Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • 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
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/16011—Herpesviridae
  • C12N2710/16211—Lymphocryptovirus, e.g. human herpesvirus 4, Epstein-Barr Virus
  • C12N2710/16222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• Epstein-Barr virus is the causative agent of infectious mononucleosis in humans, one of the most common infectious diseases in young adults.
• EBV-positive people can later develop diseases such as nasopharyngeal carcinomas, B-cell lymphomas, T-cell lymphomas, certain forms of Hodgkin's lymphoma, and possibly other malignant diseases. Chronic forms of the course and, in rare cases, even acute fatal cases are described directly in connection with the primary infection or later.
• Epstein-Barr virus Since the infection with Epstein-Barr virus is clinically normal in young children and because vaccination with recombinant vaccine viruses which contain the Epstein-Barr virus membrane antigen gp 250/350 (BLLF-1 reading frame, Baer et al., 1984) express a protection against infection or disease (Gu et al., 1993), it can be assumed that vaccination against Epstein-Barr virus-corrected diseases is possible.
• the invention relates to mammalian cell lines, in particular hamster cell lines, which stably contain the glycoprotein (gp 250 (220)), the glycoprotein (gp 350) and / or the mixed glycoprotein (gp 250 (220) / (gp) 350 ) as Epstein-Barr virus antigens, especially in a protein-free medium. This prevents the risk of viral or bacterial contamination from media supplements.
• the invention further relates to a method for obtaining the glycoproteins mentioned.
• Hamster cell lines which stably express gp 250 (220), gp 350 and or gp 250 (220) / gp 350 as EBV antigens are known; compare, for example, Motz et al. in Gene, 44 (1986) 353-359 and Vaccines, 87 (1987) 374-379 and Gu Shyan et al. in 100 years blood serum therapy 1890-1990, Halle (Saale), (1991) 93-100. So far, however, no hamster cell lines have become known with which the glycoproteins mentioned can be stably expressed. According to Motz et al. in Vaccines, 87 (1987) 374-379, 376, the integration of the viral foreign glycoproteins in CHO cell membranes can be toxic to a certain extent.
• a hamster cell line which stably expresses and secretes the glycoproteins mentioned and is obtainable by:
• the cell line is infected by transfection with a vector expressing the glycoproteins mentioned, but which does not express the membrane anchor (Motz et al., 1987), the vector comprising a selection marker which the cell never lacks, the cell line is cultivated and selected with the aid of the selection marker for cells which, after omitting the selection factor (inhibitor), stably express and secrete the glycoproteins.
• a hamster cell line was also made available through the use of a method which stably expresses the glycoproteins mentioned in a protein-free medium and is obtainable by:
• (A) starts from a hamster cell line (host cell line) which lacks a selection marker which a vector to be integrated chromosomally later according to (A) (d) or a vector integrated according to (B) (recombinant cell line) has,
• the starting cell line or the host cell line is therefore grown in a culture medium containing serum, for example in MEM alpha " or a " + 5% FCS, since this cell line cannot yet be transferred into media and cultured in media which are free of serum.
• culture media are DIF 1000, RPMI 1640, ASF 103 and HDB.
• the old medium is repeatedly only partially replaced in order to guarantee good self-conditioning of the medium during the thinning of the serum.
• SMIF Sudden-F12 Medium
• Iscove's-F12 Medium Iscove's-F12 Medium
• IF F12 nutrient solution
• putrescine for example 1, 2 micromoles 1
• L-hydroxyproline for example 153 micromoles 1 "1
• chelators such as aurintricarboxylic acid (ATA, for example 3 micromoles 1 " 1 ) (Bertheussen, 1993), EDTA (at Example 4/3 micromole 1) and citric acid (for example 40 micromole 1), for complexing divalent ions and for better availability of inorganic iron as a replacement for the protein component customary in serum-free media to add transferrin (SMIF2) .
• ATA aurintricarboxylic acid
• EDTA at Example 4/3 micromole 1
• citric acid for example 40 micromole 1
• CHO Chinese hamster ovary cell line
• BHK baby hamster kidney cell line
• BHK 21C13 ATCC CCL 10.
• hamster cell lines with generation times in the range of, for example, 15 to 40 and in particular 20 to 30 hours can be made available, with which the glycoproteins mentioned can be stably expressed
• this performance is to be regarded as inventive in that it has so far been it has not yet been possible to cultivate hamster cell lines in protein-free medium without targeted genetic manipulation (genetic engineering).
• gene manipulation gene manipulation
• DHFR gene dihydrofolate reductase gene
• MTX methotrexate
• Gp 250 (220), gp 350 and / or gp 250 (220) / gp 350 can be obtained according to the invention by: (a) cultivating a cell line according to the invention and having the desired glycoproteins expressed,
• the cells are separated from the culture medium, in particular by microfiltration,
• the glycoprotein can be obtained natively and glycosylated by the process according to the invention. This gives them their natural antigenic potential.
• the vaccine obtained in this way accordingly has antigenic properties which correspond to those of the corresponding proteins of the active EB virus.
• the vaccine obtained is thus a safe vaccine with a high potential of antigenic protection without pathogenic potential.
• the recombinant vaccine is thus superior to an inactivated or weakened vaccine in terms of safety, since neither a potential for reversing weakened viruses nor the residual risk and the Toxicology of inactivated viruses are to be feared.
• a clone derived from the cell line CHO Kl ATCC CCL61, which lacks the dihydrofolate reductase gene (dhfr-1), with the plasmid pMDIIIGP according to Motz et al. in Gene, 44 (1986) 353-359, Motz et al. in Gene, 58, (1987) 149-154 and Vaccines, 87 (1987) 374-379.
• the cells were cultivated and selected with the aid of methotrexate (MTX) for clones which stably expressed gp 250/350 even after the inhibitor had been omitted.
• a clone with stable expression of gp 250/350 was designated as CHO C6.
• Example 2 Cultivation of CHO C6 in protein-free medium
• FCS fetal calf serum
• the point in time of the exchange was determined by the vitality and the state of the culture and was determined on a case-by-case basis, at the latest when the culture supernatant contained insufficient amounts of nutrients.
• a good guideline for a change is a glucose content of about 0.7 to 1 g / 1.
• a smaller part was exchanged to supplement decayed nutrient substances.
• the cells were cultivated in the spinner bottle until generation times of less than 40 hours were obtained and the culture grew in small spheroids. Passenging was carried out with a guideline value of about 0.5 to 1 g of residual glucose / 1 in the medium. Small spheroids and single cells were targeted. For this, the cell culture suspension was left to stand for a short time so that large spheroids could settle, after which the supernatant (100 g) was centrifuged. The cells in the pellet were then transferred to the next passage.
• the culture medium was conditioned by adding 20 to 30% by volume of medium from the previous passage, which had previously been freed from dead cells and cell fragments by means of filtration or centrifugation.
• Example 3 Processing and purification of the recombinant proteins from culture supernatants
• microfiltered harvests of fermentations in protein-free SMIF1 or SMIF2 were used, namely from continuously perfused culture up to the 2-1 scale or from batch culture in a 10-1 airlift fermenter.
• Filter cartridges with a nominal cut-off of 100 kD (Milipore or Filtron) were used for the filtration.
• Guide values during cross-flow ultrafiltration with a relatively free choice were: Transmembrane pressure not above approx.
• the total harvest volume was determined and about 15% by volume of filtrate (based on the volume flows under normal filtration conditions) was produced in order to condition the membrane, ie. H. to set a constant effective filtration exclusion limit by covering and polarizing the membrane.
• the buffer change and washing process of the primary retentate took place after the final volume was reached.
• the retentate was filled three to four times to twice the volume (including the dead volume of the pump and UF system) with phosphate buffer (20 mM of pH 7) and each time again concentrated to the minimum retentate volume and for the further processing planned. This step was carried out to remove low molecular weight foreign proteins and to reduce the ionic strength for the subsequent purification step.
• Buffer system Buffer A: 20 mM phosphate buffer (pH 5.1)
• Buffer B 20 mM phosphate buffer (pH 5.1) with 1 M NaCl
• the ultrafiltration retentate was diluted to a conductivity of less than 3 S with MiliQ process water and adjusted to a pH of 5.1 with HC1 (for example first to pH 4.2 in order to precipitate a larger proportion of foreign proteins and then to ⁇ back to pH 5.1).
• the resulting turbidity was precipitated by centrifugation. Then the clear supernatant was added via an FPLC pump with the addition of a 5 percent.
• Portion of Buffer B applied to the column to elute first foreign protein. The maximum loading was about 8 mg total protein / ml gel. After the application, the protein was eluted through a gradient up to 0.4 M NaCl (increase over 22 column volumes).
• the desired gp 250/350 protein eluted between 0.15 and 0.35 M NaCl content, the exact selection of the fractions to be purified further being decided after polyacrylamide gel electrophoresis (PAGE).
• PAGE polyacrylamide gel electrophoresis
• a protein application and a separation could also be achieved without the addition of buffer B and also at other pH values (eg pH 7).
• the low pH and the Zudo- Buffer B during the application increases the performance in relation to the gp proteins in chromatography.
• phosphate buffer was used, there was no need for a re-buffering step before the subsequent work-up steps.
• other buffer systems such as citrate buffers, are also possible.
• the selected and combined protein fractions from step II were concentrated by a factor of about 10 to 20 by ultrafiltration before application to a gel filtration column.
• the final protein concentrations were, for example, in the range of 1 to 2 mg / ml.
• Minicon CS15 chambers (Amicon) were used for the ultrafiltration. (For larger solution volumes, think of correspondingly larger filtration devices with larger capacities, for example stirred cells or crossflow ultrafiltration modules.)
• Buffer system Isocratic 100 or 200 mM NaCl in a 20 mM phosphate buffer (pH 7).
• Pure gp 250/350 can be stored in a stable manner at about -20 ° C. in the collected gp fractions of the gel filtration. Overall, about 25-40% of the crude product was obtained purely through the purification process.
• Example 2 was repeated with the exception that no cell confluence was permitted in the serum weaning phase and the FCS thinning was carried out with cell adherence removed. In this procedure, no cells that could be cultivated in a protein-free medium could be obtained.

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Citations (4)

• 1993
  • 1993-04-26 DE DE4313620A patent/DE4313620A1/de not_active Ceased
• 1994
  • 1994-04-26 AU AU67220/94A patent/AU6722094A/en not_active Abandoned
  • 1994-04-26 JP JP6523875A patent/JPH08509369A/ja not_active Ceased
  • 1994-04-26 WO PCT/EP1994/001318 patent/WO1994025599A1/de not_active Application Discontinuation
  • 1994-04-26 CA CA002161517A patent/CA2161517A1/en not_active Abandoned
  • 1994-04-26 CN CN94192251A patent/CN1124502A/zh active Pending
  • 1994-04-26 EP EP94915547A patent/EP0695357A1/de not_active Withdrawn
  • 1994-04-26 KR KR1019950704749A patent/KR100255228B1/ko not_active IP Right Cessation
  • 1994-04-26 NZ NZ266146A patent/NZ266146A/en unknown

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