EP0666907A1 - CELLULES DE CULTURE DE $i(QUILLAJA SP.) - Google Patents

CELLULES DE CULTURE DE $i(QUILLAJA SP.)

Info

Publication number
EP0666907A1
EP0666907A1 EP94901055A EP94901055A EP0666907A1 EP 0666907 A1 EP0666907 A1 EP 0666907A1 EP 94901055 A EP94901055 A EP 94901055A EP 94901055 A EP94901055 A EP 94901055A EP 0666907 A1 EP0666907 A1 EP 0666907A1
Authority
EP
European Patent Office
Prior art keywords
quillaja
dialysable
cultured cells
active substances
cells
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Withdrawn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
EP94901055A
Other languages
German (de)
English (en)
Inventor
Kristian Dalsgard
Max Henry
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.)
SEED CAPITAL INVESTMENTS(SCI) BV
Original Assignee
SEED CAPITAL INVESTMENTS(SCI) BV
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
Priority claimed from NL9202117A external-priority patent/NL9202117A/nl
Application filed by SEED CAPITAL INVESTMENTS(SCI) BV filed Critical SEED CAPITAL INVESTMENTS(SCI) BV
Priority to EP94901055A priority Critical patent/EP0666907A1/fr
Priority claimed from PCT/NL1993/000220 external-priority patent/WO1994010291A1/fr
Publication of EP0666907A1 publication Critical patent/EP0666907A1/fr
Withdrawn legal-status Critical Current

Links

Definitions

  • the present invention relates to cultured cells of Quillaja sp.. a method for preparing active substances from Quillaja sp.. various products comprising the active sub ⁇ stances, a method for preparing Immune-Stimulating COMplexes (ISCOM's) from the active substances and vaccins and adju ⁇ vants comprising the ISCOM's, the adjuvant ISCOM-matrix (L ⁇ vgren, K. thesis, ISBN 91-576-3202-2), the adjuvant Quil A (Dalsgaard, K. , Arch. ges. Virusforsch. 44, 243-254
  • ISCOM's Immune-stimulating complexes
  • IVS's Immune-stimulating complexes
  • IVS's are negatively charged pentagonal dodecahedra that form sponta- neously on mixing cholesterol and the saponins of Quillaja sp.. During their formation proteins and other lipids can be incorporated. ISCOM's have been found to strongly enhance immune responses and are therefore used as an immunological adjuvant and carrier/delivery system in e.g. vaccins.
  • Quil A, QS 21, and other saponin adjuvants are products derived from the natural bark having immunological adjuvant activity in a variety of vaccines.
  • Quil A is a mixture of the active substances (saponins) originating from the bark of the Quillaja sp. tree growing mainly in Chile.
  • the natural sources of Quillaja bark are limited. In fact, old trees are already rare today and yet about 1000 tons of bark per year are exported from Chile. Because the increasing demand for active substances (saponins) for various purposes a shortage of material is to be expected in the future.
  • the invention thus provides cultured cells of Quillaja sp. for the preparation of active substances from Quillaja sp.
  • the cultured cells may originate from a callus cell culture, wherein tissues or organs are grown on a solid medium.
  • the term "callus” refers to an amorphous lump of cells having lost their organ-forming capacity, and which lump is formed when a fragment of the plant body is tissue cultured on a solid medium. The so obtained callus shows an external form resembling the agglutination tissue of the plant body.
  • the cultured cells may also originate from a suspension cell culture.
  • tissue cell culture refers herein to a fine flocky dispersion of the cells formed when pieces of callus are further inoculated and cultured in a liquid medium under aerobic conditions.
  • various kinds of Quillaja plants can be used for tissue or suspension culture, for example Quillaja saponaria Molina, Quillaja sme ⁇ adermos, Quillaja brasiliensis and the like.
  • the conventional medium-compositions for tissue cultures mentioned in the literature for example, the media called White's medium (White P.R. Growth 2:53 (1943)), Hel ⁇ ler's Medium (Heller R. These Sc. Nat. Paris (1953)), Muras- hige and Skoog's medium (Murashige T. and Skoog F. Physiol. Plant. JL5:473 (1962)), Linsmaier and Skoog's medium (Lin- smaier E.M. and Skoog F. Physiol. Plant. 18., 100 (1965)), and Gamborg, Miller and Ojima's medium (Gamborg O.L. , Miller R.A. and Ojima K. , Exp.
  • Cell. Res. 5_0, 151 (1968) can be used in the present invention.
  • These known media consist of inorganic substances and other trace elements which have hitherto been used in the media for the water-culture method for plants, such as saccharide, auxins (growth-promoting substance) , cytokinins, vitamins and amino acids.
  • inorganic salts selected from potassium chloride, calcium chloride, potassium nitrate, calcium nitrate, sodium nitrate, ammonium nitrate, sodium nitrate, magnesium sulfate, potassium phosp ⁇ hate, sodium sulfate, magnesium nitrate, ammonium nitrate, sodium sulfate, magnesium sulfate, potassium phosphate, sodium phosphate, ferric chloride, ferric sulfate, NA 2 -EDTA (NA 2 -ethylenediamine tetra-acetic acid) , manganese sulfate, zinc sulfate, boric acid, potassium iodide, copper sulfate, sodium molybdats, aluminium chloride, cobalt chloride, and the like, saccharide selected from sucrose, glucose, fructo ⁇ se, mannose, and the like, auxins such as 2,4-dichlorophe- n
  • the plant body of Quillaja sp. plant, fragments of leaf, stem, root, flower, seed or other organs or tissues of the plant are washed, surface-sterilized, placed on the sterile agar medium for tissue-culture which is contained in a tube or flask plugged with cotton wool, cellulose or plastic cap and has one of the compositions as described in the above Table 1, and are incubated at 25- 30°C.
  • Said fragments or organs or tissues swell and white, yellowish-white or greenish-yellow callus is derived in 2-6 weeks.
  • Such callus can be gradually purified by means of repeating the similar solid medium-culturing, that is, by inoculating fresh solid medium by turns with small pieces of callus formed in the previous solid medium-culturing.
  • the callus thus reformed and refined on the solid medium by subculture in then inoculated into a liquid medium having one of the compositions as described in Table 1, and cultured on a shaker at temperature of 25-30°C for 2-3 weeks in order to obtain a suspension cell culture.
  • the inoculum is e.g.
  • the quantity of inoculum is one tenth of the quantity of whole medium, and intensive agitation is unfavorable because the membranes of Quillaja cells are broken thereby.
  • the amount of air to aerate is 0.2-30 liters/liter of medium/minute and the culturing period is 2-3 weeks, i.e. the same as that of the above shake-culture.
  • the yield of dried weight of these Quillaja cells is 30-35 percent of sugar consumed in the suspension culture and amounts to 6.9 g per liter of the medium.
  • the active substances prepared from the cultured cells of Quillaja sp. are mainly saponins.
  • Saponins are a type of glycosides widely distributed 1 in plants. Saponins consist of a sapogenin which constitutes the aglucon moiety of the molecule, and several sugars. The sapogenin is in this case a triterpene and the sugar moiety may consist of rhamnose, fucose, arabinose, xylose, galactose, glucose, glucuronic acid, and possibly other minor sugars.
  • the invention further relates to a method for preparing active substances from Quillaja sp. comprising the steps of: a) culturing cells from Quillaja sp. in vitro: and b) preparing a cell culture extract comprising the active substances.
  • dialy ⁇ sable refers to compounds removed from a dialysis sack after dialysing a crude extract of a Quillaja sp. cell culture against saline for about 24 hours.
  • non-dialysable refers to compounds retained in a dialysis sack after dialysing a crude extract of a
  • the dialysable fractions are also capable of binding ammonia.
  • the dialysable fraction from the active substances obtained from the cultured cells of Quillaja sp. therefore have properties which can be utilized for similar technical purposes as extracts of the natural bark. Examples of said technical purposes are their use as emulsifiers in food and beverages and in photographic film emulsions.
  • dialysable active fraction breaks down surface crusts and reduces ammonia- and odor formation in waste water plants, slurry tanks for liquid manure in pig production stables, slurry containers and the like, thus facilitating microbial and/or enzymatic breakdown and redu ⁇ cing odor of industrial-, household-, farm- and animal waste.
  • They may also be used as an additive to animal feeds to reduce odor of their excrements, and to increase the utilization of feed.
  • Their foaming ability renders them useful as additives in beverages, such as soft drinks, or as a foam producing agent in fire extinguishers. They may also be used as detergents in e.g. shampoos and the like.
  • the non-dialysable and possibly some of the dialysa ⁇ ble fraction contains the active substances that are useful as ISCOM-forming and adjuvant agents.
  • the substances are non-dialysable because they have a strong intrinsic charac- teristic of forming micelles. These micelles have the capa ⁇ city to complex with cholesterol and other lipids leading to ISCOM formation. But these properties also means that the retained substances are hemolytic to red blood cells such as SRBC (sheep red blood cells) . This hemolytic property can be used for their assay in the cell culture extracts.
  • the matrix of immune-stimulating complexes of the invention is preferrably constructed by: a) preparing a non-dialysable fraction from an extract of cultured cells of Quillaja sp. : b) adding at least one lipid and at least one detergent to the non-dialysable fraction; c) allowing the ISCOM's or ISCOM-matrix to form; and d) removing the detergent.
  • the ISCOM's thus prepared are very well suited to be used in various vaccins as immunological adjuvants.
  • the cultured cells of the pre'sent invention are advantageous in many respects for preparing active substan ⁇ ces of Quillaja sp..
  • the natural source for the immuno- logical and ISCOM-forming substances is a highly variable material, the individual components of which are difficult to separate.
  • the cultured cells of the invention are a much more reliable way of producing the active substances.
  • Anot- her advantage is that the culture cell extracts can be validated under good manufacturing principles.
  • the cultured cell products are free from many of the e.g. coloured sub ⁇ stance in the natural extract from the plant. The resulting product is much more homogeneous.
  • the cell cultures can be subcloned to establish cell lines which will produce individual substances rather than a group of related substances, which is the case in the natural plant.
  • one of the obtained suspension culture cells produce a much more restricted saponine pattern in HPTLC than the callus culture from which it is derived, which itself is already more restricted than natural extracts.
  • the cultured cells for preparing active substances will be cheaper when scaled up because the fermentation of plant suspension cell culture is straight forward and the medium is inexpensive in large volumes.
  • the active substances will be much easier to purify and validate than substances from the natural plant, because all parame ⁇ ters governing the production and purification can be moni- tered in a reliable way.
  • This medium was previously used with success for tissue culturing Saponaria officinalis and Gypsophilla paniculata.
  • To this basal medium were added two phytohormones: one auxin selec- ted from 2,4 D (2,4-dichlorophenoxyacetic) , NAA (naphtalene- acetic acid) or IBA (indolbutyric acid) in three concentra ⁇ tions: 10 "5 M, 10 "6 M or 10 '7 M; and one cytokinin selected from kinetine (K) or benzylaminopurine (BAP or BA) .
  • K kinetine
  • BAP benzylaminopurine
  • test-tubes 25 cm length and 2.5 cm width (diameter) . These tubes were put in a culture room at 25°C and a 12 hour photoperiod of classical white neons.
  • internodes are used directly instead of other parts of the stem (nodes and 1 leaves) because it was found by Japanese scientists in using Panax ginseng (Kubo et al, J. Nat. Prod. 1980, 43., 278) and in using Bupleurum falcatum (Tani et al, J. Chromatogr. 1986, 360. 407) and by the inventors in using Gypsophila paniculata (Henry et al, Phytochemistry 1991, 3i), 1819) that the saponin biosynthesis occurred in the phloem part of the stem in the plants that were studied.
  • both callus and suspension cell culture solids were lyophilized after harvesting.
  • the lyophilized solids were kept in the freezer at -20°C until use.
  • the plates were dried in a fume hood for 5 min. , sprayed by a mixture of concentrated sulp ⁇ huric acid in methanol 1:1, and heated in an oven for 10 min. at 120°C.
  • the separated bands were recorded densitome- trically using a 256 grey scale scanner and the Apple Macin ⁇ tosh software ScanAnalysis.
  • the results 1 are shown in Figure 4.
  • the positive suspension culture extract “L” herein also called “Ls” shows a substantially higher hemoglobin release than the low- producer suspension cell culture extract "K” and than the non-producer suspension cell culture “M” herein also called “Ms”.
  • the loyphilized callus culture "Lc” contained about 1% of hemolytic/ISCOM matrix forming substances.
  • the lyophilized suspension cell culture “Ls” contained about 0.5%.
  • the "Lc” suspension cell culture was produced from the “Lc” callus culture, and since both contained substantial amounts (about the same as in the natural bark) of hemolytic and ISCOM forming saponins, the extracts of these two forms of cultures were tested for immunological adjuvant activity.
  • a suspension cell culture extract "Ms” not producing these substances was included as a negative control.
  • the adjuvant Quil A extracted from the natural bark served as a positive control.
  • Group 2 + Quil A, 50 micrograms, positive control.
  • Group 3 + callus extract "Lc", 50 micrograms.
  • Group 4 + suspension extract "Ls", 50 micrograms.
  • Group 5 + suspension extract "Ms", 50 micrograms, negative control.
  • the present invention provides cultured cells of Quillaja sp. and a method for preparing active substances therefrom, which active substances may be used for various purposes such as ISCOM formation and as emulsifiers, deter ⁇ gents, foaming agents and the like.

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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention se rapporte à des cellules de culture de Quillaja sp. destinées à la préparation de substances actives à partir de Quillaja sp., telles que des saponines. Les cellules peuvent provenir soit d'une culture de tissus de callus, soit d'une culture de cellules en suspension. Les Quillaja sp. préférés sont des espèces choisies dans le groupe comprenant Quillaja saponaria Molina, Quillaja smegmadermos, Quillaja brasiliensis. De plus, l'invention se rapporte à des substances actives extraites de cellules de culture de Quillaja sp. et à des préparations comprenant ces substances actives, ou une fraction pouvant être dialysée ou non, à des procédés de préparation des substances actives et à divers agents, comprenant la fraction pouvant être dialysée et la fraction ne pouvant pas être dialysée d'un extrait de cellules de culture de Quillaja sp. et possédant diverses propriétes.
EP94901055A 1992-10-30 1993-10-29 CELLULES DE CULTURE DE $i(QUILLAJA SP.) Withdrawn EP0666907A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP94901055A EP0666907A1 (fr) 1992-10-30 1993-10-29 CELLULES DE CULTURE DE $i(QUILLAJA SP.)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
EP92203365 1992-10-30
EP92203365 1992-10-30
NL9202117A NL9202117A (nl) 1992-10-30 1992-12-07 Gekweekte cellen van Quillaja sp.
NL9202117 1992-12-07
EP94901055A EP0666907A1 (fr) 1992-10-30 1993-10-29 CELLULES DE CULTURE DE $i(QUILLAJA SP.)
PCT/NL1993/000220 WO1994010291A1 (fr) 1992-10-30 1993-10-29 CELLULES DE CULTURE DE $i(QUILLAJA SP.)

Publications (1)

Publication Number Publication Date
EP0666907A1 true EP0666907A1 (fr) 1995-08-16

Family

ID=27234655

Family Applications (1)

Application Number Title Priority Date Filing Date
EP94901055A Withdrawn EP0666907A1 (fr) 1992-10-30 1993-10-29 CELLULES DE CULTURE DE $i(QUILLAJA SP.)

Country Status (1)

Country Link
EP (1) EP0666907A1 (fr)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9410291A1 *

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