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EP0827536A1 - Procede de purification de plasmide effectuee a grande echelle - Google Patents

Procede de purification de plasmide effectuee a grande echelle

Info

Publication number
EP0827536A1
EP0827536A1 EP96916486A EP96916486A EP0827536A1 EP 0827536 A1 EP0827536 A1 EP 0827536A1 EP 96916486 A EP96916486 A EP 96916486A EP 96916486 A EP96916486 A EP 96916486A EP 0827536 A1 EP0827536 A1 EP 0827536A1
Authority
EP
European Patent Office
Prior art keywords
plasmid
plasmid dna
dna
large scale
anion exchange
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.)
Withdrawn
Application number
EP96916486A
Other languages
German (de)
English (en)
Inventor
Ann L. Lee
Sangeetha Sagar
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.)
Merck and Co Inc
Original Assignee
Merck and Co Inc
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
Application filed by Merck and Co Inc filed Critical Merck and Co Inc
Publication of EP0827536A1 publication Critical patent/EP0827536A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/101—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by chromatography, e.g. electrophoresis, ion-exchange, reverse phase
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1017—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by filtration, e.g. using filters, frits, membranes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Definitions

  • the classical techniques for isolating plasmid DNA from microbial fermentations are suitable for small or laboratory scale plasmid preparations.
  • One such procedure involves the alkaline lysis of microbial host cells containing the plasmid, followed by acetate neutralization causing the precipitation of host cell genomic DNA and proteins which are then removed by, for example, centrifugation.
  • the liquid phase contains the plasmid DNA which is alcohol precipitated and then subjected to isopycnic centrifugation using CsCl in the presence of ethidium bromide.
  • the ethidium bromide is required in order to separate the total plasmid DNA into the three different forms, supercoiled (form I), nicked circle (form II), and linearized (form HI), and the desired plasmid form is collected. Further extraction with butanol is required to remove residual ethidium bromide followed by DNA precipitation using alcohol. Additional purification steps follow to remove host cell proteins. The removal of host proteins is performed by repeated extractions using phenol or a mixture of phenol and chloroform. The plasmid DNA is alcohol precipitated and residual phenol is removed by repeated isoamyl/chloroform extractions. The final alcohol precipitated plasmid DNA is dissolved in water or a suitable buffer solution.
  • phenol extraction is used to remove residual proteins and DNase, a process that would require a centrifuge to break phenol/water emulsion;
  • scalability of the chemical lysis step is an obstacle i.e., lysozyme/alkaline/KOAc treatment step is efficient in lysing cells on a small scale, however, the increase in viscosity makes large scale processing very difficult; and
  • RNA The mixture is then neutralized by addition of acid which results in precipitation of the high molecular weight chromosomal DNA.
  • the high molecular weight RNA and protein-SDS complexes precipitate with the addition of high concentration of KOAc salt.
  • the plasmid product remains in the clarified supernatant following centrifugation. Limitations here include the need to process quickly and on ice in order to retard the activity of nucleases which are not removed until phenol extraction.
  • the main contaminant remaining in the supernatant with the product is RNA.
  • lysozyme may enhance plasmid release during lysis.
  • An advantage is that heat treatment of the cells would also denature the DNase.
  • this technique is not suitable for scale up to a high volume of microbial fermentations and is meant for fermentations less than five liters.
  • Alternatives to isopycnic centrifugation using CsCl for plasmid purification have been published. These alternatives are suitable only for laboratory scale plasmid isolation and include:
  • Unprecedented technology is required for developing/- implementing a large scale commercially viable process for fermentation, isolation, purification and characterization of DNA as a biopharmaceutical.
  • the current laboratory method used to isolate and purify plasmid DNA consists of a series of classical laboratory techniques that are not suitable for a manufacturing process. For example, density gradient centrifugations are not scaleable; the purification procedure necessitates the use of hazardous and expensive chemicals/solvents such as ethydium bromide, a known mutagen, and is labor intensive and time consuming. Therefore, a scaleable alternative process was developed, and is disclosed herein. In addition, an HPLC assay was established to track the plasmid product through the process steps and to distinguish between the plasmid forms.
  • the microbial cells harboring the plasmid are suspended and optionally incubated with lysozyme in a buffer containing detergent, heated using a flow-through heat exchanger to lyse the cells, followed by centrifugation. After centrifugation the clarified lysate, which contains predominately RNA and the plasmid product, is filtered through a 0.45 micron filter and then diafiltered, prior to loading on the anion exchange column.
  • the plasmid product may optionally be treated with RNase before or after filtration, or at an earlier or later step.
  • the anion exchange product fraction containing the plasmid is loaded onto the reversed phase column, and is eluted with an appropriate buffer, providing highly pure plasmid DNA suitable for human use.
  • Figure 1 A schematic of a suitable heat exchanger apparatus is shown.
  • Figure 2. The relationship between the outlet temperature and the flow rate is shown, graphically.
  • Figure 3. Comparative chromatograms of total plasmid in clarified supernatant with 50 mMEDTA and 100 mMEDTA are shown.
  • FIG. 7 An elution profile of plasmid DNA from cell lysate is shown.
  • FIG. 8 An agarose gel electrophoresis analysis of the DNA product obtained at various intermediate steps of purification is shown.
  • Figure 9 A tracing of the anion exchange HPLC analysis of the DNA product demonstrating the purity of the product is shown.
  • This invention offers a number of advantages including higher product recovery than by chemical lyses, inactivation of DNases, operational simplicity and scaleability.
  • the present invention is drawn to a process for the large scale isolation and purification of plasmid DNA from microbial fermentations.
  • Large scale microbial cell fermentations as used herein are considered to be total cell fermentation volumes of greater than about 5 liters, or the cells harvested from a fermentation volume greater than about 5 liters.
  • the present invention is also drawn to providing plasmid DNA in a highly purified form suitable for human use.
  • DNA for human use includes, but is not limited to, polynucleotide vaccines and DNA for human gene therapy.
  • Polynucleotide vaccines are intended for direct injections into humans [Montgomery, D.L. et al., 1993, Cell Biol., 169, pp. 244-247; Ulmer, J.B. et al, 1993, Science, 259, pp. 1745-1749].
  • the present invention is also drawn to an in-line monitoring process for the tracking of the various forms of plasmid DNA through the isolation and purification steps.
  • the various forms of plasmid DNA referred to above which can be individually isolated by the process of the present invention are form I (supercoiled plasmid), form II (nicked or relaxed plasmid), and form in (linearized plasmid).
  • the process of the present invention is suitable for use with microbial fermentations in general. It is readily apparent to those skilled in the art that a wide variety of microbial cells are suitable for use in the process of the present invention, including but not limited to, fungal cells including yeast, and bacterial cells.
  • a preferred microbial fermentation is a bacterial fermentation of cells containing the plasmid to be isolated and purified.
  • a preferred bacterial fermentation is a fermentation of E. coli containing the plasmid to be isolated and purified. It is readily apparent to those skilled in the art that bacterial fermentations other than E. coli fermentations are suitable for use in the present invention.
  • the microbial fermentation may be grown in any liquid medium which is suitable for growth of the bacteria being utilized.
  • the plasmid to be isolated and purified by the process of the present invention can be any extrachromosomal DNA molecule.
  • the plasmids can be high copy number per cell or low copy number per cell.
  • the plasmids can also be of virtually any size. It is readily apparent to those skilled in the art that virtually any plasmid in the microbial cells can be isolated by the process of the present invention.
  • Microbial cells containing the plasmid are harvested from the fermentation medium to provide a cell paste, or slurry. Any conventional means to harvest cells from a liquid medium is suitable, including, but not limited to centrifugation or microfiltration.
  • Isolation of the plasmid DNA from harvested microbial cells using the current lab scale procedures consist mainly of enzymatic treatment of microbial cells to weaken the cell wall followed by cell lysis.
  • the purification steps include repetitive CsCl/EtBr centrifugations followed by organic solvent extractions and precipitation to remove tRNA, residual proteins, EtBr and other host contaminants. These steps are not scaleable and therefore not suitable for use in large- scale processing.
  • preparative scale chromatography is a powerful purification tool that provides high resolution, operational ease and increased productivity for purifying DNA plasmid products. Two different modes of chromatography, reversed phase and anion exchange, show suitability in purifying DNA plasmid to the stringent levels required for human use.
  • harvested microbial cells are resuspended in modified STET buffer which is comprised of about 50 mM TRIS, about 50-100 mM EDTA, about 8% Sucrose, about 2% TRITON X-100, and optionally sub-microgram concentrations of lysozyme, at a pH in the range of 6.0-10.0.
  • concentration of lysozyme optionally used in the process of the present invention is substantially less than the concentration of lysozyme used in the procedures known in the art. It is readily apparent to those skilled in the art that modifications of this basic buffer formula can be made and are suitable for use in the present invention.
  • the pH range may be adjusted according to the best results provided for the particular strain of bacteria being used.
  • the preferred pH range is about 8.0-8.5.
  • the suspension is then heated to about 70-100°C, with about 70-77°C preferred, in a flow-through heat exchanger.
  • the lysate is centrifuged to pellet large cell debris, protein and most genomic DNA.
  • a prototype heat exchanger was built to demonstrate the feasibility of flow-through heat lysis of microbial cells containing plasmid.
  • the particular heat exchanger consisted of a 10 ft. x 0.25 inch O.D. stainless steel tube shaped into a coil. The coil was completely immersed into a constant high temperature water bath. The hold-up volume of the coil was about 50 mL. Thermocouples and a thermometer were used to measure the inlet and exit temperatures, and the water bath temperature, respectively.
  • the product stream was pumped into the heating coil using a Masterflex peristaltic pump with silicone tubing. Cell lysate exited the coil and was then centrifuged in a Beckman J-21 batch centrifuge for clarification.
  • Figure 1 provides a schematic of this particular apparatus, however other types of heat exchanger construction are suitable for use in the present invention, including but not limited to a shell and tube construction, which is preferrable.
  • the clarified lysate can optionally be treated with RNase, and the plasmid product can be filtered to further remove small debris.
  • a wide variety of filtration means are suitable for use in this process, including but not limited to filtration through a membrane having a small pore size.
  • a preferred filtration method is filtration through a 0.45 micron filter.
  • the material can be diafiltered.
  • Standard, commercially available diafiltration materials are suitable for use in this process, according to standard techniques known in the art.
  • a preferred diafiltration method is diafiltration using an ultrafilter membrane having a molecular weight cutoff in the range of 30,000 to 500,000, depending on the plasmid size.
  • the DNA preparation described above is diafiltered using an ultrafiltration membrane (about 100,000 molecular weight cutoff) against column buffer prior to loading on the anion exchange column. Diafiltration prior to the anion exchange column is preferred, and it greatly increases the amount of lysate that can be loaded onto the column.
  • anion exchange matrices are suitable for use in the present invention, including but not limited to those available from POROS Anion Exchange Resins, Qiagen, Toso Haas, Sterogene, Spherodex, Nucleopac, and Pharmacia.
  • the column (Poros II PI/M, 4.5 mm x 100) is initially equilibrated with 20 mM Bis/TRIS Propane at pH 7.5 and 0.7 M NaCl. The sample is loaded and washed with the same initial buffer. An elution gradient of 0.5 M to 0.85 M NaCl in about 25 column volumes is then applied and fractions are collected.
  • FIG. 5 shows a sample elution profile of filtered clarified cell lysate from the anion exchange column. Agarose gel analysis revealed that the second peak which appears after the flow-through is composed of the plasmid product. The earlier large peak is due to RNA. This is confirmed by incubating the clarified cell lysate with ribonuclease prior to loading on the column, which showed that the large peak disappears and is replaced by several smaller more rapidly eluting peaks, due to the degradation products of ribonuclease digestion.
  • the anion exchange product fraction is loaded onto a reversed phase column.
  • a wide variety of commercially available matrices are suitable for use in the present invention, including but not limited to those available from POROS, Polymer Labs, Toso Haas, Pharmacia, PQ Corp., Zorbax, BioSepra resins, BioSepra Hyper D resins, BioSepra Q-Hyper D resins and Amicon.
  • the matrices can also be polymer based or silica based.
  • the reversed phase column (Poros R/H), is equilibrated with about 100 mM Ammonium Bicarbonate at pH 8.5. A gradient of 0-11 % isopropanol is then used to elute bound material.
  • the three forms of plasmids, forms I, II and III described above, can be separated by this method.
  • the eluted plasmid DNA can then be concentrated and/or diafiltered to reduce the volume or to change the buffer.
  • DNA intended for human use it may be useful to diafilter the DNA product into a pharmaceutically acceptable carrier or buffer solution.
  • any method suitable for concentrating a DNA sample is suitable for use in the present invention. Such methods includes diafiltration, alcohol precipitation, lyophilyzation and the like, with diafiltration being preferred. Following diafiltration the final plasmid DNA product may then be sterilized. Any method of sterilization which does not affect the utility of the DNA product is suitable, such as sterilization by passage through a membrane having a sufficiently small pore size, for example 0.2 microns and smaller. The following examples are provided to illustrate the process of the present invention without, however, limiting the same thereto. EXAMPLE 1
  • the cell suspension was pumped through the heat exchanger at 81 mL/min which corresponded to a residence time of the cell solution in the heat exchanger of about 35 seconds.
  • the bath temperature was maintained at 92°C.
  • the inlet and outlet temperatures of the cell solution were measured to be about 24°C and about 89°C (average), respectively.
  • About 1 liter of sample was run through the heat exchanger. No visible clogging of the tube was observed although the lysate was somewhat thicker than the starting material.
  • the lysate was cooled to room temperature and its viscosity was measured to be about 40 cp.
  • the cell lysate was clarified by batch centrifugation at 9000 RPM for 50 minutes using the Beckman J-21.
  • Microbial cells and lysates were prepared as described in Examples 1 and 2, and the following analyses were performed.
  • Figure 3 shows comparative chromatograms of the total plasmid in the clarified supernatant with 50 mM EDTA vs 100 mM EDTA.
  • the cell suspension was prepared as described in Example 1.
  • the operating flow rate for these runs was approximately 186 ml/min.
  • the temperatures of the inlet, outlet and bath are 24°C, 92°C and 96°C respectively.
  • An acceptable range of lysis temperatures was determined by measuring the percentage of supercoiled plasmid generated for each run.
  • Figure 4 illustrates the concentration of supercoiled plasmid as a function of exit temperature.
  • An acceptable range of lysis temperatures is between 75°C and 92°C. At temperatures below 75°C, more relaxed circle plasmid was generated, most likely due to increased DNase activity. Above 93 °C, the levels of supercoiled plasmid appear to diminish, possibly due to heat denaturation.
  • FIG. 5 A comparison of the anion exchange column eluate from clarified lysate treated with RNase (thin line) and untreated (thick line) is shown in Figure 5.
  • the peak at about 10 minutes is plasmid DNA, and is followed by a large peak in the untreated sample which is RNA.
  • the large RNA peak has been eliminated and a greater separation of the plasmid peak from contaminant peaks is produced.
  • the plasmid DNA eluted from the anion exchange column was separated into the individual forms by reversed phase HPLC analysis.
  • the separation of supercoiled plasmid (form 1) from nicked circle (form 2) is shown in Figure 7. The two forms were easily separated and allowed the isolation of individual forms of the plasmid.
  • the anion exchange product was then loaded onto a reversed phase chromatography column (Poros R/H) which had been previously equilibrated with lOOmM ammonium bicarbonate at pH 8.0, and a gradient of 0% to 80% methanol was used to elute the bound material.
  • the highly purified supercoiled plasmid DNA eluted at 22% methanol.
  • agarose gel of the product fractions from each of the major steps of the purification process is shown in Figure 8. Based on the agarose gels and the colorimetric and HPLC assays described in Example 3, the final product, shown in Figure 9, is highly pure.
  • the product consists of greater than 90% supercioled and less than 10% open circle plasmid.
  • RNA was below the limits of detection of the assay used.
  • Genomic DNA and protein contaminant levels were also below the limits of detection in the assays used.
  • the overall supercoiled plasmid yield at the end of the process was approximately 60% of the supercoiled plasmid in the clarified lysate.
  • the batch temperature was maintained at 100°C and the inlet and outlet temperatures of the cell suspension were measured to be about 24°C and between 70-77°C, respectively.
  • the cell lysate exiting the heat exchanger was collected in Beckman centrifuge bottles (500 mis each) and the material was centrifuged immediately in Beckman J-21 centrifuges for 50 minutes at 9000 RPM. Following centrifugation, the supernatant was found to contain 4-5 times more plasmid product than in the case without lysozyme incubation. The supernatant product of the centrifugation was immediately diafiltered against 3 volumes of TE buffer (25 mM Tris- EDTA at pH 8.0) and then incubated with 20xl0 5 units of E.
  • TE buffer 25 mM Tris- EDTA at pH 8.0
  • the filtered lysate was diluted to 0.7 M NaCl with a 20 mM Bis/Tris Propane-NaCl buffer at pH 7.5, which prepares the diluted filtrate for loading onto the anion exchange column.
  • the anion exchange column (3.6 L of POROS PI/M) was previously equilibrated with 20 mM Bis/Tris Propane and 0.7M NaCl. The filtered lysate was loaded to column capacity.
  • the diluted anion exchange supercoiled fraction was then loaded onto a 7 L reversed phase column (POROS R2/M) which had been previously equilibrated with 100 mM Ammonium Bicarbonate containing 1.2% IPA.
  • POROS R2/M reversed phase column
  • 3.2 grams of supercoiled plasmid were loaded onto the reversed phase column and then the column was washed with 6-10 column volumes of 1.2% IPA in 100 mM Ammonium Bicarbonate. This extensive wash was performed to clear impurities.
  • a gradient of 1.2% IPA to 11.2% IPA in 5 column volumes was performed.
  • the supercoiled plasmid fraction elutes at about 4% IPA.
  • the supercoiled product fraction from the reversed phase column was then concentrated and diafiltered into normal saline using a 30 kD MWCO membrane.
  • the final product bulk was filtered through a 0.22 micron filter.
  • Table 1 provides a purification table describing clearance of impurities and yields across each of the major process steps.
  • the overall product yield of the process was more than 50% of the supercoiled plasmid in the clarified cell lysate as indicated by the anion exchange HPLC assay described in EXAMPLE 3.
  • the purity of the product was very high with less than 1% E. coli RNA and protein, and less than 2.9% E. coli genomic DNA.

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Abstract

L'invention concerne un procédé d'isolation et de purification à grande échelle d'ADN plasmidique à partir de fermentations microbiennes importantes. Ce procédé permet d'isoler individuellement les trois formes d'ADN plasmidique: superhélice (forme I), cercle crénelé ou relâché (forme II) et ligne droite (forme III). Il permet également d'obtenir de l'ADN extrêmement purifié qu'on peut introduire dans une composition pharmaceutique.
EP96916486A 1995-05-19 1996-05-15 Procede de purification de plasmide effectuee a grande echelle Withdrawn EP0827536A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US44611895A 1995-05-19 1995-05-19
US446118 1995-05-19
PCT/US1996/007083 WO1996036706A1 (fr) 1995-05-19 1996-05-15 Procede de purification de plasmide effectuee a grande echelle

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EP0827536A1 true EP0827536A1 (fr) 1998-03-11

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EP96916486A Withdrawn EP0827536A1 (fr) 1995-05-19 1996-05-15 Procede de purification de plasmide effectuee a grande echelle

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EP (1) EP0827536A1 (fr)
JP (1) JPH11505707A (fr)
KR (1) KR19990014924A (fr)
CN (1) CN1190435A (fr)
AR (1) AR003122A1 (fr)
AU (1) AU709003B2 (fr)
CA (1) CA2220867A1 (fr)
CZ (1) CZ366197A3 (fr)
EA (1) EA000785B1 (fr)
HR (1) HRP960222A2 (fr)
HU (1) HUP9802557A3 (fr)
MX (1) MX9708967A (fr)
NO (1) NO975280L (fr)
NZ (1) NZ309231A (fr)
PL (1) PL323475A1 (fr)
SK (1) SK155797A3 (fr)
WO (1) WO1996036706A1 (fr)
YU (1) YU29796A (fr)

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JPH11505707A (ja) 1999-05-25
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YU29796A (sh) 1999-06-15
AU5921996A (en) 1996-11-29
CA2220867A1 (fr) 1996-11-21
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AR003122A1 (es) 1998-07-08
HUP9802557A3 (en) 2000-04-28
HUP9802557A2 (hu) 1999-02-01
CN1190435A (zh) 1998-08-12
AU709003B2 (en) 1999-08-19
NO975280L (no) 1998-01-16
WO1996036706A1 (fr) 1996-11-21

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