EP0000651A1 - Method of separating a factor IX preparation from blood plasma - Google Patents

Method of separating a factor IX preparation from blood plasma Download PDF

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Publication number
EP0000651A1
EP0000651A1 EP78300177A EP78300177A EP0000651A1 EP 0000651 A1 EP0000651 A1 EP 0000651A1 EP 78300177 A EP78300177 A EP 78300177A EP 78300177 A EP78300177 A EP 78300177A EP 0000651 A1 EP0000651 A1 EP 0000651A1
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plasma
factor
factors
preparation
copolymer
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EP0000651B1 (en
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Jacques Jean Joseph Delente
Richard Alan Schoenfeld
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Monsanto Co
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Monsanto Co
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/644—Coagulation factor IXa (3.4.21.22)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21022—Coagulation factor IXa (3.4.21.22)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL 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
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/827—Proteins from mammals or birds
    • Y10S530/829—Blood
    • Y10S530/83—Plasma; serum

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  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
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  • Biotechnology (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Peptides Or Proteins (AREA)

Abstract

A blood coagulation Factor IX preparation is separated from plasma by selective adsorption of Factors II, IX and X, to the substantial exclusion of Factors VII and VIII, with 0.025-0,1 % by weight of a water-insoluble, crosslinked polyelectrolyte copolymer of ethylene and maleic anhydride containing pendant diloweralkylaminoloweralkyl functional groups.

Description

  • This-invention relates to blood fractionation and more particularly to the separation of a blood coagulation Factor IX preparation from plasma by the selective adsorption cf Factors II, IX and X.
  • The process of blood coagulation is a complicated physiological activity that involves the interaction of numerous substances found in normal whole blood. It is known that certain factors associated with the blood coagulation mechanism are absent or seriously deficient in certain individuals. In those patients suffering from classical hemophilia, antihemophilic factor A (AHF, Factor VIII) is deficient. In those patients afflicted with hemophilia B, plasma thromboplastin component (PTC, Factor IX) is missing from the blood.
  • Several other factors which are important in the coagulation mechanism are Factors II, VII and X. As with Factors VIII and IX, these other factors also are deficient or absent in certain individuals. Factors II, VII and X are usually associated with Factor IX in the fractionation of blood plasma into various fractions, and a concentrate of these four factors has come to be known.as the prothrombin complex.
  • In the development of modern blood banking programs involving the collection and storage of large quantities of blood and blood components, the establishment of adequate preservation systems is critical. Since World War II it has been common practice to collect blood in a solution of citric acid, sodium citrate and dextrose known as ACD blood. The problem of preserving blood is much simplified, however, when it is reduced to preservation of various blood components since it is easier to meet the environmental requirements of the separate components than of whole blood.
  • Moreover, it is wasteful and even detrimental to the patient to administer more blood components than required. Thus,the hemophiliac needing certain blood coagulation factors ideally should be given only those factors required or at least a purified concentrate of these factors containing a reduced level of unneeded factors.
  • The fractionation of blood to obtain blood coagulation Factors VIII and IX and the prothrombin complex is well known. Most fractionation methods require the separation of Factor VIII from the plasma or other starting material prior to the separation of Factor IX or the prothrombin complex. For example, Factor VIII is frequently first separated from plasma as a cryoprecipitate or by precipitation with glycine or polyethylene glycol as described in U.S. Patents 3,631,018 and 3,652,530 and references cited therein.
  • Various prior methods of blood fractionation for the preparation of the prothrombin complex include the barium sulfate adsorption method described by Fowell in U.S. Patent 2,999,791 and the tricalcium phosphate adsorption method disclosed by Soulier et al, La Presse Medicale 72, 1223-28 (1964). Tullis discloses the use of DEAE-cellulose ion exchanger for the production of a prothrombin complex, New England Journal of Medicine 273, 667-74 (1965) while the corresponding use of DEAE-Sepizadex is described by Wado and Mozen in U.S. Patent 3,717,705. Andersson et al in U.S. Patent 3,920,625 further describe the use of DEAE-Sephadex specifically for the preparation of Factor IX concentrates. Use of polyethylene glycol for the production of prothrombin complex is taught by Fekete and Shanbrom in U.S. Patents 3,560,475 and 3,682,881. Aluminum hydroxide and other such gel materials also are known as useful in the concentration of prothrombin complex factors as seen from Bidwell, U.S. Patent 2,867,567.
  • As distinguished from all of the foregoing methods, in the present invention a Factor IX preparation is separated from plasma such that it also contains Factors II and X but not Factor VII which is usually contained in the prothrombin complex concentrates of the prior art. Moreover, the Factor IX preparation of this invention advantageously can be separated from the plasma prior to the separation of Factor VIII.
  • The method of the present invention is one of separating a Factor IX preparation from plasma characterized by contacting liquid blood plasma at a pH of from about 7.5 to about 8.5 with from about 0.025% to about 0.1% by weight of the plasma of a water-insoluble, cross-linked polyelectrolyte copolymer of ethylene and maleic anhydride containing pendant diloweralkylaminoloweralkyl functional groups whereby Factors II, IX and X are selectively adsorbed by the polyelectrolyte copolymer to the substantial exclusion of Factors VII and VIII which are unadsorbed and remain in the liquid plasma.
  • In accordance with the present invention, a Factor IX preparation containing Factors II, IX and X is separated from liquid plasma with a water-insoluble, cross-linked polyelectrolyte copolymer of ethylene and maleic anhydride containing pendant diloweralkylaminoloweralkyl functional groups. By use of the polyelectrolyte copolymer at a relatively low concentration of from about 0.025% to about 0.1% by weight of the plasma and a pH of from about 7.5 to about 8.5, a Factor IX preparation containing Factors II, IX and X surprisingly is selectively adsorbed by.the polyelectrolyte copolymer to the substantial exclusion of Factors VII and VIII which are unadsorbed and remain in the liquid plasma.
  • If desired, the adsorbed Factor IX preparation can then be eluted from the polyelectrolyte by washing witn an aqueous solution of a physiologically acceptable salt sucn as NaCl, for example a solution of about one to three molar NaCl. The elution preferably is carried out at a pH of from about 5.5 to about 6.5 although nigher ph's also can be used.
  • The smarting plasma used in the fractionation Method of this invention is generally obtained fresh frozen. This plasma should be thawed before fractionation with the polyelectrolyte copolymer, preferably by heating to a temperature of at least about 35°C. The appropriate polyelectrolyte copolymer can then be admixed with the plasma at a concentration of from about 0.025% to about 0.1% and preferably about 0.035% to about 0.05%. and the pH adjusted to a range of from about 7.5 to about 8.5. The mixture is stirred for a suitable time, for example at least about 10 minutes, during which time the Factor IX preparation is selectively adsorbed by tne polyelectrolyte copolymer and the remaining liquid plasma is made deficient in Factors II, IX and
  • In general, the water-insoluble, cross-linked polyelectrolyte copolymers employed in this invention are copolymers of ethylene and maleic anhydride containing pendant diloweralky laminoloweralkyl functional groups. By the term "loweralkyl" is meant an alkyl having from about 1 to about 4 carbon atoms.
  • The base copolymer of ethylene and maleic anhydride (EMA) can be prepared, for example, by reacting ethylene and maleic anhydride in the presence of a peroxide catalyst in a suitable solvent. The copolymer will preferably contain substantially equimolar quantities of the ethylene residue and the anhydride residue.
  • The base EMA copolymer can be reacted with a loweralkyliazinobisloweralkylamine which has two primary amine groups and leads to a cross-linked EMA copolymer. The desired pendant diloweralkylaminoloweralkyl functional groups can then be incorporated into the cross-linked copolymer by reaction of diioweralkylaminoloweralkylamine with part of all of the remaining anhydride groups of the EMA polymer. The polyelectrolyte copolymer also desirably is converted to the HCl salt form to provide better handling characteristics. Further details on the preparation of these polyelectrolyte copolymers can be had by reference to the disclosure in U.S. Patent 3,554,985 which is incorporated herein by reference. Use of these polyelectrolyte ccpolymers in blood fractionation is described in U.S. Patent 3,555,001.
  • A preferred diloweralkylaminoloweralkyl Functional group is dimethylaminopropyl and a preferred cross-linking agent is methyliminobispropylamine.
  • A preferred polyelectrolyte copolymer for use Ln this invention contains about five methyliminobispropylamine cross-linking groups and about 90 pendant dimethylaminopropylamine functional groups per 100 maleic anhydride units in the EMA copolymer.
  • Other cross-linking agents, for example, divinylbenzene and ethylene diamine, and other functional groups, for example, dimethylaninoethyl and diethylaminobutyl, also can be used in the polyelectrolyte copolymers which are employed in the method of separating the Factor IX preparation herein.
  • Following the adsorption of the Factor IX preparation, the Factor VIII remaining in the plasma solution can be further ccncentrated and recovered by known techniques. The adsorbed Factor IX preparation can be conveniently recovered by elution from the polyelectrolyte copolymer by washing with an aqueous solution of HaCl having a molarity of from about one to about three.
  • In a preferred embodiment of the invention, about 0.035% by weight of the polyelectrolyte copolymer containing about five methyliminobispropylamine cross-linking groups and about 90 dimethylaminopropylamine functional groups per 100 maleic anhydride units in the EMA copolymer is employed for selective adsorption of the Factor IX preparation at a pH of about 8. The adsorbed Factor IX preparation is then eluted from the polyelectrolyte copolymer by washing with 1.7 molar NaCl at pH 6. The eluant can then be dialyzed against 0.1 molar NaCl at 40C and freeze dried for storage.
  • Tne following examples will further illustrate the invention although it will be appreciated that the invention is not limited to these specific examples.
    • EXAMPLE I
  • In this example, the polyelectrolyte copolymer ccnsisted of the reaction product of substantially equimolar parts of ethylene and maleic anhydride (EMA) cross-linked with methyliminobispropylamine (HIBPA) and then further reacted with dimethylaminopropylamlne (DMAPA) such as to provide about five MIBPA cross-linking groups and about 90 DMAPA pendant groups per 100 maleic anhydride units in the EMA copolymer and converted to the HC1 salt form. One liter of normal human plasma was adjusted to pH 8 with 1 molar HaOH and 0.35 grams of the aforesaid polyelectrolyte copolymer was added thereto and the mixture was stirred for 20 minutes. The mixture was then filtered and the filtrate was retained as a Factor IX depleted plasma. The filter cake was washed with distilled water to remove entrained protein.
  • A Factor IX preparation containing Factors II, IX and X was then eluted from the polyelectrolyte copolymer by washing with 25 ml. of 1.7 molar NaCl at pH 6.0 (the pH being adjusted with 0.1 molar citric acid) for 20 minutes. The copolymer slurry was then filtered and the filtrate was retained as the desired Factor IX preparation. In a series of seven one-liter replicate fractionations using the above procedure, an average of 483 t 48 units of Factor IX per liter were obtained having a purification index of 178± 33. One unit of Factor IX is defined as the amount of said factor in one ml of pooled normal whole plasma. The purification index is calculated as the ratio of the amount of total protein in the starting plasma to the amount of total protein in the final Factor IX preparation multiplied by the ratio of the units of Factor IX in the final Factor IX preparation to the units of Factor IX in the starting plasma.
    • EXAMPLE 2
  • Using 0.4 mg/ml of the polyelectrolyte copolymer of Example 1 for admixture with normal human plasma for 20 minutes at pH 7.4 to separate a Factor IX preparation, as in Example 1, the adsorption of Factors II, VII, VIII, IX and X was measured with the following results:
    Figure imgb0001
  • These results show a high selectivity for adsorption of Factors II, IX and X to the substantial exclusion of Factors VII and VIII based on the corresponding amounts of these factors in the starting plasma.
  • Conventional one-stage assays were used for determining tne coagulation factors in the foregoing examples. The one-stage assay system for Factor VIII sold commercially by Dade Division of American Hospital Supply Corporation was employed in tnese examples. This assay system is based on the activated partial thromboplastin time (FTT) used to determine deficiencies in factors necessary for the intrinsic method of clot formation. The PTT test was devised by Brinkhous and co-workers and reported in J. Lab. Clin, Med. 41, 637 (1953). In these assays for the various coagulation factors, the unknown sample was reacted with a partial thromboplastin reagent and the appropriate factor- deficient substrate plasma which did not contain the factor to be determined, and the time for clotting was observed. The partial thromboplastin reagent contains crude cephalin obtained from rabbit brain which is known to clot normal plasma faster than it clots hemophilic plasma. Such reagents are well-known and described, for example, in U.S. Patents 3,395,210, 3,486,981 and 3,522,148.

Claims (6)

1. A method of separating a Factor IX preparation from plasma characterized by contacting liquid blood plasma at a pH of from about 7.5 to about 8.5 with from about 0.025% to about 0.1% by weight of the plasma of a water-insoluble, cross-linked polyelectrolyte copolymer of ethylene and maleic anhydride containing pendant diloweralkylaminoloweralkyl functional groups whereby Factors II, IX and X are selectively adsorbed by the polyelectrolyte copolymer to the substantial exclusion of Factors VII and VIII which are unadsorbed and remain in the liquid plasma,
2. A method of Claim 1 characterized in that the diloweralkylaminoloweralkyl functional group is dimethylaminopropyl.
3. A method of Claim 1 or Claim 2 characterized in that the copolymer of ethylene and maleic anhydride is cross-linked with methyliminobispropylamine.
4. A method of Claim 1 characterized in that the polyelectrolyte copolymer contains about five methyliminobispropylamine cross-linking groups and about 90 dimethylaminopropyl pendant groups per 100 maleic anhydride- groups.
5. A method of any of the preceding claims characterized in that the absorbed Factor IX preparation is eluted from the polyelectrolyte copolymer by washing with an aqueous solution of NaCl having a molarity of from about one to about three.
6. A method of any of the preceding claims characterized in that the concentration of the polyelectrolyte copolymer is from about 0.035% to about 0.05% by weight of the plasma.
EP78300177A 1977-07-25 1978-07-21 Method of separating a factor ix preparation from blood plasma Expired EP0000651B1 (en)

Applications Claiming Priority (2)

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US05/818,920 US4081432A (en) 1977-07-25 1977-07-25 Method of separating a Factor IX preparation from plasma using ethylene-maleic anhydride polymers
US818920 1977-07-25

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EP0000651A1 true EP0000651A1 (en) 1979-02-07
EP0000651B1 EP0000651B1 (en) 1982-01-27

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JP (1) JPS5426322A (en)
AT (1) AT359645B (en)
AU (1) AU517885B2 (en)
CA (1) CA1107649A (en)
DE (1) DE2861573D1 (en)
ES (1) ES471857A1 (en)
HU (1) HU180882B (en)
IL (1) IL55194A0 (en)
IT (1) IT1097302B (en)
MX (1) MX5405E (en)
PT (1) PT68337B (en)
RO (1) RO75338A (en)
SU (1) SU841572A3 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0098256A2 (en) * 1982-06-28 1984-01-11 Monsanto Company Production of blood coagulation factor VIII:C
EP0256522A2 (en) * 1986-08-16 1988-02-24 BEHRINGWERKE Aktiengesellschaft Process for the manufacture of a low-prothrombin preparation of vitamin K-dependent coagulation factors and an agent obtained therefrom

Families Citing this family (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1603244A (en) * 1977-05-20 1981-11-18 Max Planck Gesellschaft Medicaments for the suppression of pathological processes
ES471858A1 (en) * 1977-07-25 1979-02-01 Monsanto Co Non-activating polyelectrolytes and their use in a method for separation of blood coagulation factors.
FR2472390A1 (en) * 1979-05-04 1981-07-03 Merieux Inst PROCESS FOR THE PREPARATION OF HIGHLY PURIFIED PROTHROMBINIC COMPLEX CONCENTRATES AND CONCENTRATES OBTAINED
SE448945B (en) * 1979-12-20 1987-03-30 Blombaeck E G B PROCEDURE FOR CLEANING AND / OR CONCENTRATION OF THE FACTOR VIII COMPLEX
AT368883B (en) 1980-07-22 1982-11-25 Immuno Ag METHOD FOR PRODUCING A NEW HUMAN PROTEIN-BASED PREPARATION PROCESSING BLOOD
DE3045153A1 (en) * 1980-11-29 1982-07-08 Behringwerke Ag, 3550 Marburg METHOD FOR THE PRODUCTION OF BLOOD COagulation FACTORS AND THE PREPARATION OF FACTORS IX AND X THEREFORE PRODUCED
US4447416A (en) * 1982-04-28 1984-05-08 American National Red Cross Plasma protein concentrates of reduced thrombogenicity and their use in clinical replacement therapy
US4382028A (en) * 1982-07-19 1983-05-03 Monsanto Company Separation of plasma proteins from cell culture systems
US5055557A (en) * 1983-03-04 1991-10-08 Scripps Clinic & Research Foundation Ultrapurification of factor IX and other vitamin K-dependent proteins
US5614500A (en) * 1983-03-04 1997-03-25 The Scripps Research Institute Compositions containing highly purified factor IX proteins prepared by immunoaffinity chromatography
US4786726A (en) * 1986-01-06 1988-11-22 Blood Systems, Inc. Factor IX therapeutic blood product, means and methods of preparing same
ZA877535B (en) * 1986-10-09 1988-04-11 F. Hoffmann-La Roche & Co. Aktiengesellschaft Factor ix-peptides
AU707697B2 (en) * 1995-08-28 1999-07-15 Sekisui Kagaku Kogyo Kabushiki Kaisha Serum or Plasma Separating Compositions

Citations (4)

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US3717708A (en) * 1968-10-24 1973-02-20 Cutter Lab Blood coagulation complex
DE2420747A1 (en) * 1973-05-13 1974-11-28 Yeda Res & Dev POLYMERIC CARRIERS FOR BINDING BIOLOGICALLY ACTIVE PROTEINS
US3920625A (en) * 1973-06-19 1975-11-18 Kabi Ab Isolation of coagulation factors from biological material using cross linked sulfated, sulfonated carbohydrates
DE2534603A1 (en) * 1975-08-02 1977-02-17 Basf Ag Copolymers having (N)-dialkyl-(N)-amido gps. - obtd. by reaction of maleic anhydride copolymers with (N)-substd. diamines having free amino gp.

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US3554985A (en) * 1963-01-02 1971-01-12 Monsanto Co Cross-linked copolymer polyelectrolytes based on alpha,beta-ethylenically unsaturated acids
US3555001A (en) * 1969-05-29 1971-01-12 Monsanto Co Process for the fractionation of plasma and serum using water-insoluble polyelectrolytes containing diloweralkylaminoloweralkylimide groups
US3682881A (en) * 1970-10-02 1972-08-08 Baxter Laboratories Inc Fractionation of plasma using glycine and polyethylene glycol

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3717708A (en) * 1968-10-24 1973-02-20 Cutter Lab Blood coagulation complex
DE2420747A1 (en) * 1973-05-13 1974-11-28 Yeda Res & Dev POLYMERIC CARRIERS FOR BINDING BIOLOGICALLY ACTIVE PROTEINS
US3920625A (en) * 1973-06-19 1975-11-18 Kabi Ab Isolation of coagulation factors from biological material using cross linked sulfated, sulfonated carbohydrates
DE2534603A1 (en) * 1975-08-02 1977-02-17 Basf Ag Copolymers having (N)-dialkyl-(N)-amido gps. - obtd. by reaction of maleic anhydride copolymers with (N)-substd. diamines having free amino gp.

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEMICAL ABSTRACTS, vol. 77, no. 5, 31 July 1972, Columbus, Ohio, (USA) G. DIKE et al.: "Preparation and clinical use of a new concentrate containing factor IX, prothrombin, and factor X and of a separate concentrate containing factor VII", page 308, column 2, abstract no. 31941b; & Brit. J. Haematol., 1972, 22(4), 469-90 (Eng.) *
CHEMICAL ABSTRACTS, vol. 85, no. 3, 19 July 1976, Columbus, Ohio, (USA) G. CASILLAS et al.: "Concentrates of blood clotting factors. Observations on the development of regular preparation of concentrates", page 309, column 1, abstract no. 16737m; & Rev. Asoc. Bioquim. Argent. 1975, 40 (222-223), 146-57 (Span.) *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0098256A2 (en) * 1982-06-28 1984-01-11 Monsanto Company Production of blood coagulation factor VIII:C
EP0098256A3 (en) * 1982-06-28 1985-10-23 Monsanto Company Production of blood coagulation factor viii:c
EP0256522A2 (en) * 1986-08-16 1988-02-24 BEHRINGWERKE Aktiengesellschaft Process for the manufacture of a low-prothrombin preparation of vitamin K-dependent coagulation factors and an agent obtained therefrom
EP0256522A3 (en) * 1986-08-16 1989-02-01 BEHRINGWERKE Aktiengesellschaft Process for the manufacture of a low-prothrombin preparation of vitamin k-dependent coagulation factors and an agent obtained therefrom

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ES471857A1 (en) 1979-02-01
IT1097302B (en) 1985-08-31
JPS5426322A (en) 1979-02-27
AU517885B2 (en) 1981-09-03
AU3824378A (en) 1980-01-24
IL55194A0 (en) 1978-09-29
HU180882B (en) 1983-05-30
US4081432A (en) 1978-03-28
SU841572A3 (en) 1981-06-23
ATA531778A (en) 1980-04-15
AT359645B (en) 1980-11-25
PT68337B (en) 1994-02-25
IT7826005A0 (en) 1978-07-21
JPS6154008B2 (en) 1986-11-20
MX5405E (en) 1983-07-13
RO75338A (en) 1980-11-30
CA1107649A (en) 1981-08-25
EP0000651B1 (en) 1982-01-27
PT68337A (en) 1978-08-01
DE2861573D1 (en) 1982-03-11

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