherein the mineral acid is hydrochloric acid with low normality. 15. Method according to any one of claims 8-14, wherein S"an alkali with low normality is used. 16. Method according to any one of claims 8-15, wherein the pH value is kept low for 2 to A 17. Method according to any one of claims 8-15 wherein 35 the pH value is kept low for 2.5 to 4h. 18. Method according to any one of claims 8-15, wherein the pH value is kept low for 3h. 8 p l 19. Method according to any one of claims 8-18, wherein the pH value is raised again to a physiologically compatible value. 20. Method according to any one of claims 8-18 wherein the pH value is raised to a value of 7.0 to 21. Method according to any one of claims 8 to performed at a temperature from 0 C to 60 0 C. 22. Method according to any one of claims 8-20 performed at a temperature from +4 0 C to +25 0 C. 23. Method for decontaminating biological material, whereby th pH value is lowered by the addition of acid and, after a given length of time, raised again by the addition of alkali, according to any one of claims 8-22, wherein the progress of the treatment and/or the end point of the treatment is monitored or determined respectively by determining the (residual) LDH (Lactate-Dehydrogenase) activity. 24. Method according to claim 23, wherein the LDH activity decreases by the factor 4 to 15 by the 25 acid/alkali treatment. gg. 25. Method according to claim 23 wherein the LDH Sactivity decreases by the factor 8 to 12 by the acid/alkali treatment. 26. Method according to any one of claims 23-25, wherein the decay of the LDH activity is determined by measuring the activity before and after the acid/alkali treatment. 35 27. Decontaminated biological material substantially as hereinbefore described with reference to any one of the examples. ^s \1 f.. k> A' "^YTJ^ 28. Method for decontaminating biological material substantially as hereinbefore described with reference to a'sy one of the examples. DATED: 16 MARCH, 1995 PHILLIPS ORMONDE FITZPATRICK Attorneys for: BURGHARD BOHN *000 0 0 0 0 0000 0 0 00* 0 0 0' 0 0 0 0 0000 00 00 0 0* 98201 S--1- 10 ABSTRACT The invention concerns decontaminated biological material, in particular liquids, which are obtainable by treatment with acid followed by alkali treatment; the treatment position and/or the end-point of treatment is monitored or rather determined by the determination of the reactivity of LDH (Lactate-dehyrogenase). The invention further concerns a procedure for the decontamination of biological material, in particular fluids, in which the pH-value is lowered by addition of acid, and after a defined time span, raised again by the addition of alkali; the progress of the treatment and/or the end-point of treatment is monitored or determined by the determination of the (residual) activity of LDH (Lactate-dehydrogenase). 3 WDN !NTERNATIONAL SEARCH REPORT International Application No PCT/DE91/00307 I. CIASSIFICATION OF SUBJECT MATTER (If several classification symbols apply, Indicate all) According to International Patent Classification (IPC) or to both National Classification ,nd IPC Int. Cl.5 A61L 2/00, A61L 2/18 II. FIELDS SEARCHED Minimum Documentation Searched 7 Classfitallon System I Classification Symbols Int. Cl. 5 A61L Documentation Searcli:d other than Minimum Documentation to the Extent that such Documents are Included In the Fields Searched llt. DOCUMENTS CONSIDERED TO BE RELEVANT* Category Citation of Document, with indication, where appropriate, of the relevant passages 1 Relevant to Claim No. 1 X GB, A, 1471336 (TOWER PHARMACEUTICALS) 1,6-9,12,13 21 April 1977, see examples 1,12; claim 1 X US, A, 3227626 BAUMGARTEN et al.) 1 4 January 1966, see column 2, lines 8-13; claim 1 A US, A, 4841023 HOROWITZ) 20 June 1989, 1 see claim 1 Special ca%)gories of cited documents: ~o later document published after the international filing date document defining the general state of the art which Is not or priority date and not In conflict with the application but considered to be of particular relevance cited to understand the principle or theory underlying the invention .i;ller document but published on or after the International docume;-t cf particular relevance: the claimed invention filing date cannot be considered novel or cannot be considered to document which may throw doubts on priorltt claim(s) or Involve an inventive step which Is cited to establish the publication datk of another document of particular relevance;' the claimed Invention citatior, or other special reason (as specified; cannot be considered to Involve an inventive step when the document refer'ring to an oral disclosure, u'e, exhibitlon or document Is combined with one or more other such docu- other means mentas, such combination being obvious to a person skilled -i document published prior to the International filing date but in the art. later than the priority date clalmr J document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 17 June 1991 (17.06.91) 12 August 1991 (12.08.91) International Searching Authority Signature of Authorized Officer European Patent Office Form PCTIISA/210 (second sheet) (January 1985) ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. DE 9100307 SA 46159 This annex lists the patent family members relating to the patent documents cited in the above-mentioned international search report. The members are as contained in the European Patent Office EDP file on 30/07/91 The European Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent document Publication Patent family Publication cited in search report date member(s) date GB-A- 1471336 21-04-77 None US-A- 3227626 None US-A- 4841023 20-06-89 None 0 M For more details about this annex see Official Journal of the European Patent Office, No. 12/82 INTERNATIONALER RECHERCHENHRICHT Inteenationales Aktonzeichen PCT/DE 91 /00307 1. KILASSIFN ATION DES ANMEILDUNGSGEGENSTANDS (bel mehreran Klassilikationssymbolen s~nd aills anzugeben) 6 Nach der Internatianalen Patentklassifikation (IPC) ader nach der natianaten Klassifikatian und der IPC Int.C1 A 61 L 2/00, A 61 L 2/18 11. RECHERCHIERTE SACHGEBIETE Recherchierier MindestpriifStoff 7 Klassifikatianssystem Klassifikationssymbole Int.I. 5 A 61 L Recherchierte nicht zurn Mindesiprtifstoff gehdrende Ver6ff enilichungen, sowelt these unter die recherchierten Sachgebiete fallen 8 III. EINSCHLAGIGE VEROFFENTLICHUNGEN 9 Art- Kennzeichnung der Verdffentlichungll 9 saweit erforderlich urIaer Angabe der matageblichen TeJIe 12 Betr. Anspruch Nr.13 X GB, A, 1471336 (TOWER PHARMACEUTICALS) 1,6-9,12,13 21. April 1977 siehe Beispielen 1,12; Anispruch 1 X US, A, 3227626 BAUMGARTJEN et 1 4. Januar 1966 siehe Spalte 2, Zeilen 8-13; Anspruch 1 A US, A, 4841023 HOROWITZ) 1 Juni 1989 siehe Anspruch 1 Besondere Kategarien von engegbenen Verdffenlchungen& 0 Ver~ffentlichung, die don aligemoilnen Stand der Technik -17 Spiters Ver~ffentlichung, die nach demn internatlonalen An- definiert, aber nicht ala besonders bedeutsam anzusehen it modcatumn odor dern Prlarititsdatum ver~ffantIicht warden let und mit der Anmeldung nicht kollidiert, sandern nur zumn alteres Dakcument, des lodoch arst am oder nach dern Interne- Verstindnis des der Erfindung zugrunbdeliegenden Prinzips- tionalen Anmeldedatum ver~ffontlicht warde-ist oder der Ihr zugtunideliegendmn Theorie arnegeben let Ver~ffentflchung, die gesigner it. ulnen Priarititianspruch 'eg nlihn vo 'odrrBduug i wpu zwyeifelhbl; erscheinon zu lassen, odor durch die dat Ver~f. t Erffntlung vorn bohtas odor aeduftundebiesr Titi- fontlichungadatum einer anderen im Racherchenboricht go kt o m nd beanncht al nr de u rldrahrTtg namnten %kr~tentlichinv beeg were-en soil odor die sus oingem atbrhmdbtahtwre andoron besonderen (Arund angege~n Is% (wvie auugefihrt) Ver~femlichung von bosonderer Sedeutung; die beanspruch- er~fentlchug. i~ sch uf ene i~nd~cho0flnb&~uto teirfindlung kann nicht als out ertinderischer Titigkelt be- "0 ie Bentzung eine Au ut en odrndereh Malnhenbi ruhend batrachtert warden, wonn die Vor6ffentliciiung mit bei eutzu on uieln dradrMaah n oeri odor rnehreron anderen Vorer~ntllchungen dieser Kate- b'3z~ht ori in erbndun gebach Wrdunddiew Verbindung filr Ver~ffentlchung, die var dem i nternationalen Anmeldeda- amnen Faichmann nshollegAnd 1st turn. aber nach damn beensp~uchtjrm Priarttsdatum verdflent- Veroffernilchung. die Mltqllad drselben Patmntfamillo it licht warden Ist& IV. BESCIHEINIGUNG Datum dlos Abschlutsm derlnterationalen Recherche Abseddawrldis Interntonalen, Recharchonberchs- 17. Juni 1991 1 .0nhitdes bovallmachtigton 8 dIonhteten Europiisches P'atentamit Mine M.v Forrnblatt PCT/lSA/21O0 (Blatt 2) (J14nusr 1985) ANHANG ZUM INTERNATIONALEN RECHERCHENBERICHT OBER DIE INTERNATIONALE PATENTANMELDUNG NR. DE 9100307 SA 46159 in diesemn Anhang sind die Mitglieder der Patentfawilien der im obengenannten internationalen Rechercbenbericht angcftihrten Patentdokumente angegeben. Die Angaben fiber die Famnhienmitglieder entsprechen dew Stand der Datel des Europaischcn Patentawts am 30/07/91 Diese Angaben dienen nur zur Unterrichtung und erfolgen obue Gewihr. 1wn Recherchenbericht Datum der Mitglied(er) der Datum der angefiffhrtes Patentdokument Verbllentlichung Patentfamilic Verbffentlichung GB-A- 1471336 21-04-77 Keine US-A- 3227626 Ke in e US-A- 484102 20-06-89 Keine Ffir nibere Einzelheiten zu diesewn Anbang siehe Aintsblatt des Europllischen Patcutamnts, Nr.1Z182