Saponin formation

Abstract

The present invention relates to a method for producing saponins containing quillaic acid triterpenoid aglycones, the method comprising at least the following steps: i) culturing plant cells capable of naturally synthesizing saponins containing quillaic acid triterpenoid aglycones in a cell culture medium containing a nitrogen source, ii) depleting the culture medium of any nitrogen source, iii) inducing the production of saponins using at least one inducer, and iv) recovering the produced saponins.

Description

[Technical field]

[0001] The present invention relates generally to the production of saponins in plant cell culture, and in particular to saponins containing quillaric acid triterpenoid aglycones. In particular, the present invention relates to plant cells capable of producing such saponins, methods of producing such saponins, and related aspects. [Background technology]

[0002] Saponins are triterpenoid glycosides. They have a wide range of applications, from fire extinguisher foams to food additives and immunostimulants (Reichert et al., 2019). Saponins have been of interest as immunostimulants for decades. Traditionally, saponins are purified from plants, such as the Quillaja saponaria Molina tree. For example, Quil A is a saponin preparation isolated from the South American tree Quillaja saponaria Molina, and was first described as having adjuvant activity by Dalsgaard et al. in 1974. Purified fractions of Quil A that retain adjuvant activity without the toxicity associated with Quil A have been isolated by HPLC (see, for example, EP03622789). Various fractions, such as fractions QS-7, QS-17, QS-18 and QS-21, have been found to have adjuvant activity, but their toxicity differs considerably. QS-18 is the most abundant saponin fraction (Kensil et al. 1991), while QS-7 and QS-21 have been found to be less toxic in mice. QS-21 is more abundant than QS-7, and the QS-21 fraction is the most widely studied saponin adjuvant (Ragupathi et al. 2011).

[0003] An example of an adjuvant formulation containing QS-21 is Adjuvant System 01 (AS01), a liposome-based adjuvant containing two immunostimulants, 3-O-desacyl-4'-monophosphoryl lipid A (3D-MPL) and QS-21 (Garcon, 2011; Didierlaurent, 2017). 3D-MPL is a non-toxic derivative of lipopolysaccharide from Salmonella minnesota. AS01 is included in vaccines against malaria (RTS,S - Mosquirix™) and varicella zoster (HZ / su - Shingrix™), as well as in several candidate vaccines. AS01 injection results in rapid and transient activation of innate immunity in animal models. Upon immunization, neutrophils and monocytes are rapidly recruited to the draining lymph nodes (dLN). In addition, AS01 inhibits MHCII high It induces the recruitment and activation of dendritic cells (DC), which is necessary for T cell activation (Didierlaurent et al., 2014). Some data are also available on the mechanism of action of the components of AS01. 3D-MPL signals via TLR4, stimulating NF-κB transcriptional activity and cytokine production, and directly activating antigen-presenting cells (APC) in both humans and mice (De Becker et al. 2000; Ismaili et al. 2002; Martin, 2003; Mata-Haro, 2007). QS-21 induces high antigen-specific antibody responses and CD8 +It promotes T cell responses (Kensil, 1998; Newman, 1992; Soltysik, 1995) as well as antigen-specific antibody responses in humans (Livingston, 1994). Due to its physical properties, it is believed that QS-21 may act as a danger signal in vivo (Lambrecht, 2009; Li, 2008). QS-21 has been shown to activate the ASC-NLRP3 inflammasome and subsequent IL-1β / IL-18 release (Marty-Roix, 2016), but the exact molecular pathways involved in the adjuvant effect of saponins have yet to be clearly defined. Another example of an adjuvant formulation that includes QS-7 is Matrix M (as part of a saponin fraction designated "fraction A" - see for example WO 2011 / 161151), an ISCOM-based formulation included in a vaccine against COVID-19 (Nuvaxovid™).

[0004] Extracts of Quillaja saponaria are commercially available and include its fractions of various purities, such as Quil A, Fraction A, Fraction B, Fraction C, QS-7, QS-17, QS-18 and QS-21. Such extracts are typically derived from harvesting the bark from the Quillaja saponaria tree. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] EP03622789 [Patent Document 2] WO 2011 / 161151 [Non-patent literature]

[0006] [Non-Patent Document 1] Reichert et al. in 2019 (“Quillaja Saponin Characteristics and Functional Properties”; Annu Rev Food Sci Technol. Mar 25;10, p43-73)

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[0007] Because current sources of saponin are dependent on natural resources, which may be limited, there is a need to develop alternative, sustainable methods of producing saponin that are less dependent on natural resources, such as producing saponin in plant cell culture.

[0008] WO 94 / 10291 discloses cultured cells of Quillaja saponaria and a method for preparing saponin for use as an active substance useful as an adjuvant. However, the inventors have observed that when using the method disclosed in WO 94 / 10291, not only is saponin not always produced, but even when produced, the levels achieved are low, unreproducible, and show some variability. Thus, there remains a need to develop a method for producing saponin in plant cell culture that is capable of producing high levels of saponin in a robust, reliable, and consistent manner. [Means for solving the problem]

[0009] Summary of the Invention In one aspect of the invention, a method for converting a non-producing plant cell naturally capable of synthesizing saponins containing quillaic acid triterpenoid aglycones into a saponin-producing plant cell comprising at least the following steps: i) culturing a plant cell naturally capable of synthesizing a saponin containing a quillaic acid triterpenoid aglycone in a culture medium containing a nitrogen source; ii) depleting the culture medium of any nitrogen sources; and iii) inducing the production of saponin with at least one inducer. In accordance with the present invention, there is provided a method (and cells obtainable by the method) comprising:

[0010] In another aspect of the present invention, there is provided a method for producing a saponin containing quillaic acid triterpenoid aglycone, comprising at least the following steps: i) culturing a plant cell naturally capable of synthesizing a saponin containing a quillaic acid triterpenoid aglycone in a culture medium containing a nitrogen source; ii) depleting the culture medium of all nitrogen sources; iii) inducing the production of saponin with at least one inducer; and iv) Recovering the produced saponin A method is provided, comprising:

[0011] In a further aspect of the invention, a suspension of plant cells is provided that is capable of naturally synthesizing a saponin containing a quillaric acid triterpenoid aglycone and producing the saponin at a volumetric productivity of at least 5 mg saponin per L of cell culture.

[0012] In a further aspect of the present invention, there is provided a suspension cell line of a plant cell capable of naturally synthesizing a saponin containing a quillaric acid triterpenoid aglycone and producing the saponin at a volumetric productivity of at least 5 mg saponin per L of cell culture.

[0013] In a further aspect of the invention, there is provided a method of preparing an adjuvant comprising a saponin, the method comprising the steps of: (a) preparing a saponin according to the method of the invention; and (b) formulating the saponin as an adjuvant. [Brief description of the drawings]

[0014] [Figure 1]FIG. 1 shows the evolution of proliferation (PCV%) of suspension cell lines of plant cells cultured in various culture media, each containing a different nitrogen source (as indicated), and then induced (as indicated). [Diagram 2] FIG. 2 shows the QS-18 volumetric productivity calculated from plant cell extracts obtained from one suspension cell line of plant cells after 3, 5, or 7 days of nitrogen deprivation (as indicated) followed by induction with various concentrations of MeJa (as indicated) for 2, 4, or 7 days. [Diagram 3] FIG. 3 shows the QS-18 volumetric productivity calculated from plant cell extracts obtained from one suspension cell line of plant cells after 5 days of nitrogen deprivation followed by induction with various concentrations of MeJa (as indicated) for 2, 4, or 7 days. [Figure 4] Figure 4 shows a comparison of QS-18 volumetric productivity calculated from plant cell extracts obtained from three different suspension cell lines of plant cells after 5 days of nitrogen deprivation followed by 4 days of induction with various concentrations of MeJa (as indicated). [Diagram 5] Panel (A) shows the HPLC-ELSD chromatogram of the QS-18 standard. A single peak is obtained at the retention time indicated. Panel (B) shows the high-resolution LCMS chromatogram using a QToF mass spectrometer. The saponin species contained and identified in the QS-18 standard (including the QS-18 saponin species) are named next to the peak corresponding to the retention time (RT) indicated. [Figure 6] Figure 6 shows the HPLC-ELSD chromatogram of the QS-21 standard. A single peak is obtained at the retention time shown. [Figure 7]Figure 7 shows an HPLC-ELSD chromatogram representing a plant cell extract obtained from one suspension cell line (CMC40B6) of plant cells cultured under conditions that allow for the production of saponins (e.g., herein, nitrogen starvation for 5 days followed by induction with 2.8 μM MeJa / PCV% for 4 days). A peak corresponding to the QS-17 family of saponins, a peak corresponding to the QS-18 family of saponins, and a peak corresponding to the QS-21 family of saponins are shown, with the retention time of each peak being similar to the retention time of the respective standard peaks. [Figure 8] FIG. 8 shows HPLC-ELSD chromatograms representing plant cell extracts obtained from one suspension cell line (CMC40B6) of plant cells cultured under conditions where saponin is not produced (e.g., herein, nitrogen deprivation for 5 days without subsequent induction). [Figure 9] FIG. 9 shows a comparison of QS-21 volumetric productivity calculated from plant cell extracts obtained from three different suspension cell lines of plant cells after 5 days of nitrogen deprivation followed by 7 days (CMC16B and CMC35A8) or 8 days (CMC40B6) of induction with various concentrations of MeJa (as indicated). [Figure 10] Figure 10 shows a comparison of QS-21 volumetric productivity calculated from plant cell extracts obtained from the CMC16B suspension cell line. Cells were cultured under various conditions: (i) no depletion / no induction, (ii) no depletion / induction, (iii) depletion / no induction, and (iv) depletion / induction (as indicated). [Figure 11] FIG. 11 shows the volumetric productivity of QS-21 calculated from plant cell extracts obtained from the CMC5B-1 suspension cell line after 5 days of nitrogen deprivation followed by 5 days of induction with 3.3 μM MeJa / PCV%. [Figure 12]Panel (A) shows a high-resolution LCMS chromatogram of the QS-21 standard using a QToF mass spectrometer. The QS-21 saponin species (including QS-21 1988) contained and identified in the standard are named next to the peak corresponding to the retention time (RT) shown. Panel (B) shows the LCMS-MS chromatogram for the content of QS-21 1988 (as A V1 and A V2 isomers) in the standard. [Figure 13] Figure 13 shows the QS-18 volumetric productivity calculated from plant cell extracts obtained from one suspension line of plant cells (CMC16B) after 5 days of nitrogen starvation followed by induction with 8 μM MeJa / PCV% for 4, 7, 10, or 14 days. A range of reduced concentrations of the nitrogen source are tested during the nitrogen starvation phase (as indicated). [Figure 14] Figure 14 shows UPLC / MS chromatograms using a QToF mass spectrometer for the detection of QS-7 1862. Comparison of plant cell extracts obtained from CMC16B suspension cell line cultivated under two different conditions: no depletion / no induction (panel A) and depletion / induction (panel B). [Figure 15] FIG. 15 shows the QS-21 volumetric productivity calculated from plant cell extracts obtained from the CMC40B6 suspension cell line (Panel A). The nitrogen source in the culture medium was allowed to be consumed naturally by the cells to residual levels, and then the cells were further maintained in the consumed medium for 5 more days, after which they were induced with 2 μM MeJa / PCV% for 7 days (D14). The levels of ammonium and nitrate as monitored in the culture medium during this experiment are shown in Panels B and C, respectively. Panel D shows the QS-21 volumetric productivity calculated from plant cell extracts obtained from the same culture of CMC40B6, but subjected to nitrogen depletion by replacing the culture medium with a culture medium without a nitrogen source and maintaining the cells in the medium for 5 days, after which the cells were induced under the same conditions (2 μM MeJa / PCV%, 7 days). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present inventors have developed culture conditions that allow for the production of saponins containing quillaic acid triterpenoid aglycones with improved yield and / or consistency, e.g., at least about 5-10 times higher than using conventional methods in the art (e.g., volumetric productivity of at least 10 mg per L of culture medium and up to 50 mg / L of saponin is achieved by the method of the present invention). Surprisingly, using these culture conditions, the production of saponins could be achieved even in plant cells that did not produce saponins using the methods of the prior art. Although it has been shown that in some plant cells that naturally synthesize some triterpenoid saponins, the production of such saponins can be triggered by induction (Yendo et al., 2010), the present inventors have observed that the physiological state of the plant cells is also an important factor in controlling saponin production. As a result, the inventors have developed a method suitable for the production of saponins containing quillaic acid triterpenoid aglycones, which comprises three distinct steps: (i) an expansion step aimed at providing a desired level of cellular biomass, (ii) a nitrogen depletion step aimed at increasing the susceptibility of the cells to subsequent induction, and (iii) an induction step aimed at triggering saponin production.

[0016] In the context of the present invention, the term "saponin" should be understood to refer to a triterpenoid glycoside whose triterpenoid core (or aglycone) is quillaic acid. Alternatively, such saponins may be referred to as "saponins containing quillaic acid triterpenoid aglycones."

[0017] In the context of the present invention, the term "plant cell culture" or "plant cell" should be understood as an in vitro culture of any plant tissue or any plant cell type. The plant cells used in the method of the present invention are derived from any plant that naturally synthesizes saponins containing Quillaja acid triterpenoid aglycones. The plant may belong to the Quillaja genus, for example, Quillaja saponaria or Quillaja brasiliensis species. Alternatively, the plant may belong to the Saponaria genus, for example, Saponaria vaccaria or Saponaria officinalis. In one embodiment, the method of the present invention uses plant cells derived from the Quillaja genus. In a further embodiment, the plant cells are derived from the Quillaja saponaria species. In a further alternative embodiment, the plant cells are derived from the Quillaja brasiliensis species.

[0018] The method of the invention is applicable to any kind of culture vessel, of any size and adapted to the type of cells to be cultured, such as, for example, a petri dish, a shake flask, or a bioreactor. The bioreactor may comprise a disposable bioreactor, typically comprising a plastic bag, or a non-disposable bioreactor, such as a stainless steel bioreactor. In one embodiment, a disposable bioreactor for the culture is used. In an alternative embodiment, a non-disposable bioreactor is used. In yet an alternative embodiment, a shake flask is used.

[0019] Conventional culture media known for plant cell culture, such as classical Murashige and Skoog (MS) medium, can be used in the method of the present invention. These media typically contain at least one or more macronutrients, e.g. selected from NH4NO3, KNO3, CaCl2, MgSO4, KH2PO4, NH4Cl, or KCl; at least one or more micronutrients, e.g. selected from KI, H3BO3, MnSO4, ZnSO4, Na2MoO4, CuSO4, CoCl2, DeSO4, or Na2EDTA; at least one or more micronutrients, e.g. myo-inositol, nicotinic acid, pyridoxine-HC1, nicotinic acid, pyridoxine-HC2, nicotinic acid, pyridoxine-HC3, nicotinic acid, pyridoxine-HC4, nicotinic acid, pyridoxine-HC5, nicotinic acid, pyridoxine-HC6, nicotinic acid, pyridoxine-HC7, nicotinic acid, pyridoxine-HC8, nicotinic acid, pyridoxine-HC9, nicotinic acid, pyridoxine-HC1, nicotinic acid, pyridoxine-HC1, nicotinic acid, pyridoxine-HC2 ... at least one or more vitamins selected from, for example, thiamine-HCl, thiamine-HCl, or thiamine-HCl; optionally one or more amino acids, such as glycine; at least one or more carbon sources, for example, selected from sucrose, glucose, or fructose; and at least one or more plant hormones, for example, selected from one or more cytokinins, such as 6-benzylaminopurine (BA), or one or more auxins, such as 2,4-dichlorophenoxyacetic acid (2,4-D) and / or 1-naphthaleneacetic acid (NAA).

[0020] Supplementation of cells undergoing growth or active biosynthesis, such as during production of saponins, with fresh culture medium, or selected nutrients that may be consumed, may also enhance production and / or be necessary, for example, supplementation of carbon and / or phosphate sources may be useful in the methods of the invention.

[0021] It is contemplated that the amount of medium exchanged or replenished, the frequency of exchange, and the composition of the replenished medium can be varied according to various embodiments of the present invention. This can vary depending on the stage of the method of the present invention. Replenishment may be performed in a continuous, semi-continuous, or fed-batch mode. In a fed-batch process, certain medium components, such as selected nutrients, are periodically or continuously supplied. Suitably, a significant portion, but not all, of the contents of the batch culture is replaced by fresh medium for continued cell growth and saponin production. Alternatively, the process is "continuous", i.e., fresh medium is continuously supplied and run-off medium is continuously or repeatedly removed. In one embodiment, fresh culture medium or replenishment of selected nutrients is supplied in the method of the present invention by fed-batch, for example during step i), during step ii), and / or during step iii) of the method of the present invention.

[0022] The method of the invention is applicable to any kind of plant material cultured in vitro, whether cultured in suspension in liquid medium or on solid medium (e.g. callus), cells, tissues or organs of a given plant body, such as archaeons, leaves, stems, hairy roots, internodes, cambium, etc. In one embodiment, the plant cells used in the method of the invention are derived from the cambium, e.g. cambium meristem cells (CMCs). In an alternative embodiment, the plant cells are derived from hairy roots.

[0023] The plant cells used in the method of the invention may be callus, e.g. derived from the cambium of a plant. In the context of the present invention, "callus" should be understood as a cluster of dedifferentiated cells cultured on a solidified medium. The generation of callus can be achieved from any plant tissue explant by any method known to the skilled artisan, e.g. the methods described in WO 94 / 10291, US 2019 / 0134128 or WO 15 / 082978. Typically, tissue explants from small sized plants can be surface sterilized, e.g. by washing thoroughly with clean water, using a disinfectant such as hypochlorite, using a wetting agent such as Tween or Triton, using an antibiotic and / or using an antifungal agent. The surface sterilized explant is then typically placed on the surface of a solidified medium such as agar and incubated in a sterile environment until the mass of undifferentiated cells grows (typically between 2 and 12 weeks, e.g. 8 weeks) in a manner similar to the plant source material. The callus may be gradually purified and further propagated by repeating the same solid medium culture, i.e., by successively inoculating small pieces of the callus formed in the previous solid medium culture into fresh solid medium, for example, every 4 weeks. In one embodiment, the callus is cultured in the presence of the hormones 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BA), for example at 0.5 mg / L.

[0024] The callus thus formed and purified on solid medium by subculture can be inoculated and cultivated in liquid medium to obtain a suspension cell culture. The terms "suspension plant culture" and "suspension of plant cells" are interchangeable and refer to an in vitro culture of plant cells dispersed in a liquid medium. The method of the invention is particularly suitable for suspension plant cultures or suspension of plant cells. Thus, in one embodiment, the plant cells for use in the method of the invention are grown in suspension in a liquid medium. In the context of the present invention, the term "cell line" refers to a plant cell derived from a given callus and adapted to grow in suspension in a liquid culture medium. Different suspension cell lines can be established from a given callus. To obtain cells in suspension culture, such as the suspension cell line of the plant cells of the invention, the cells are for example removed from the callus and transferred to a sterile culture vessel containing a nutrient culture medium. It is understood that the medium optimized for the suspension cell line may be different from the optimal medium for the callus. It is within the skill of the art to determine the suitable and optimal culture medium.

[0025] The transition from callus to suspension cell lines is also known to those skilled in the art (for example, as described in WO 94 / 10291 or US 2019 / 0134128). The inventors have observed that the use of conditioned medium (i.e., culture medium in which some cells have previously grown and therefore contains components secreted by the previous cells) and / or phytosulfokine alpha (PSK) can aid in the transition from callus to suspension cultures. Thus, in one embodiment, conditioned medium and / or PSK are included in the culture medium when transitioning from callus to suspension cell lines and / or when subculturing suspension cell lines. Once initiated and adapted to growth in suspension, the suspension cell lines can be subcultured or propagated, for example by dilution, for example every 4 weeks, to keep them in a state of growth and proliferation, prior to step i) of the method of the invention.

[0026] Step i) - culturing plant cells in a cell culture medium comprising a nitrogen source. In one embodiment, the plant cells in step i) of the method of the invention are callus. In a preferred embodiment, the plant cells in step i) of the method of the invention grow in suspension or are a suspension cell line.

[0027] The plant cells in step i) are cultured and maintained in conditions that allow proliferation and growth until the desired cell biomass is achieved. This is the expansion phase. For example, if the plant cells grow in suspension or are suspension cell lines, the cell biomass may be assessed by measuring the PCV. The term "PCV" stands for Packed Cell Volume and refers to the volume that the cells occupy in the culture medium. It may be calculated as follows: PCV(%)=(Volume of cell pellet / Volume of sample)×100. A preferred PCV range achieved at the end of step i) may be between 10%-70%, more preferably between 20%-60%, even more preferably between 30%-50%, for example about 40%. In other words, the plant cells in step i) may be cultured until a PCV in the range of preferably between 10%-70%, more preferably between 20%-60%, even more preferably between 30%-50%, for example 40% is reached. In one embodiment, the PCV at the end of step i) is about 15%, about 20%, about 30%, about 40%, about 50%, or about 60%. Alternatively, the above PCV ranges or values ​​can be achieved by appropriate dilution of the plant cells cultured and maintained in step i) before starting step ii). For example, the plant cells cultured in a culture medium containing a nitrogen source (as described below) in step i) are centrifuged and the desired cell biomass is directly resuspended in a culture medium containing no or a reduced nitrogen source to obtain the desired PCV range or value at the start of step ii).

[0028] The duration of step i) may vary from cell line to cell line depending on the proliferation rate of the cell line and depending on the desired PCV range or value to be reached. Preferably, step i) may last for 4 to 8 days, more preferably 5 to 7 days, even more preferably 4 to 5 days or longer.

[0029] Suitable culture media for use in step i) may be variations of classical MS medium, such as increasing the concentration of a phosphate source (e.g., KH2PO4) and / or modifying the sugar balance (e.g., glucose and fructose versus sucrose).

[0030] In some embodiments, the culture medium in step i) comprises at least between 2 mM and 4 mM, or between 0.6 mM and 5 mM, or between 1.5 mM and 5 mM KH2PO4. In further embodiments, the culture medium in step i) comprises at least about 2.5 mM or about 1.25 mM KH2PO4.

[0031] In the context of the present invention, the term "nitrogen source" refers to nitrates (i.e., NO 3- A source of ions, e.g., KNO3 or NH4NO3) and / or ammonium (i.e., NH 4+ The source of the ion includes, for example, NH4Cl or NH4NO3.

[0032] Both nitrate and ammonium are known to support plant cell growth.

[0033] Advantageously, the nitrogen source in the culture medium in step i) suitably comprises at least a nitrate, such as KNO3. In some embodiments, the nitrogen source comprises at least KNO3. In further embodiments, the nitrogen source comprises at least KNO3 and NH4NO3. In further embodiments, the nitrogen source may optionally comprise NH4Cl. In further embodiments, the nitrogen source does not comprise NH4Cl as the only nitrogen source.

[0034] The total concentration of nitrogen sources in the culture medium in step i) may be in the range of 10 mM to 50 mM, preferably 15 mM to 40 mM, more preferably 20 mM to 30 mM, for example about 25 mM, about 30 mM or about 40 mM. The concentration of KNO3 (if present) may be in the range of 5 mM to 30 mM, preferably 10 mM to 20 mM, more preferably about 15 mM or about 20 mM. The concentration of NH4NO3 (if present) may be in the range of 5 mM to 30 mM, preferably 10 mM to 20 mM, more preferably about 10 mM or about 20. The concentration of NH4Cl (if present) may be in the range of 5 mM to 30 mM, preferably 5 mM to 20 mM, more preferably 10 mM to 20 mM, more preferably about 10 mM or about 15 mM.

[0035] The carbon source in the culture medium in step i) may be one or more of sucrose, glucose and fructose, and in particular may be a combination of sucrose, glucose and fructose. The total concentration of the carbon sources may be in the range of 40 mM to 100 mM, preferably 50 mM to 90 mM, more preferably 60 mM to 80 mM, for example about 60 mM or about 70 mM. The concentration of sucrose (if present) may be in the range of 5 mM to 100 mM, preferably 10 mM to 80 mM, more preferably 20 mM to 60 mM, for example about 10 mM. The concentration of glucose or fructose (if present) may be in the range of 5 mM to 60 mM, 15 mM to 60 mM, preferably 10 mM to 80 mM, more preferably 20 mM to 40 mM, for example about 30 mM or about 60 mM. In one embodiment, the culture medium in step i) comprises at least glucose at a concentration in the range of 5 mM to 60 mM, 15 mM to 60 mM, 10 mM to 80 mM, 20 mM to 40 mM, or about 30 mM, or about 60 mM.

[0036] The inventors have observed that controlling the osmolality of the culture medium in step i) is advantageous for subsequent saponin production. A preferred osmolality range to be maintained during step i) and / or step ii) and / or step iii) (preferably during all three steps) of the method of the invention may be between 100 and 220 mOsm, more preferably between 180 and 200 mOsm. Preferably, the osmolality is below 200 mOsm.

[0037] Osmolality may be controlled by the carbon source included in the culture medium. An osmolality between 180 and 200 mOsm may be achieved, for example, by setting a target glucose concentration in the culture medium at 60 mM. The level of glucose in the medium may be continuously or periodically monitored and adjusted. The inventors have observed that classical media, such as MS medium, typically containing about 80 mM sucrose, result in peaks of osmolality higher than 200 mOsm. Thus, in some embodiments, the culture medium used in step i) of the method of the invention preferably contains between 2.5 mM and 40 mM sucrose, more preferably between 5 mM and 20 mM sucrose, for example 10 mM. Alternatively, the culture medium used in step i) does not contain sucrose.

[0038] In some embodiments, the medium used in step i) comprises one or more hormones selected from auxins and / or cytokinins. Preferably, the medium used in step i) comprises one or more hormones selected from NAA, 2,4-D and BA. More preferably, the medium used in step i) comprises at least 2,4-D. In some embodiments, the medium used in step i) comprises NAA and 2,4-D. Preferably, the concentrations of NAA and / or 2,4-D in the medium used in step i) may be between 0.2 mg / L and 0.8 mg / L, for example, they may be about 0.4 mg / L, about 0.5 mg / L, or about 0.6 mg / L. In further embodiments, the medium used in step i) comprises NAA, 2,4-D and BA.

[0039] In some embodiments, the culture medium of step i) comprises further micronutrients and / or vitamins. Suitably, the further micronutrients are one or more of KI, H3BO3, MnSO4, ZnS04, Na2MoO4, CuSO4, CoCl2, DeSO4, or Na2EDTA, and the vitamins are one or more of myo-inositol, nicotinic acid, pyridoxine-HCl, or thiamine-HCl.

[0040] In a further embodiment, the culture medium in step i) comprises CaCl2 and / or MgSO4. Preferably, in the medium in step i), the concentration of CaCl2 is 1 to 5 mM, such as 2 mM to 4 mM, for example about 3 mM. Preferably, in the medium in step i), the concentration of MgSO4 is 0.5 to 3 mM, such as 1 mM to 2.5 mM, for example about 1.5 mM or about 2 mM.

[0041] Examples of suitable culture media to be used in step i) are medium 4 or medium 6 as described in the Examples section (the composition of which is provided in Table 1 below). In some embodiments, the culture medium in step i) is medium 4 or is medium 6. The composition of the culture medium in step i) may require some adaptation to different plant cells or cell lines. For example, the inventors have observed that controlling the levels of nutrients such as glucose and phosphate affects saponin production. Advantageously, the target for the glucose concentration in the culture medium in step i) may be between 40 mM and 70 mM, e.g. 60 mM, and / or the target for the phosphate concentration may be between 1 mM and 5 mM, e.g. 2.5 mM or 5 mM. Such levels of glucose and / or phosphate concentration are further advantageously targeted in the culture medium of step ii) and / or in the culture medium of step iii). It is within the skill of the art to adjust the concentration of nutrients in the culture medium in either step i), step ii) or step iii) by monitoring the concentration and consumption of the nutrients at any given time, for example by sampling the plant cells in the cell culture method by any method known in the art and measuring the concentration of the nutrients.

[0042] A facile method that we have used to assess whether any changes in culture conditions subsequently affect saponin production is to examine the ability of plant cells cultured in any given condition to produce foam after nitrogen depletion, induction and mechanical disruption of the cells (e.g., as described below). Indeed, saponin is also known for its detergent activity. Thus, if the cells are producing saponin, some foam will be visible after mechanical disruption. The occurrence and observation of such foam appears to correlate with the production of saponin, as confirmed by subsequent measurement of saponin using suitable analytical methods (as described below). This offers the advantage of an easy readout (visible to the naked eye) that predicts the final desired result. By sampling a small amount of suspended plant cells at various time points and various conditions and then examining the production of foam, the production of saponin can be predicted.

[0043] In the method of the present invention, the plant cells may be cultured at any temperature known to be suitable for plant cell culture, which can be adjusted by the skilled artisan. For example, the temperature may be in the range of 15°C to 35°C, preferably in the range of 20°C to 30°C, such as 25°C. In one embodiment, the method of the present invention is operated at about 25°C.

[0044] In the case of suspension cell culture, the plant cell culture may be agitated. A preferred range of agitation is between 30 rpm and 80 rpm, more preferably between 40 and 60 rpm, and even more preferably about 50 rpm. In one embodiment, the method of the invention is operated at about 50 rpm.

[0045] Step ii) - Depleting the culture medium of all nitrogen sources. While searching for the appropriate conditions to induce saponin production, the inventors observed that in order to achieve optimal saponin production, the physiological state of the cells is important prior to inducing saponin production.

[0046] The inventors have observed that depleting any nitrogen source from the culture medium used in step i) prior to inducing the cells to produce saponin results in an appropriate physiological state of the cells, which results in increased yields of saponin after subsequent induction (e.g., as described below). Without wishing to be bound by theory, it is believed that nitrogen depletion alters the physiological state of the cells, which makes them more responsive to subsequent induction.

[0047] As described above, it has been reported that nitrogen source is important for the growth of plant cells cultured in vitro. However, surprisingly, the inventors have observed that plant cells can grow in the absence of nitrogen source or in the presence of a reduced concentration of nitrogen source. Furthermore, surprisingly, the inventors have observed that culturing plant cells in the absence of nitrogen source or in the presence of a reduced concentration of nitrogen source (or allowing the cells to naturally consume the nitrogen source present in the culture medium) before inducing the cells promotes the subsequent production of saponin. This is the nitrogen depletion step. Although nitrogen depletion may not be sufficient to obtain saponin production, the inventors have observed that it is a necessary condition for obtaining saponin production after induction.

[0048] Thus, the term "depleting the culture medium of any nitrogen source" means in the context of the present invention reducing the level of any nitrogen source contained in the culture medium in step i) and maintaining the cells in said culture medium with a reduced level of the nitrogen source.

[0049] Nitrogen depletion may be performed by (i) allowing the cells to naturally consume the nitrogen source contained in the culture medium in step i) to a residual level without further supplementing the culture medium with the nitrogen source (i.e., "natural depletion"), and / or (ii) by replacing the culture medium at the end of step i) with a culture medium without the nitrogen source or with a culture medium containing a reduced concentration of the nitrogen source. It is within the skill of the art to monitor and measure the residual level of the nitrogen source in the culture, especially in the case of natural depletion, in order to determine the optimal duration of step ii).

[0050] In the context of the present invention, the term "reduced concentration of nitrogen source" should be understood to relate to the concentration of the nitrogen source used during step i), i.e. the reduced concentration in the replacement culture medium or in the spent culture medium during step ii) is lower. Preferably, the reduced concentration of the nitrogen source in the culture medium during step ii) is between 0 mM and 5 mM, or between 1.25 mM and 5 mM, and may be about 1.25 mM, about 2.5 mM or about 5 mM, and the nitrogen source may be one or more of KNO3, NH4NO3 and NH4Cl. If the nitrogen source is NH4Cl, the reduced concentration is preferably about 1.25 mM or about 2.5 mM.

[0051] Preferably, at the end of step ii) and before inducing the cells, the concentration of the nitrogen source in the culture medium is reduced by a factor of 2, more preferably by a factor of 4, even more preferably by a factor of 8, compared to the concentration of the nitrogen source contained in the culture medium used in step i), e.g. the culture medium described in the previous section.

[0052] In some embodiments, at the end of step ii) and before inducing the cells, the residual level of the nitrogen source in the culture medium is less than 10 mM, less than 5 mM, less than 2.5 mM, or less than 1 mM. In further embodiments, at the end of step ii), the residual level of the nitrogen source in the culture medium is between 1 mM and 2 mM.

[0053] In some embodiments, at the end of step ii) and before inducing the cells, the residual level of nitrate in the culture medium is less than 5 mM, less than 2 mM, less than 1.5 mM, or less than 1 mM. In further embodiments, at the end of step ii), the residual level of nitrate in the culture medium is between 0.5 mM and 1.5 mM. In yet other embodiments, the level of nitrate in the culture medium is undetectable.

[0054] In some embodiments, at the end of step ii) and before inducing the cells, the residual level of ammonium in the culture medium is less than 5 mM, less than 2 mM, less than 1.5 mM, less than 1 mM, or less than 0.5 mM. In further embodiments, at the end of step ii), the residual level of ammonium in the culture medium is between 0.5 mM and 1.5 mM. In yet other embodiments, the level of ammonium in the culture medium is undetectable.

[0055] The residual level of the nitrogen source in the culture medium may be measured by any method known in the art. If the nitrogen source contains nitrates, such as KNO3 and / or NH4NO3, the residual level can be measured by measuring the NO3 in the culture medium using, for example, a colorimetric-based assay that relies on an enzymatic reaction that converts nitrates to nitrites, producing a colored compound that can be quantified using a spectrophotometer. - If the nitrogen source contains ammonium, such as NH4Cl and / or NH4NO3, residual levels may be assessed by measuring the presence of NH4Cl in the culture medium using, for example, a colorimetric-based assay that relies on an enzymatic reaction to convert ammonium into a colored compound that can be quantified using a spectrophotometer. + It may be assessed by measuring the presence of ions.

[0056] It will be clear to the skilled artisan that the transition between steps i) and ii) of the method of the invention may differ depending on how nitrogen depletion is performed, e.g. by replacing the culture medium in step i) or by natural depletion, i.e. by allowing the cells to consume the nitrogen source contained in the culture medium in step i) to residual levels without further replenishing the culture medium with the nitrogen source (see the figures at the top of Example 3 and in Experiment 10 in the Examples section, respectively, which provide schematic diagrams for illustrative purposes only).

[0057] When nitrogen depletion is performed by replacing the culture medium in step i) with a culture medium that does not contain a nitrogen source or with a culture medium that contains a reduced concentration of the nitrogen source, the replacement marks the start of step ii). For example, if the plant cells grow in suspension, the suspension may be centrifuged and the culture medium replaced with a culture medium that does not contain a nitrogen source or with a reduced concentration of the nitrogen source (e.g., the reduced range or value described previously). The inventors have observed that in order to obtain saponin production during step iii), it is not necessary for the nitrogen source in the replacement culture medium in step ii) to be completely absent. An example of a suitable replacement culture medium to be used in step ii) is Medium 1 (whose composition is provided in Table 1 below), as described in the Examples section. In some embodiments, the replacement culture medium in step ii) is Medium 1. Alternatively, the composition of the replacement culture medium in step ii) may be a variation of Medium 1 and may be as described in the previous section regarding "step i"), apart from the composition of the nitrogen source. The nitrogen source in the replacement culture medium in step ii) is preferably as described previously in this section. In some embodiments, the replacement medium comprises one or more hormones, one or more nutrients, and / or one or more vitamins, as described previously in the section relating to "step i"). If the replacement culture medium does not comprise a nitrogen source or does not comprise KNO3, then preferably KCl is added as a potassium source. Thus, in some embodiments, the replacement culture medium comprises KCl, for example at a concentration in the range of 5-30 mM, 10-20 mM, for example about 15 mM.

[0058] When nitrogen depletion is performed by replacing the culture medium in step i) (e.g., by centrifugation) with a culture medium that does not contain a nitrogen source or with a culture medium that contains a reduced concentration of a nitrogen source, the cells cultured in step i) are centrifuged and the given cell biomass is directly resuspended in the replacement culture medium to obtain the desired PCV. Preferably, the PCV ranges between 10% and 40%, more preferably between 15% and 30%, even more preferably between 25% and 30%, for example about 15%, about 20% or about 30%. Alternatively, the cells are cultured in step i) until such a PCV range or value is reached, and the culture medium is simply replaced with a replacement culture medium that does not contain a nitrogen source or contains a reduced concentration of a nitrogen source. In other words, in some embodiments, the range or value of the PCV at the end of step i) and at the start of step ii) is the same.

[0059] When nitrogen depletion is carried out by natural depletion, the last replenishment of the culture medium in step i) marks the start of step ii). Then, during step ii), the cells are allowed to naturally consume the nitrogen source contained in the culture medium to a residual level by culturing the cells in the same culture medium. Thus, the culture medium in step ii) may be the culture medium previously described in the section on "step i") in the case of natural depletion.

[0060] Thus, when nitrogen depletion is performed by natural depletion, the range or value of the PCV at the start of step ii) may be the same as the range or value at the end of step i), i.e. preferably between 10% and 40%, more preferably between 15% and 30%, even more preferably between 25% and 30%, for example about 15%, about 20% or about 30%. Alternatively, the cells may be cultured in step i) until a given range or value of the PCV is reached and then diluted with fresh culture medium (final replenishment), so that the range or value of the PCV at the start of step ii) (after dilution) may be lower than at the end of step i).

[0061] Nitrogen depletion by replacing the culture medium in step i) with a culture medium that does not contain a source of nitrogen source may have the advantage of shortening the duration of step ii). Since the nitrogen source is not present from the beginning of step ii), no time is required to reach a residual level (compared to natural depletion). However, especially at large scale, when using bioreactors, operating a large volume of bioreactors is more restrictive and it is not always possible to easily remove the entire culture medium. Furthermore, when operating a process at large scale, there is a desire to reduce the number of operations required during the process. Natural depletion may then be advantageous in such large scale situations.

[0062] It will also be apparent to those skilled in the art that the duration of step ii) may therefore vary depending on how the nitrogen depletion is carried out.

[0063] Preferred ranges for the duration of step ii) are 1-9 days, 2-7 days, 5-7 days, more preferably 3-6 days, e.g. 4 days, 5 days or 7 days. These ranges are particularly preferred when nitrogen depletion is performed by replacing the culture medium in step i) with a culture medium that does not contain a source of nitrogen source or with a reduced concentration of the nitrogen source. In some embodiments, after replacing the culture medium at the end of step i) with a culture medium that does not contain a source of nitrogen source or with a reduced concentration of the nitrogen source, the cells are maintained in the replacement culture for 1-9 days, 2-7 days, 5-7 days, 3-6 days, 4 days, 5 days or 7 days.

[0064] Alternatively, preferred ranges for the duration of step ii) are 5-20 days, more preferably 6-19 days, even more preferably 7-18 days, 8-16 days, 9-15 days, e.g. 10, 11, 12, 13 or 14 days. These ranges are particularly suitable when nitrogen depletion is performed by natural depletion. In some embodiments, after natural depletion of the nitrogen source to residual levels, the cells are maintained in the depleted medium for 1-9 days, 2-7 days, 5-7 days, 3-6 days, 4 days, 5 days, or 7 days.

[0065] It is within the skill of the art to determine the optimal duration of step ii) depending on the plant cell culture used and / or the type of depletion used. As described previously, a convenient method used by the inventors is to examine the ability of plant cells to produce foam in any given condition after nitrogen depletion, induction and mechanical disruption of the cells (e.g., as described below). By sampling small amounts of plant cells at various times after nitrogen depletion and subsequent induction (testing various depletion conditions and / or various depletion periods), and then examining foam production, saponin production can be predicted.

[0066] During the development of the method of the present invention, the inventors occasionally observed "starvation" of the cells in glucose and / or phosphate during the nitrogen depletion stage. This may have a negative impact on the level of saponin production after subsequent induction of the cells. Therefore, during step ii), the glucose and / or phosphate levels in the culture medium may be advantageously monitored and replenished, if necessary. For example, when the culture medium reaches a residual level of glucose of 15 mM or less, the culture medium may be fed with a solution of 60 mM glucose. Similarly, when the culture medium reaches a residual level of phosphate of 0.6 mM or less, the culture medium may be fed with a solution of 2.5 mM phosphate. Feeding may be performed periodically or continuously. Since the consumption rate of nutrients may vary from cell line to cell line and from depletion condition to depletion condition, it is within the skill of the art to determine the optimal conditions and modes of nutrient feeding, e.g. glucose or phosphate, for each cell line and each depletion condition.

[0067] Step iii) - Inducing the production of saponin using at least one inducer. Saponins are naturally occurring, structurally and functionally diverse phytochemicals that are widely distributed in plants. Saponins are generally believed to play an important role in plant defense against pathogens, pests and herbivores due to their antimicrobial, antifungal, antiparasitic and insecticidal properties (refs). Many plants synthesize and accumulate saponins during normal growth and development. The distribution of these natural products varies widely among plant species, individual plants, organs and tissues during development and maturation, and shows seasonal variations. Some studies suggest that variations in the distribution, composition and amount of saponins in plants may reflect variations in the need for plant protection. In some plant species, the production of saponins is induced in response to biotic stresses, including herbivore and pathogen attack. Abiotic stress factors such as humidity, nutrient starvation, light and temperature can affect both the quality and quantity of saponin content. Increased saponin levels in response to stress are often mediated by transcriptional activation of biosynthetic genes through a complex signaling cascade involving the hormones jasmonate and salicylate. Thus, the biosynthesis of these molecules can be induced using elicitors, and this feature has been exploited in several plant species to improve saponin yields (Yendo et al., 2010).

[0068] However, the inventors have observed that induction may not be sufficient to consistently obtain high yields of quillaic acid-based triterpenoid saponins, and a prior step of nitrogen depletion (as described previously) is required to obtain high yields.

[0069] Thus, induction is carried out after the plant cells have been depleted of all nitrogen sources as previously described.

[0070] Suitable inducers for use in the present invention are monocarboxylic acid compound type inducers, such as 5-chlorosalicylic acid, salicylic acid, acetylsalicylic acid, methyl esters, such as methyl jasmonate (MeJa), or chemically synthesized 2-HEJ. In one embodiment, at least the inducer used in step iii) is MeJa.

[0071] In one embodiment, a preferred range for the concentration of the at least one inducer in step iii) is in the range of 0.5-12 μM, more preferably 1-8 μM, even more preferably 2-6 μM, even more preferably 3-5 μM. Other preferred concentrations are 1-3 μM, for example about 2 μM. In some embodiments, the at least inducer used in step iii) is MeJa, used at a concentration in the range of 1-3 μM. In further embodiments, the at least inducer used in step iii) is MeJa, used at a concentration in the range of 2-6 μM, for example about 2 μM, about 3 μM, or about 6 μM.

[0072] While inducer concentrations are typically referred to in terms of the volume of culture medium, an alternative method for defining concentration used by the inventors is in terms of % PCV.

[0073] A preferred range for the inducer, e.g. MeJa, for use in step iii) is 0.5-12 μM / PCV%, more preferably 1-8 μM / PCV%, even more preferably 2-6 μM / PCV%, even more preferably 3-5 μM / PCV%. A particularly preferred range for MeJa is 1-3 μM / PCV%, e.g. 2 μM / PCV%. In some embodiments, at least the inducer used in step iii) is MeJa used in a concentration in the range of 1-3 μM / PCV%. In a further embodiment, at least the inducer used in step iii) is MeJa used in a concentration in the range of 2-6 μM / PCV%, e.g. 2 μM / PCV%, 3 μM / PCV% or 6 μM / PCV%.

[0074] The inducer may be added directly to the culture medium. Thus, the inducer may be added directly to the culture medium at the end of step ii), which marks the start of step iii). The inducer may be added once or may be added further, for example every other day during the period of step iii). Alternatively, the culture medium may be replaced by a culture medium containing the inducer(s) at the end of step ii), which marks the start of step iii). Such a culture medium containing the inducer may advantageously further contain nutrients that may have been consumed, such as a carbon source or a phosphate source, but does not contain a nitrogen source.

[0075] It is within the skill of the art to monitor nutrient consumption levels (eg, by sampling) along the various stages of the methods of the invention and replenish nutrients that may have been consumed.

[0076] Preferably, induction may be carried out for between 1 and 14 days, such as 10 days, more preferably between 2 and 10 days, even more preferably between 3 and 8 days, such as 7 days, even more preferably between 4 and 6 days, such as 5 days. In other words, the plant cells may be harvested 1 to 14 days, such as 10 days, 2 to 10 days, 3 to 8 days, such as 7 days, 4 to 6 days, such as 5 days, after addition of the first shot of at least one inducer, before proceeding with step iv) and harvesting the saponin produced.

[0077] It is within the skill of the art to determine the optimal concentration of inducer(s) and the optimal duration of induction for a given cell line. This may be assessed, for example, by running a time point experiment with various concentrations of inducer(s) while measuring the levels of saponin (as described below). Thus, a person skilled in the art will be able to determine the optimal conditions for any given plant cell culture. As described above, after mechanical disruption, the occurrence and observation of bubbles in the plant cells can be examined by sampling as a predictor of the level of saponin production achieved.

[0078] Elements / nutrients contained in the culture medium, especially carbon and phosphate sources, may have been consumed and / or may be consumed during induction, and therefore it may be necessary to restore the composition of the culture medium at the time of induction and / or during induction. This may be done by continuously or periodically supplementing the culture medium, e.g. with glucose and phosphate, as appropriate. As previously described, during the development of the method of the invention, the inventors sometimes observed "starvation" of the cells in glucose and / or phosphate during the induction phase. This may have a negative effect on the level of saponin production. Therefore, during step iii), the levels of glucose and / or phosphate in the culture medium may be advantageously monitored and supplemented as necessary. For example, when the culture medium reaches a residual level of glucose below 15 mM, the culture medium may be fed with a solution of 60 mM glucose. The feeding may be done periodically or continuously. Since the rate of consumption of nutrients may vary from cell line to cell line and from induction condition to induction condition, it is within the skill of the art to determine the optimal conditions and modes of nutrient feeding, e.g. glucose or phosphate, for each cell line and each induction condition.

[0079] Step iv) - Recovering the saponin At any suitable time after induction, the saponin is recovered. The saponin may be recovered by any method known in the art, such as extraction with a non-aqueous polar solvent, extraction with an acidic or basic medium, or recovery by resin absorption, or extraction by mechanical disruption of the plant cells, for example by ball milling or sonication. Alternatively, the saponin may be extracted by freezing the cell pellet obtained after centrifugation of the cell culture (leading to cell lysis). Suitably, the cell pellet is frozen at -20°C, more suitably at -70°C, for example for at least 24 hours.

[0080] Any method known in the art for analyzing and quantifying the saponin content of any composition or any extract may be used, for example, UPLC-UV-MS absorbance at 214 nm. Alternatively, the saponin content may be determined by HPLC-ELSD (Evaporative Light Scattering Detector) or by LCMS-MS. The use of appropriate standards for the saponins of interest to be investigated makes it possible to quantify the respective saponin content in the plant cell extract of the present invention. For example, suitable standards may be saponin fractions isolated from the crude bark extract of Quillaja saponaria tree, such as fraction QS-21 (e.g., as described and reported in Kensil et al. 1991 or WO 19 / 10692), fraction QS-18, fraction QS-17, and fraction QS-7 (e.g., as described and reported in Kensil et al. 1991).

[0081] Adjuvant formulations The saponin produced according to the method of the present invention can be suitably used as an adjuvant, for example to be included in a vaccine. Any type of adjuvant formulation known in the art can be used. For example, one or more saponins produced, such as QS-21 saponin, can be formulated into liposomes (see, for example, WO 2019 / 106192 or WO 2013 / 041572).

[0082] saponin As mentioned above, the saponins of the present invention are quillajaic acid-based triterpenoid glycosides. Using the crude bark extract of the Quillaja saponaria tree as a reference, such saponins are traditionally known and regrouped as fractions, such as QS-17, QS-7, QS-21, or QS-18 fractions. The fractions usually contain mixtures of structurally related saponin species (see, for example, Kensil et al. 1991), which are detailed below and grouped by "family". QS-21 and QS-7 are particularly interesting saponin families due to their immunostimulatory activity.

[0083] In some embodiments, the saponins produced by the methods of the present invention are one or more saponin species from the QS-7 saponin family, the QS-17 saponin family, the QS-18 saponin family, and / or the QS-21 saponin family.

[0084] The inventors observed that all Quillaja acid-based triterpenoid glycosides naturally synthesized in plant cells were produced to moderate extent when the method of the present invention was used, although some variability in production capacity was observed between different plant cells or cell lines, which may reflect some inherent ability of a given plant cell or a given cell line to synthesize saponins. Also, consistent with what has been observed with bark from Quillaja saponaria trees, the inventors observed that very often the most abundant saponins produced by the plant cells or cell lines of the present invention were saponins from the QS-18 saponin family (as further detailed below) (see FIG. 7), while the respective proportions of each family may vary from cell line to cell line (as shown in the Examples). The data disclosed in the Examples herein primarily provide QS-18 volumetric productivity and QS-21 volumetric productivity. However, QS-17 volumetric productivity was also analyzed and observed to increase similarly when the method of the present invention was used (see FIG. 7, data not shown).

[0085] Suspension cell lines of plant cells capable of naturally synthesizing Quillajaic acid-based triterpenoid saponins and producing said saponins with a volumetric productivity of at least 5 mg / L, at least 10 mg / L, at least 20 mg / L, at least 40 mg / L or at least 50 mg / L also form an object of the present invention. In a particular embodiment, the saponins produced by the suspension cell lines of plant cells of the present invention are one or more saponin species from the QS-7 saponin family, the QS-17 saponin family, the QS-18 saponin family and / or the QS-21 saponin family.

[0086] Each saponin family has one or more common structural features that characterize it from other families. Individual species within each family also exhibit certain structural features that characterize it from other species in the family, including: xylose or rhamnose species - the presence of a xylose or rhamnose residue in the trisaccharide at C3 position of Quillaja acid; A or B isomers - A has an acyl chain linked through the 4 position of D-fucose at C28 position of Quillaja acid, and B has an acyl chain linked through the 3 position of D-fucose; V1 and V2 - the presence of a terminal apiose or xylose residue, respectively, in the sugar at C28 position of Quillaja acid (in other species of the family, this terminal residue may also be absent).

[0087] Those skilled in the art will also recognize that the structures described herein contain ionizable groups and may exist in dissociated form or as salts under appropriate circumstances. The structures are generally depicted with the glucuronic acid moiety in ionized form, and the molecular weights depicted are calculated directly from the depicted ions (corresponding to the monoisotopic m / z observed in negative mode electrospray mass spectrometry), however, all undissociated, dissociated and salt forms are intended to be encompassed by the described definition. It is desirable that the salts be pharmaceutically acceptable, although pharmaceutically unacceptable salts may nevertheless be useful during the manufacture of pharmaceutical products or for non-pharmaceutical applications.

[0088] Group I – QS-7 saponin family The term "QS-7 saponin family" (triterpenoid glycosides having an acetyl group linked through the 4-position of D-fucose at the C28 position of Quillajaic acid), as used herein, means: (i) the xylose species QS-7 with a monoisotopic molecular weight (m / z) of 1862 by negative mode electrospray mass spectrometry ("QS-7 1862") (which can exist as V1 apiose and V2 xylose isomers):

[0089] - QS-7 1862 V1

[0090] [ka]

[0091] - QS-7 1862 V2

[0092] [ka]

[0093] (ii) the QS-7 xylose species with monoisotopic molecular weights (m / z) of 1730, 1700, 1568, 1554, and 1716 by negative mode electrospray mass spectrometry:

[0094] - Xyl-QS-7 1730

[0095] [ka]

[0096] - QS-7 1700

[0097] [ka]

[0098] - QS-7 1568

[0099] [ka]

[0100] - QS-7 1554

[0101] [ka]

[0102] - QS-7 1716:

[0103] [ka]

[0104] (iii) the rhamnose species QS-7 with a monoisotopic molecular weight (m / z) of 1876 by negative mode electrospray mass spectrometry ("QS-7 1876") (which may exist as V1 apiose and V2 xylose isomers):

[0105] - QS-7 1876 V1

[0106] [ka]

[0107] - QS-7 1876 V2

[0108] [ka]

[0109] (iv) the rhamnose species QS-7 with a monoisotopic molecular weight (m / z) of 1714 by negative mode electrospray mass spectrometry ("QS-7 1714") (which may exist as V1 apiose and V2 xylose isomers):

[0110] - QS-7 1714 V1

[0111] [ka]

[0112] - QS-7 1714 V2

[0113] [ka]

[0114] (v) the rhamnose species QS-7 having a monoisotopic molecular weight (m / z) of 1568 by negative mode electrospray mass spectrometry ("QS-7 1568") (which may exist as V1 apiose and V2 xylose isomers):

[0115] - QS-7 1568 V1

[0116] [ka]

[0117] - QS-7 1568 V2

[0118] [ka] (vi) the rhamnose species QS-7 having a monoisotopic molecular weight (m / z) of 1730 by negative mode electrospray mass spectrometry:

[0119] - Rha-QS-7 1730

[0120] [ka]

[0121] and (vii) the rhamnose species QS-7 having a monoisotopic molecular weight (m / z) of 1582 by negative mode electrospray mass spectrometry:

[0122] - QS-7 1582

[0123] [ka]

[0124] In one embodiment, the saponin produced by the methods of the present invention is one or more of the above QS-7 saponin species from the QS-7 saponin family.

[0125] In a further embodiment, the invention provides suspension cell lines of plant cells capable of producing one or more of the above QS-7 saponin species from the QS-7 saponin family.

[0126] Group II – QS-18 saponin family The term "QS-18 saponin family" (triterpenoid glycosides having a beta-O-glucopyranosylation at the C3 position of the rhamnose residue of the sugar at the C28 position of Quillaja acid), as used herein, means: (i) the xylose species QS-18 having a monoisotopic molecular weight (m / z) of 2150 by negative mode electrospray mass spectrometry ("QS-18 2150") (which can exist as A and B isomers, as well as V1 apiose and V2 xylose isomers):

[0127] - QS-18 2150A V1

[0128] [ka]

[0129] - QS-18 2150A V2

[0130] [ka]

[0131] - QS-18 2150B V1

[0132] [ka]

[0133] - QS-18 2150B V2

[0134] [ka]

[0135] (ii) the xylose species QS-18 ("QS-18 2018") having a monoisotopic molecular weight (m / z) of 2018 by negative mode electrospray mass spectrometry (which may exist as A and B isomers):

[0136] - QS-18 2018 A

[0137] [ka]

[0138] - QS-18 2018 B

[0139] [ka]

[0140] (iii) the rhamnose species QS-18 having a monoisotopic molecular weight (m / z) of 2164 by negative mode electrospray mass spectrometry ("QS-18 2164") (which can exist as A and B isomers, as well as V1 apiose and V2 xylose isomers):

[0141] - QS-18 2164A V1

[0142] [ka]

[0143] - QS-18 2164A V2

[0144] [ka]

[0145] - QS-18 2164 B V1

[0146] [ka]

[0147] - QS-18 2164B V2

[0148] [ka]

[0149] and (iv) the rhamnose species QS-18 having a monoisotopic molecular weight (m / z) of 2032 by negative mode electrospray mass spectrometry ("QS-18 2032") (which may exist as A and B isomers):

[0150] - QS-18 2032A

[0151] [ka]

[0152] - QS-18 2032 B

[0153] [ka]

[0154] In some embodiments, the saponin produced by the methods of the present invention is one or more of the above QS-18 saponin species from the QS-18 saponin family. In certain embodiments, the saponin produced by the methods of the present invention is one or more of QS-18 2150 A V1, QS-18 2150 A V2, QS-18 2150 B V1, and QS-18 2150 B V2.

[0155] In further embodiments, the present invention provides suspension cell lines of plant cells capable of producing one or more of the above QS-18 saponin species from the QS-18 saponin family. In certain embodiments, the saponins produced by the suspension cell lines of plant cells of the present invention are one or more of QS-18 2150 A V1, QS-18 2150 A V2, QS-18 2150 B V1 and QS-18 2150 B V2.

[0156] Group III – QS-17 saponin family The term "QS-17 saponin family" (triterpenoid glycosides having a beta-O-glucopyranosylation at the C3 position of the rhamnose residue in the sugar at the C28 position of quillaric acid and an alpha-O-rhamnosylation at the C2 position of the arabinofuranose moiety of the acyl chain linked to the fucose residue in the sugar at the C28 position of quillaric acid), as used herein, means: (i) the xylose species QS-17 having a monoisotopic molecular weight (m / z) of 2296 by negative mode electrospray mass spectrometry ("QS-17 2296") (which may exist as A and B isomers, as well as V1 apiose and V2 xylose isomers):

[0157] - QS-17 2296A V1

[0158] [ka]

[0159] - QS-17 2296A V2

[0160] [ka]

[0161] - QS-17 2296 B V1

[0162] [ka]

[0163] - QS-17 2296B V2

[0164] [ka]

[0165] (ii) the xylose species QS-17 having a monoisotopic molecular weight (m / z) of 2134 by negative mode electrospray mass spectrometry ("QS-17 2134") (which can exist as A and B isomers, as well as V1 apiose and V2 xylose isomers):

[0166] - QS-17 2134A V1

[0167] [ka]

[0168] - QS-17 2134A V2

[0169] [ka]

[0170] - QS-17 2134 B V1

[0171] [ka]

[0172] - QS-17 2134B V2

[0173] [ka]

[0174] (iii) the xylose species QS-17 ("QS-17 2164"), which can exist as A and B isomers, having a monoisotopic molecular weight (m / z) of 2164 by negative mode electrospray mass spectrometry:

[0175] - QS-17 2164A

[0176] [ka]

[0177] - QS-17 2164 B

[0178] [ka]

[0179] (iv) the rhamnose species QS-17 having a monoisotopic molecular weight (m / z) of 2310 by negative mode electrospray mass spectrometry ("QS-17 2310") (which can exist as A and B isomers, as well as V1 apiose and V2 xylose isomers):

[0180] - QS-17 2310A V1

[0181] [ka]

[0182] - QS-17 2310A V2

[0183] [ka]

[0184] - QS-17 2310B V1

[0185] [ka]

[0186] - QS17-2310B V2

[0187] [ka]

[0188] and (v) the rhamnose species QS-17 having a monoisotopic molecular weight (m / z) of 2148 by negative mode electrospray mass spectrometry ("QS-17 2148") (which can exist as A and B isomers, as well as V1 apiose and V2 xylose isomers):

[0189] - QS-17 2148A V1

[0190] [ka]

[0191] - QS-17 2148A V2

[0192] [ka]

[0193] - QS-17 2148 B V1

[0194] [ka]

[0195] - QS-17 2148B V2

[0196] [ka]

[0197] In one embodiment, the saponin produced by the method of the invention is one or more of the above QS-17 saponin species from the QS-17 saponin family. In a further embodiment, the invention provides a suspension cell line of a plant cell capable of producing one or more of the above QS-17 saponin species from the QS-17 saponin family.

[0198] Group IV – QS-21 saponin family The term "QS-21 saponin family" (triterpenoid glycosides having an acyl chain linked by a fucose residue in the sugar at C28 of the quillaric acid core terminated by an arabinofuranose residue), as used herein, means: (i) the xylose species QS-21 having a monoisotopic molecular weight (m / z) of 1988 by negative mode electrospray mass spectrometry ("QS-21 1988") (which can exist as A and B isomers, as well as V1 apiose and V2 xylose isomers):

[0199] - QS-21 1988 A V1

[0200] [ka]

[0201] - QS-21 1988 A V2

[0202] [ka]

[0203] - QS-21 1988 B V1

[0204] [ka]

[0205] - QS-21 1988 B V2

[0206] [ka]

[0207] (ii) the xylose species QS-21 having a monoisotopic molecular weight (m / z) of 1856 by negative mode electrospray mass spectrometry ("QS-21 1856") (which may exist as A and B isomers):

[0208] - QS-21 1856 A

[0209] [ka]

[0210] - QS-21 1856 B

[0211] [ka]

[0212] (iii) the rhamnose species QS-21 ("QS-21 2002") having a monoisotopic molecular weight (m / z) of 2002 by negative mode electrospray mass spectrometry (which can exist as A and B isomers, as well as V1 apiose and V2 xylose isomers):

[0213] - QS-21 2002 A V1

[0214] [ka]

[0215] - QS-21 2002 A V2

[0216] [ka]

[0217] - QS-21 2002 B V1

[0218] [ka]

[0219] - QS-21 2002 B V2

[0220] [ka]

[0221] In some embodiments, the saponin produced by the methods of the present invention is one or more of the above QS-21 saponin species from the QS-21 saponin family. In certain embodiments, the saponin produced by the methods of the present invention is one or more of QS-21 1988 A V1, QS-21 1988 A V2, QS-21 1988 B V1, and QS-21 1988 B V2.

[0222] In further embodiments, the present invention provides suspension cell lines of plant cells capable of producing one or more of the above QS-21 saponin species from the QS-21 saponin family. In certain embodiments, the saponins produced by the suspension cell lines of plant cells of the present invention are one or more from QS-21 1988 A V1, QS-21 1988 A V2, QS-21 1988 B V1 and QS-21 1988 B V2.

[0223] The invention will be further described with reference to the following items:

[0224] Item 1. A method for converting a non-producing plant cell capable of naturally synthesizing a saponin containing a quillaic acid triterpenoid aglycone into a saponin-producing plant cell, comprising at least the following steps: i) culturing non-producing plant cells in a culture medium containing a nitrogen source; ii) depleting the culture medium of any nitrogen sources; and iii) inducing the production of saponin with at least one inducer. A method comprising:

[0225] Item 2. A method for producing a saponin containing quillaic acid triterpenoid aglycone, comprising at least the following steps: i) culturing a plant cell capable of naturally synthesizing a saponin containing a quillaic acid triterpenoid aglycone in a culture medium containing a nitrogen source; ii) depleting the culture medium of all nitrogen sources; iii) inducing the production of saponin with at least one inducer; and iv) Recovering the produced saponin A method comprising:

[0226] Item 3. The method according to item 1 or 2, wherein the plant cells are grown in suspension.

[0227] Item 4. The method according to any one of Items 1 to 3, wherein the plant cells are suspension cell lines.

[0228] Item 5. The method according to any one of Items 1 to 4, wherein the nitrogen source in the culture medium in step i) contains at least nitrate.

[0229] Item 6. The method according to Item 5, wherein the nitrate is KNO3 and / or NH4NO3.

[0230] Item 7. The method according to Item 6, wherein the nitrate is KNO3.

[0231] Item 8. The method according to any one of Items 1 to 6, wherein the nitrogen source comprises KNO3 and NH4NO3.

[0232] Item 9. The method according to any one of Items 5 to 8, wherein the nitrogen source further comprises NH4Cl.

[0233] Item 10. The method according to any one of Items 1 to 8, wherein the nitrogen source does not contain NH4Cl.

[0234] Item 11. The method according to Items 1 to 10, wherein the total concentration of the nitrogen source is 10 mM to 50 mM.

[0235] Item 12. The method according to Item 11, wherein the total concentration of the nitrogen source is 15 mM to 40 mM.

[0236] Item 13. The method according to Item 12, wherein the total concentration of the nitrogen source is 20 mM to 30 mM.

[0237] Item 14. The method according to Items 11 to 13, wherein the total concentration of the nitrogen source is about 25 mM.

[0238] Item 15. The method according to Items 11 and 12, wherein the total concentration of the nitrogen source is about 40 mM.

[0239] Item 16. The method according to any one of Items 5 to 15, wherein the KNO3 concentration, if present, is 5 mM to 30 mM.

[0240] Item 17. The method according to Item 16, wherein the KNO3 concentration is 10 mM to 20 mM.

[0241] Item 18. The method according to item 17, wherein the KNO3 concentration is about 15 mM.

[0242] Item 19. The method according to item 17, wherein the KNO3 concentration is about 20 mM.

[0243] Item 20. The method according to Items 5 to 19, wherein the NH4NO3 concentration, if present, is 5 mM to 30 mM.

[0244] Item 21. The method according to Item 20, wherein the NH4NO3 concentration is 10 mM to 20 mM.

[0245] Item 22. The method according to Item 21, wherein the NH4NO3 concentration is about 10 mM.

[0246] Item 23. The method according to Item 20, wherein the NH4NO3 concentration is about 20 mM.

[0247] Item 24. The method according to Items 5 to 23, wherein the NH4Cl concentration, if present, is 5 to 20 mM.

[0248] Item 25. The method according to Item 24, wherein the NH4Cl concentration is about 10 mM.

[0249] Item 26. The method according to items 1 to 8, wherein the nitrogen source comprises 15 mM KNO3 and 10 mM NH4NO3.

[0250] Item 27. The method according to any one of Items 1 to 26, wherein the culture medium in step i) contains one or more of sucrose, glucose and fructose as a carbon source.

[0251] Item 28. The method according to Item 27, wherein the carbon source is sucrose, glucose and fructose.

[0252] Item 29. The method according to Item 27, wherein the carbon source does not contain sucrose.

[0253] Item 30. The method according to Items 27 to 29, wherein the total concentration of the carbon source is in the range of 40 mM to 100 mM.

[0254] Item 31. The method according to Item 30, wherein the total concentration of the carbon source is in the range of 50 mM to 90 mM.

[0255] Item 32. The method according to Item 31, wherein the total concentration of the carbon source is in the range of 60 mM to 80 mM.

[0256] Item 33. The method according to Item 31, wherein the total concentration of the carbon source is about 60 mM.

[0257] Item 34. The method according to Item 31, wherein the total concentration of the carbon source is about 70 mM.

[0258] Item 35. The method according to Items 27 to 34, wherein the concentration of sucrose, if present, is in the range of 5 mM to 100 mM.

[0259] Item 36. The method according to Item 35, wherein the concentration of sucrose is in the range of 5 mM to 20 mM.

[0260] Item 37. The method according to Item 35, wherein the concentration of sucrose is in the range of 10 mM to 80 mM.

[0261] Item 38. The method according to Item 37, wherein the concentration of sucrose is in the range of 20 mM to 60 mM.

[0262] Item 39. The method according to Item 36, wherein the concentration of sucrose is about 10 mM.

[0263] Item 40. The method according to Items 27 to 39, wherein the concentration of glucose, if present, is in the range of 5 mM to 60 mM.

[0264] Item 41. The method according to Item 40, wherein the glucose concentration is in the range of 15 mM to 60 mM.

[0265] Item 42. The method according to Item 40, wherein the glucose concentration is in the range of 20 mM to 40 mM.

[0266] Item 43. The method according to Items 28 to 39, wherein the concentration of glucose, if present, is in the range of 10 mM to 80 mM.

[0267] Item 44. The method according to Items 40 to 43, wherein the glucose concentration is about 30 mM.

[0268] Item 45. The method according to Item 43, wherein the concentration of glucose is about 60 mM.

[0269] Item 46. The method according to Items 27 to 45, wherein the concentration of fructose, if present, is in the range of 5 mM to 60 mM.

[0270] Item 47. The method according to Item 46, wherein the fructose concentration is in the range of 15 mM to 60 mM.

[0271] Item 48. The method according to Item 47, wherein the fructose concentration is in the range of 20 mM to 40 mM.

[0272] Item 49. The method according to Items 27 to 45, wherein the concentration of fructose, if present, is in the range of 10 mM to 80 mM.

[0273] Item 50. The method according to Items 46 to 49, wherein the concentration of fructose is about 30 mM.

[0274] Item 51. The method according to any one of items 1 to 50, wherein the culture medium in step i) contains a hormone.

[0275] Item 52. The method according to Item 51, wherein the hormone is one or more auxins and / or one or more cytokinins.

[0276] Item 53. The method according to item 52, wherein the one or more auxins are NAA or 2,4-D.

[0277] Item 54. The method of item 52, wherein the one or more cytokinins are BA.

[0278] Item 55. The method according to Items 1 to 54, wherein the culture medium in step i) contains at least 2,4-D.

[0279] Item 56. The method according to Items 1 to 54, wherein the culture medium in step i) contains at least 2,4-D and NAA.

[0280] Item 57. The method according to Items 1 to 56, wherein the culture medium in step i) contains 2,4-D, NAA and BA.

[0281] Item 58. The method according to Items 53 to 57, wherein the concentrations of NAA, 2,4-D and BA, if present, are in the range of 0.2 mg / L to 0.8 mg / L.

[0282] Item 59. The method according to Item 58, wherein the concentrations of NAA, 2,4-D and BA are about 0.5 mg / L.

[0283] Item 60. The method according to items 1 to 59, wherein the culture medium in step i) contains an additional macronutrient selected from CaCl2, MgSO4 and KH2PO4.

[0284] Item 61. The method according to Items 1 to 60, wherein the culture medium in step i) contains at least KH2PO4.

[0285] Item 62. The method according to Item 61, wherein the concentration of KH2PO4 is in the range of 0.6 mM to 5 mM.

[0286] Item 63. The method according to Item 62, wherein the concentration of KH2PO4 is in the range of 1.5 mM to 5 mM.

[0287] Item 64. The method according to Item 62, wherein the concentration of KH2PO4 is about 1.25 mM.

[0288] Item 65. The method according to Item 63, wherein the concentration of KH2PO4 is about 2.5 mM.

[0289] Item 66. The method according to Item 63, wherein the concentration of KH2PO4 is 5 mM.

[0290] Item 67. The culture medium in step i) is KI, HBO 3、 67. The method according to items 1 to 66, comprising a trace nutrient selected from MnSO4, ZnS04, Na2MoO4, CuSO4, CoCl2, DeSO4, or Na2EDTA.

[0291] Item 68. The method according to any one of Items 1 to 67, wherein the culture medium in step i) contains a vitamin selected from myo-inositol, nicotinic acid, pyridoxine-HCl, or thiamine-HCl.

[0292] Item 69. The method according to Items 1 to 68, wherein the culture medium in step i) contains CaCl2, KH2PO4, KNO3, MgSO4, NH4NO3, sucrose, glucose, fructose, NAA and 2,4-D.

[0293] Item 70. The method according to Items 1 to 69, wherein the culture medium in step i) is medium 4.

[0294] Item 71. The method according to Items 1 to 68, wherein the culture medium in step i) contains CaCl2, KH2PO4, KNO3, MgSO4, NH4NO3, sucrose, NAA and 2,4-D.

[0295] Item 72. The method according to item 71, wherein the culture medium is medium 6.

[0296] Item 73. The method according to any one of Items 1 to 72, wherein the osmolality of the culture medium in step i) and / or step ii) and / or step iii) is in the range of 100 to 220 mOsm.

[0297] Item 74. The method according to Items 1 to 73, wherein the osmolality is between 180 and 200 mM.

[0298] Item 75. The method according to items 1 to 74, wherein the plant cells in step i) are cultured until a PCV in the range of 10% to 70% is reached.

[0299] Item 76. The method according to Item 75, wherein the PCV is in the range of 15% to 30%.

[0300] Item 77. The method according to Item 75, wherein the PCV is in the range of 20% to 60%.

[0301] Item 78. The method according to Item 77, wherein the PCV is in the range of 30% to 50%.

[0302] Item 79. The method according to Item 75, wherein the PCV is about 15%.

[0303] Item 80. The method according to Item 75, wherein the PCV is about 20%.

[0304] Item 81. The method according to Item 77, wherein the PCV is about 30%.

[0305] Item 82. The method according to Item 77, wherein the PCV is about 40%.

[0306] Item 83. The method according to any one of Items 1 to 82, wherein step i) is performed for 4 to 8 days or longer.

[0307] Item 84. The method according to Item 83, wherein step i) is for 5 to 7 days.

[0308] Item 85. The method according to Item 83, wherein step i) is for 4 to 5 days.

[0309] Item 86. The method according to items 1 to 85, wherein step ii) is carried out by replacing the culture medium at the end of step i) with a culture medium that does not contain a nitrogen source and maintaining the cells in the replaced culture medium.

[0310] Item 87. The method according to any one of items 1 to 86, wherein step ii) is carried out by replacing the culture medium at the end of step i) with a culture medium containing 5 mM or less of a nitrogen source and maintaining the cells in the replaced culture medium.

[0311] Item 88. The method according to item 87, wherein the replacement culture medium contains between 0.5 mM and 5 mM of a nitrogen source.

[0312] Item 89. The method of item 88, wherein the replacement culture medium contains between 1.25 mM and 2.5 mM of a nitrogen source.

[0313] Item 90. The method of item 88, wherein the replacement culture medium contains about 1.25 mM of a nitrogen source.

[0314] Item 91. The method of item 88, wherein the replacement culture medium contains about 2.5 mM of a nitrogen source.

[0315] Item 92. The method of item 88, wherein the replacement culture medium contains about 5 mM of a nitrogen source.

[0316] Item 93. The method according to Items 87 to 92, wherein the nitrogen source in the replacement medium is one or more of KNO3, NH4NO3 or NH4Cl.

[0317] Item 94. The method according to Items 87 to 93, wherein the nitrogen source in the replacement medium is KNO3.

[0318] Item 95. The method according to Items 87 to 94, wherein the nitrogen source in the replacement medium is NH4NO3.

[0319] Item 96. The method according to Items 87 to 95, wherein the nitrogen source in the replacement medium is NH4Cl.

[0320] Item 97. The method according to Items 86 to 96, wherein the replacement culture medium contains KCl.

[0321] Item 98. The method according to Item 97, wherein the KCl concentration is 10 to 20 mM.

[0322] Item 99. The method according to items 86 to 98, wherein the replacement culture medium comprises one or more nutrients according to items 27 to 50 and 60 to 67, one or more hormones according to items 51 to 59, and one or more vitamins according to item 68.

[0323] Item 100. The method according to Items 86 to 99, wherein the replacement culture medium is Medium 1.

[0324] Item 101. The method according to Items 1 to 100, wherein step ii) is 1 to 9 days.

[0325] Item 102. The method according to Item 101, wherein step ii) is performed for 2 to 7 days.

[0326] Item 103. The method according to Item 101, wherein step ii) is 5 to 7 days.

[0327] Item 104. The method according to Item 103, wherein step ii) is for 3 to 6 days.

[0328] Item 105. The method according to Item 104, wherein step ii) is 4 days.

[0329] Item 106. The method according to Item 104, wherein step ii) is 5 days.

[0330] Item 107. The method according to Item 104, wherein step ii) is 7 days.

[0331] Item 108. The method according to items 86 to 100, wherein the cells are maintained in the replacement culture medium for 1 to 9 days.

[0332] Item 109. The method of item 105, wherein the cells are maintained in the replacement culture medium for 2 to 7 days.

[0333] Item 110. The method of item 109, wherein the cells are maintained in the replacement culture medium for 5 to 7 days.

[0334] Item 111. The method of item 110, wherein the cells are maintained in the replacement culture medium for 3 to 6 days.

[0335] Item 112. The method of item 111, wherein the cells are maintained in the replacement culture medium for 4 days.

[0336] Item 113. The method of item 111, wherein the cells are maintained in the replacement culture medium for 5 days.

[0337] Item 114. The method of item 109, wherein the cells are maintained in the replacement culture medium for 7 days.

[0338] Item 115. The method according to items 1 to 85, wherein step ii) is carried out by allowing the cells to naturally consume the nitrogen source contained in the culture medium used in step i) to a residual level without further supplementing the culture medium with a nitrogen source, and maintaining the cells in the consumed culture medium.

[0339] Item 116. The method according to Item 115, wherein the residual level of the nitrogen source is half, quarter, or eighth of the concentration of the nitrogen source contained in the culture medium in step i).

[0340] Item 117. The method according to Item 115, wherein the residual level of the nitrogen source is less than 5 mM.

[0341] Item 118. The method according to Item 117, wherein the residual level of the nitrogen source is less than 2.5 mM.

[0342] Item 119. The method according to Item 118, wherein the residual level of the nitrogen source is less than 1 mM.

[0343] Item 120. The method according to Item 117, wherein the residual level of the nitrogen source is between 1 and 2 mM.

[0344] Item 121. The method according to Items 115 to 119, wherein the residual level of the nitrogen source is undetectable.

[0345] Item 122. The method according to any one of Items 115 to 121, wherein the nitrogen source in the spent culture medium is one or more of KNO3, NH4NO3 or NH4Cl.

[0346] Item 123. The method according to Items 115 to 122, wherein the nitrogen source in the spent medium is KNO3.

[0347] Item 124. The method according to Items 115 to 123, wherein the nitrogen source in the consumed medium is NH4NO3.

[0348] Item 125. The method according to Items 115 to 123, wherein the nitrogen source is KNO3 and NH4NO3.

[0349] Item 126. The method according to Items 115 to 125, wherein the nitrogen source in the spent medium is NH4Cl.

[0350] Item 127. The method according to Items 1 to 85 and 115 to 126, wherein step ii) is performed for 5 to 20 days.

[0351] Item 128. The method according to Item 127, wherein step ii) is 6 to 19 days.

[0352] Item 129. The method according to Item 128, wherein step ii) is 7 to 18 days.

[0353] Item 130. The method according to Item 129, wherein step ii) is 8 to 16 days.

[0354] Item 131. The method according to Item 130, wherein step ii) is 9 to 15 days.

[0355] Item 132. The method according to Item 131, wherein step ii) is 10 days.

[0356] Item 133. The method according to Item 131, wherein step ii) is 11 days.

[0357] Item 134. The method according to Item 131, wherein step ii) is 12 days.

[0358] Item 135. The method according to Item 131, wherein step ii) is 13 days.

[0359] Item 136. The method according to Item 131, wherein step ii) is 14 days.

[0360] Item 137. The method according to items 115 to 136, wherein the cells are maintained in the spent culture medium for 2 to 7 days.

[0361] Item 138. The method according to item 137, wherein the cells are maintained in the spent culture medium for 5 to 7 days.

[0362] Item 139. The method according to item 138, wherein the cells are maintained in the spent culture medium for 3 to 6 days.

[0363] Item 140. The method of item 139, wherein the cells are maintained in the spent culture medium for 4 days.

[0364] Item 141. The method of item 139, wherein the cells are maintained in the spent culture medium for 5 days.

[0365] Item 142. The method of item 138, wherein the cells are maintained in the spent culture medium for 7 days.

[0366] Item 143. The method according to items 1 to 142, wherein the culture medium in step ii) is monitored and supplemented with glucose to maintain a minimum level of about 15 mM.

[0367] Item 144. The method according to items 1 to 143, wherein the culture medium in step ii) is monitored and supplemented with KH2PO4 to maintain a minimum level of about 2.5 mM.

[0368] Item 145. The method according to Items 1 to 144, wherein at least one inducer in step iii) is a monocarboxylic acid compound type inducer.

[0369] Item 146. The method according to item 145, wherein the at least one inducer is one or more of 5-chlorosalicylic acid, salicylic acid, acetylsalicylic acid, and methyl ester.

[0370] Item 147. The method according to Items 145 and 146, wherein at least one inducer is methyl jasmonate (MeJa).

[0371] Item 148. The method according to any one of Items 1 to 147, wherein the concentration of at least one inducer in step iii) is in the range of 0.5 to 12 μM.

[0372] Item 149. The method according to Item 148, wherein the concentration of the at least one inducer in step iii) is 1 to 8 μM.

[0373] Item 150. The method according to Item 149, wherein the concentration of the at least one inducer in step iii) is 2 to 6 μM.

[0374] Item 151. The method according to Item 150, wherein the concentration of the at least one inducer in step iii) is 3-5 μM.

[0375] Item 152. The method according to Item 149, wherein the concentration of the at least one inducer in step iii) is 1 to 3 μM.

[0376] Item 153. The method according to Item 152, wherein the concentration of the at least one inducer in step iii) is about 2 μM.

[0377] Item 154. The method according to Item 152, wherein the concentration of the at least one inducer in step iii) is about 3 μM.

[0378] Item 155. The method according to Item 150, wherein the concentration of the at least one inducer in step iii) is about 6 μM.

[0379] Item 156. The method according to items 1 to 147, wherein the concentration of at least one inducer in step iii) is in the range of 0.5 to 12 μM / PCV%.

[0380] Item 157. The method according to Item 156, wherein the concentration of the at least one inducer in step iii) is 1-8 μM / PCV%.

[0381] Item 158. The method according to Item 157, wherein the concentration of at least one inducer in step iii) is 2-6 μM / PCV%.

[0382] Item 159. The method according to Item 158, wherein the concentration of the at least one inducer in step iii) is 3-5 μM / PCV%.

[0383] Item 160. The method according to Item 157, wherein the concentration of the at least one inducer in step iii) is 1-3 μM / PCV%.

[0384] Item 161. The method according to Item 160, wherein the concentration of at least one inducer in step iii) is 2 μM / PCV%.

[0385] Item 162. The method according to Item 160, wherein the concentration of at least one inducer in step iii) is 3 μM / PCV%.

[0386] Item 163. The method according to Item 158, wherein the concentration of at least one inducer in step iii) is 6 μM / PCV%.

[0387] Item 164. The method according to items 1 to 163, wherein at least one inducer in step iii) is added directly to the cells at the end of step ii).

[0388] Item 165. The method according to any one of Items 1 to 164, wherein step iii) is for 1 to 14 days.

[0389] Item 166. The method according to Item 165, wherein step iii) is for 2 to 10 days.

[0390] Item 167. The method according to Item 166, wherein step iii) is for 3 to 8 days.

[0391] Item 168. The method according to Item 167, wherein step iii) is for 4 to 6 days.

[0392] Item 169. The method according to Item 168, wherein step iii) is 5 days.

[0393] Item 170. The method according to Item 167, wherein step iii) is 7 days.

[0394] Item 171. The method according to Item 166, wherein step iii) is 10 days.

[0395] Item 172. The method according to items 1 to 164, wherein the plant cells are harvested between 1 and 14 days after addition of at least one inducer.

[0396] Item 173. The method according to item 172, wherein the plant cells are harvested between 2 and 10 days after addition of the at least one inducer.

[0397] Item 174. The method according to item 173, wherein the plant cells are harvested between 3 and 8 days after addition of the at least one inducer.

[0398] Item 175. The method according to Item 174, wherein the plant cells are harvested between 4 and 6 days after addition of the at least one inducer.

[0399] Item 176. The method according to item 173, wherein the plant cells are harvested 5 days after addition of the at least one inducer.

[0400] Item 177. The method according to item 173, wherein the plant cells are harvested 7 days after addition of the at least one inducer.

[0401] Item 178. The method according to item 173, wherein the plant cells are harvested 10 days after addition of the at least one inducer.

[0402] Item 179. The method according to items 164 to 178, wherein at least one inducer is further added every other day.

[0403] Item 180. The method according to items 1 to 179, wherein the culture medium in step iii) is monitored and supplemented with glucose to maintain a minimum level of about 15 mM.

[0404] Item 181. The method according to items 1 to 180, wherein the culture medium in step iii) is monitored and supplemented with KH2PO4 to maintain a minimum level of about 2.5 mM.

[0405] Item 182. The method according to any one of Items 1 to 181, wherein the plant cells in step i) and / or step ii) and / or step iii) are cultured at a temperature in the range of 20° C. to 30° C.

[0406] Item 183. The method according to any one of Items 1 to 182, wherein the plant cells in step i), step ii) and step iii) are cultured at a temperature of about 25°C.

[0407] Item 184. The method according to any one of Items 1 to 183, wherein the plant cells in step i) and / or step ii) and / or step iii) are stirred at a speed in the range of 40 to 60 rpm.

[0408] Item 185. The method according to Item 184, wherein the cells in step i), step ii) and step iii) are stirred at a speed of about 50 rpm.

[0409] Item 186. The method according to items 1 to 185, wherein the plant cells are cultured in a shake flask.

[0410] Item 187. The method according to items 1 to 185, wherein the plant cells are cultured in a bioreactor.

[0411] Item 188. A plant cell obtainable by the method according to items 1 and 3 to 187.

[0412] Item 189. A suspension of plant cells obtainable by the methods described in Items 1 and 3 to 187.

[0413] Item 190. A suspension cell line obtainable by the methods described in Items 1 and 3 to 187.

[0414] Item 191. A suspension of plant cells capable of naturally synthesizing a quillaric acid-based triterpenoid saponin and producing said saponin with a volumetric productivity of at least 5 mg / L.

[0415] Item 192. The plant cell suspension according to Item 191, having a volumetric productivity of at least 10 mg / L.

[0416] Item 193. The plant cell suspension according to Item 192, having a volumetric productivity of at least 20 mg / L.

[0417] Item 194. The plant cell suspension according to Item 193, having a volumetric productivity of at least 40 mg / L.

[0418] Item 195. The plant cell suspension according to Item 194, having a volumetric productivity of at least 50 mg / L.

[0419] Item 196. A suspension of plant cells according to Items 191 to 195, wherein the suspension produces quillaic acid-based triterpenoid saponins at the volumetric productivity.

[0420] Item 197. A suspension cell line of a plant cell capable of naturally synthesizing a quillaric acid-based triterpenoid saponin and producing said saponin with a volumetric productivity of at least 10 mg / L.

[0421] Item 198. The suspension cell line of the plant cell according to Item 197, having a volumetric productivity of at least 20 mg / L.

[0422] Item 199. The suspension cell line of a plant cell according to Item 198, having a volumetric productivity of at least 40 mg / L.

[0423] Item 200. The suspension cell line of plant cells according to Item 199, having a volumetric productivity of at least 50 mg / L.

[0424] Item 201. The suspension cell line of the plant cell according to Items 197 to 200, wherein the suspension cell line produces quillaic acid-based triterpenoid saponins at said volumetric productivity.

[0425] Item 202. The method according to Items 1 to 187, the plant cell, the plant cell suspension, or the suspension cell line according to Items 189 to 201, wherein the plant cell is derived from the genus Quillaja.

[0426] Item 203. The method according to Item 202, the plant cell, the suspension of the plant cell, or the suspension cell line, wherein the plant cell is derived from the species Quillaja Saponaria.

[0427] Item 204. The method according to Item 202, the plant cell, the suspension of the plant cell, or the suspension cell line, wherein the plant cell is derived from the species Quillaja brasiliensis.

[0428] Item 205. The method according to Items 202 to 204, the plant cell, the plant cell suspension, or the suspension cell line, wherein the plant cell is a cambium meristem cell (CMC).

[0429] Item 206. The method, plant cell, plant cell suspension, or suspension cell line according to Items 202 to 206, wherein the saponin is one or more saponin species from the QS-7 saponin family, the QS-17 saponin family, the QS-18 saponin family, and / or the QS-21 saponin family.

[0430] Item 207. The method according to Item 206, the plant cell, the plant cell suspension, or the suspension cell line, wherein the saponin is one or more saponin species from the QS-7 saponin family.

[0431] Item 208. The method according to item 207, the plant cell, the plant cell suspension or the suspension cell line, wherein the saponin is one or more saponin species selected from QS-7 1862 V1, QS-7 1862 V2, Xyl-QS-7 1730, QS-7 1700, Xyl-QS-7 1568, QS-7 1554, QS-7 1716, QS-7 1876 V1, QS-7 1876 V2, QS-7 1714 V1, Rha-QS-7 1568 V1, Rha-QS-7 1730.

[0432] Item 209. The method according to item 207, the plant cell, the plant cell suspension or the suspension cell line, wherein the saponin is one or more saponin species selected from QS-7 1862 V1, QS-7 1862 V2, Xyl-QS-7 1730, QS-7 1700, Xyl-QS-7 1568, QS-7 1554, QS-7 1716, QS-7 1876 V1, QS-7 1876 V2, QS-7 1714 V1, QS-7 1714 V2, Rha-QS-7 1568 V1, Rha-QS-7 1568 V2, Rha-QS-7 1730, QS-7 1582.

[0433] Item 210. The method according to Item 206, the plant cell, the plant cell suspension, or the suspension cell line, wherein the saponin is one or more saponin species from the QS-17 saponin family.

[0434] Item 211. The method according to item 210, the plant cell, the plant cell suspension or the suspension cell line, wherein the saponin is one or more saponin species from QS-17 2296 A V1, QS-17 2296 A V2, QS-17 2296 B V1, QS-17 2296 B V2, QS-17 2164 A, QS-17 2164 B, QS-17 2310 A V1, QS-17 2310 A V2, QS-17 2310 B V1, QS-17 2310 B V2.

[0435] Item 212. Saponin is QS-17 2296 A V1, QS-17 2296 A V2, QS-17 2296 B V1, QS-17 2296 B V2, QS-17 2164 A, QS-17 2164 B, QS-17 2310 A V1, QS-17 2310 A V2, QS-17 2310 B V1, QS-17 2310 B V2, QS-7 2134 A V1, QS-7 2134 A V2, QS-7 2134 B V1, QS-7 2134 B V2, QS-7 2148 A V1, QS-7 2148 A V2, QS-7 2148 B V1, QS-7 2148 B V2.

[0436] Item 213. The method according to Item 206, the plant cell, the plant cell suspension, or the suspension cell line, wherein the saponin is one or more saponin species from the QS-18 saponin family.

[0437] Item 214. The method according to item 213, the plant cell, the plant cell suspension or the suspension cell line, wherein the saponin is one or more of QS-18 2150 A V1, QS-18 2150 A V2, QS-18 2150 B V1, QS-18 2150 B V2, QS-18 2018 A, QS-18 2018 B, QS-18 2164 A V1, QS-18 2164 A V2, QS-18 2164 B V1, QS-18 2164 B V2.

[0438] Item 215. The method according to item 213, the plant cell, the plant cell suspension or the suspension cell line, wherein the saponin is one or more of QS-18 2150 A V1, QS-18 2150 A V2, QS-18 2150 B V1, QS-18 2150 B V2, QS-18 2018 A, QS-18 2018 B, QS-18 2164 A V1, QS-18 2164 A V2, QS-18 2164 B V1, QS-18 2164 B V2, QS-18 2032 A, QS-18 2032 B.

[0439] Item 216. The method according to items 214 and 215, the plant cell, the plant cell suspension or the suspension cell line, wherein the saponin is one or more of QS-18 2150 A V1, QS-18 2150 A V2, QS-18 2150 B V1 and QS-18 2150 B V2.

[0440] Item 217. The method according to Item 206, the plant cell, the plant cell suspension, or the suspension cell line, wherein the saponin is one or more saponin species from the QS-21 saponin family.

[0441] Item 218. The method according to item 217, the plant cell, the plant cell suspension or the suspension cell line, wherein the saponin is one or more of QS-21 1988 A V1, QS-21 1988 A V2, QS-21 1988 B V1 and QS-21 1988 B V2, QS-21 1856 A, QS-21 1856 B, QS-21 2002 A V1, QS-21 2002 B V1, QS-21 2002 A V2, QS-21 2002 B V2.

[0442] Item 219. The method according to 218, the plant cell, the plant cell suspension, or the suspension cell line, wherein the saponin is one or more of QS-21 1988 A V1, QS-21 1988 A V2, QS-21 1988 B V1 and QS-21 1988 B V2.

[0443] Item 220. A method for preparing an adjuvant containing saponin, comprising the steps of: a) producing saponin according to the method described in Items 2 to 187 and 202 to 219; and b) formulating the recovered saponin as an adjuvant. A method comprising:

[0444] Item 221. The method of item 220, wherein the saponin adjuvant formulation is a liposomal formulation.

[0445] Item 222. The method according to items 220 and 221, wherein the saponin is one or more of the QS-21 saponin family.

[0446] term Unless otherwise explained in the context of this disclosure, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0447] The singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. The term "plurality" refers to two or more. Furthermore, it should be understood that all base or amino acid sizes, and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for purposes of illustration.

[0448] With respect to numerical values, the terms "approximately," "around," or "about" typically mean a value within plus or minus 10 percent of the stated value, particularly within plus or minus 5 percent of the stated value, especially the stated value.

[0449] Furthermore, numerical limitations given for concentrations or levels of substances such as antigens are intended to be approximate. Thus, if a concentration is stated to be at least (for example) 200 pg, it is intended that the concentration is understood to be at least approximately (or "about" or "≈") 200 pg. The term "comprises" means "includes." Thus, unless the context requires otherwise, the term "comprises," and variations such as "comprise" and "comprising," are understood to mean the inclusion of a described compound or composition (e.g., nucleic acid, polypeptide, antigen) or step, or group of compounds or steps, but not the exclusion of any other compound, composition, step, or group thereof. EXAMPLES

[0450] The invention will now be further illustrated by the following non-limiting examples.

[0451] Working Example Example 1 – Callus Production Callus was established from cambial meristem cells of selected Quillaja saponaria plants.

[0452] Young shoots from growing plants were cut into small pieces. The outer layers were removed by surface sterilization to expose the cambium. The cambium was then placed on agar plates containing Murashige and Skoog (MS) medium supplemented with the plant hormones 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BA) at 0.5 mg / L each.

[0453] The plates were then incubated in the dark at 25° C. for 4 weeks, after which they were subcultured onto fresh solid MS medium (with the addition of the plant hormones listed above) and incubated in the dark at 25° C. After 8 weeks, the growing cambium cells were separated from the solid callus and transferred to fresh MS medium (with the addition of the plant hormones listed above). To maintain viability, the resulting callus was continuously subcultured every 4 weeks as described above.

[0454] Example 2 – Initiation and maintenance of suspension plant cells Suspension plant cell cultures, i.e. suspension cell lines, were initiated by inoculating callus material into liquid medium in shake flasks. A 10% inoculation material was used, e.g., 3 g of callus material was inoculated into 30 ml of liquid MS medium containing NAA and 2,4-dichlorophenoxyacetic acid (2,4-D) at 0.5 mg / L each. The liquid volume as a percentage of the total flask volume was fixed at 20% or less. The liquid suspension flasks were incubated at a temperature of 25° C. on a shaker set at 200 rpm for 14 days, and then subcultured again into liquid medium. Subculture was achieved by allowing the large aggregates in the cell suspension to settle, and then sucking off the liquid containing the fine cells in suspension into a centrifuge tube. The suspension was centrifuged, the supernatant poured off, and the remaining cell pellet (packed cell volume - PCV) was resuspended in fresh MS medium (plus NAA and 2,4-D) (see medium 6 in Table 1 below). The volume of fresh medium added was such that the final cell concentration was 10% (as PCV) of the final volume. The subcultured suspension flasks were incubated at a temperature of 25°C on a shaker set at 200 rpm for 14 days, after which they were subcultured one more time as described above. The suspension plant cell cultures are maintained by further subculturing every 9-14 days. Subcultures can also be referred to as "passages" (P), with P0 corresponding to the time when a given cell line has transitioned from the callus stage to being able to grow in suspension in liquid medium.

[0455] Example 3 – Effects of nitrogen depletion and induction The following figures provide schematic diagrams illustrating embodiments of the methods of the invention that were carried out in Experiments 1-9 below.

[0456] TIFF2024538802000056.tif57160

[0457] 3.1 Experiment 1 Five different cultures of the same suspension plant cell line named "CMC40B6" (established as described in Examples 1 and 2) were grown in parallel in maintenance culture media such as Medium 4 below until the PCV of the cultures reached 20%. At this point (day 0), the culture medium of all cultures was removed and replaced with either Medium 1, Medium 2, Medium 3, Medium 4 or Medium 5 containing various levels of nitrogen source and variable nitrogen source (as described in Table 1 below) and grown for an additional 5 days. On day 5, the PCV% of each culture was measured and 3 μM / PCV% MeJa was added directly to the culture medium of each of the five cultures and grown for an additional 4 days. The growth of the cultures was analyzed by sampling the cultures on days 5, 7 and 8 and measuring the PCV. On day 9 (i.e., 4 days after induction), the cells were harvested, disrupted (as described in Example 4) and the QS-18 saponin content was analyzed and measured (as described in Example 5).

[0458] [Table 1]

[0459] result The results are presented in Figure 1. Before induction, the medium providing the best conditions for growth was medium 3 (containing NH4Cl as the only nitrogen source). However, immediately after induction with Meja, the cells died. Before induction, the medium providing the lowest growth was medium 2 (containing KNO3 as the only nitrogen source). The absence of a nitrogen source (medium 1) did not prevent the cells from growing.

[0460] Only cells grown in medium 1 (without nitrogen source) before induction were able to produce saponin (volumetric productivity of QS-18 reached approximately 5 mg / L when measured 4 days after induction) (data not shown).

[0461] 3.2 Experiment 2 – QS-18 volumetric productivity (no nitrogen source during nitrogen starvation) Medium 1 (without nitrogen source) as described above was then selected to test various induction conditions (concentrations and durations). Five separate cultures of cell line CMC40B6 inoculated with 10% cell biomass (PCV) in medium 4 were grown until the PCV reached approximately 30%. At this point (day 0), the culture medium of all cultures was replaced with medium 1. On D3, D5 and D7, the PCV% of each culture was measured and MeJa was added directly to the culture medium at various concentrations (0.35, 0.7, 1.4, 2.8, 5.6 or 11.2 μM / PCV%). At 2, 4 and 7 days after induction (i.e. after adding MeJa) and before adding MeJa, cells were harvested and disrupted (as described in Example 4) and the saponin content in plant cell extracts was measured (as described in Example 5). QS-18 volumetric productivity was investigated. The results are presented in Figure 2. Data (not shown) were obtained as described for Experiment 4 below in Table 2.

[0462] The presence of QS-21 and QS-17 saponins was also examined and confirmed for the above conditions: 5 days of nitrogen starvation followed by 4 days of challenge with 2.8 μM / PCV%, and the results are presented in FIG.

[0463] result Regardless of the MeJA concentration used, the best productivity is reached when plant cells are starved for 5 days (above 20 mg / L). Starvation for 3 days produced almost no QS-18, but starvation for 7 days still provided good QS-18 productivity (above 10 mg / L). After 5 days of starvation, QS-18 productivity peaked between 2 and 7 days after induction, with the highest productivity achieved at approximately 4 to 5 days (irrespective of Meja concentration). After 5 days of starvation, the concentrations of MeJa giving the highest productivity range between 0.7 and 2.8 μM / PCV%. After 7 days of starvation, the concentrations of MeJa giving the highest productivity range between 1.4 and 5.6 μM / PCV unit (see Figure 2).

[0464] Furthermore, the chromatograms shown in FIG. 7 indicate that after 5 days of nitrogen depletion followed by 4 days of induction with 2.8 μM / PCV%, QS-17 saponin and QS-21 saponin were produced as well, as reflected by the presence of peaks at the retention times corresponding to the respective standards.

[0465] The chromatograms shown in FIG. 8 indicate that in this experiment, 5 days of nitrogen depletion without induction was not sufficient to achieve detectable levels of saponin production, as reflected by the absence of peaks at the expected retention times corresponding to the respective standards.

[0466] 3.3 Experiment 3 – QS-18 volumetric productivity (no nitrogen source during nitrogen starvation) A second suspension cell line (CMC16B) (established as described in Examples 1 and 2) was tested. Five different cultures of this suspension cell line were grown in parallel in medium 4 until the PCV of the cultures reached 30%. At this point (day 0), the culture medium of all cultures was removed and replaced with medium 1 (without nitrogen source). After 5 days, the PCV% of each culture was measured on day 5, and various MeJa concentrations were added directly to the culture medium of each culture (0.35, 0.7, 1.4, 2.8, or 5.6 / PCV%). At 2, 4, and 7 days after induction (i.e., after adding Meja), cells were harvested and disrupted (as described in Example 4), and the saponin content in the plant cell extracts was measured (as described in Example 5). QS-18 volumetric productivity was investigated. The results are presented in Figure 3. Data (not shown) were obtained as described for experiment 4 below in Table 2.

[0467] result Regardless of the MeJa concentration used, maximum productivity was obtained approximately 4 days after Meja addition (i.e., 4 days after induction), with the highest productivity (approximately 40 mg / L) being obtained at a 2.8 μM / PCV% MeJa concentration.

[0468] 3.4 Experiment 4 – QS-18 volumetric productivity (no nitrogen source during nitrogen starvation) Three different suspension cell lines (CMC16B, CMC40B6 and CMC35A8) (established as described in Examples 1 and 2) were tested. CMC16B was used at passage P19, CMC40B6 was used at passage P39 and CMC35A8 was used at passage P14. Five separate cultures of each suspension cell line were grown in Medium 4 until the PCV of the cultures reached 30%. At this point (day 0), the culture medium of all cultures was removed and replaced with Medium 1 (no nitrogen source). After 5 days, the PCV% of each culture was measured on day 5 and various MeJa concentrations were added directly to the culture medium of each culture of each cell line (0.35, 0.7, 1.4, 2, 2.8 or 5.6 μM / PCV%, where applicable, as summarized in Tables 2 and 3 below. Four days after induction (i.e., after addition of Meja), cells were harvested and disrupted (as described in Example 4) and saponin content in plant cell extracts was measured (as described in Example 5). QS-18 volumetric productivity was determined. The data are shown in Tables 2 and 3 below, and the results are presented in graphical form in Figure 4.

[0469] [Table 2]

[0470] The calculation is as follows (assuming a "cell culture density" of 1000 g / L):

[0471]

number

[0472] [Table 3]

[0473] The calculation is as follows (assuming a "cell culture density" of 1000 g / L):

[0474]

number

[0475] result Figure 4 shows some cell line variability, which may reflect some inherent variability in the ability of different cell lines to synthesize saponin. However, in all three cell lines, nitrogen depletion and subsequent induction by the method of the present invention resulted in saponin production. Furthermore, the effectiveness and reproducibility of the method of the present invention is confirmed by the data obtained for cell lines CMC40B6 and CMC16B, as the levels of QS-18 volumetric productivity obtained in experiments 2 and 3 are within the same range as those shown in Figures 2 and 3.

[0476] 3.5 Experiment 5 – QS-21 volumetric productivity (no nitrogen source during nitrogen starvation) The same suspension cell lines used in experiment 4 above (CMC16B, CMC40B6, and CMC35A8) were tested. CMC16B was used at passage P19, CMC40B6 was used at passage P27, and CMC35A8 was used at passage P15. A parent culture of each suspension cell line grown in medium 4 was split into five separate cultures by centrifuging the cells of the parent culture and resuspending them in medium 1 (without nitrogen source) at a PCV between 20-25% (this is day 0). After 5 days, the PCV% of each culture was measured at day 5, and various MeJa concentrations were added directly to the culture medium of each culture of each cell line (0.35, 0.7, 1.4, 2.8, or 5.6 μM / PCV%) (where applicable, as summarized in Table 4).

[0477] Glucose and phosphate (PO4) supply conditions during the nitrogen depletion and induction phases: The minimum targets for glucose and phosphate (PO4) levels during both the nitrogen depletion and induction phases were 15 mM and 0.6 mM, respectively. Glucose and phosphate (PO4) levels were measured at the following time points: (i) before starting nitrogen depletion, 2 and 5 days after depletion, and (ii) before starting induction, 2, 4 and 7 days after induction, or 8 days after induction (if applicable). If the measured level of glucose was lower than 15 mM, the culture medium was fed with a 60 mM glucose solution. If the measured level of phosphate (PO4) levels was lower than 0.6 mM, the culture medium was fed with a 2.5 mM phosphate solution.

[0478] Note During this experiment, phosphate (PO4) starvation was observed during the nitrogen depletion and / or induction phases for all three cell lines: (i) for CMC40B6, at D5 after depletion and D2 / D4 / D8 after induction, (ii) for CMC16B, at D2 / D5 after depletion and D4 / D7 after induction, and (iii) for CMC35A8, at D2 / D5 after depletion. "Starvation" means that phosphate (PO4) levels measured on the indicated days were below the detection limit.

[0479] Seven days after induction, i.e. after addition of Meja (CMC16B and CMC35A8), or eight days after induction (CMC40B6), cells were harvested and disrupted as described below.

[0480] Saponin Extract Before harvesting the cells, the PCV of the cell culture was measured. A sample of the cell culture was collected and centrifuged. The supernatant was discarded and the cell pellet was dried in a freeze dryer and weighed ("cell FW"). Once dry, 1 mL ("total extraction volume") of 80% methanol was added to the cell pellet. Extraction was performed by vortexing the sample at 2500 rpm for 90 minutes or by ball milling. The mixture was then centrifuged at 1000 g for 5 minutes. After centrifugation, 1 μL of the collected supernatant was analyzed according to the method described in Example 6.

[0481] QS-21 volumetric productivity was examined and saponin content in plant cell extracts was measured (as described in Example 6).

[0482] The data is shown in Table 4 and the results are presented in graphical form in FIG.

[0483] [Table 4]

[0484] The calculation is as follows (assuming a "cell culture density" of 1000 g / L):

[0485]

number

[0486] result As with QS-18 volumetric productivity (Figure 8), some cell line variability was observed (Figure 9), which may reflect some inherent variability in the ability of different cell lines to synthesize saponin. However, in all three cell lines, nitrogen depletion and subsequent induction by the methods of the present invention resulted in QS-21 saponin production.

[0487] 3.6 Experiment 6 – QS-21 volumetric productivity (no nitrogen source during nitrogen starvation) The same suspension cell line (CMC16B) used in experiments 4 and 5 above was tested at passage P40. A parental culture of CMC16B grown in medium 6 was split into four separate cultures by centrifuging the cells of the parental culture and resuspending them at a PCV between 20-25% (this is day 0). Two cultures were resuspended in medium 1 (no nitrogen source) and two cultures were resuspended in medium 6 (containing nitrogen). After 5 days, on day 5, all four cultures were either left untreated (i.e., no MeJa addition) or had 6 μM / PCV% MeJa added directly to the culture medium (as summarized in Table 5 below and shown in Figure 10).

[0488] Glucose and phosphate (PO4) supply conditions during the nitrogen depletion and induction phases: The minimum targets for glucose and phosphate (PO4) levels during both the nitrogen depletion and induction phases were 15 mM and 1.5 mM, respectively. Glucose and phosphate (PO4) levels were measured at the following time points: (i) before the start of nitrogen depletion, 2 and 5 days after depletion, and (ii) before the start of induction, 2, 4 and 7 days after induction. If the measured level of glucose was lower than 15 mM, the culture medium was fed with a 60 mM glucose solution. If the measured level of phosphate (PO4) was lower than 1.5 mM, the culture medium was fed with a 5 mM phosphate solution. Phosphate (PO4) starvation was not observed during this experiment.

[0489] Seven days after induction (i.e., after addition of Meja), both untreated and Meja-treated cells were harvested and disrupted (as described in Experiment 5) and the saponin content in plant cell extracts was measured (as described in Example 6). Measurements were performed in duplicate or triplicate, where applicable. QS-21 volumetric productivity was examined. The data are shown in Table 5 and the results are presented in graphical form in Figure 10 (each bar represents the average volumetric productivity).

[0490] [Table 5]

[0491] The calculation is as follows (assuming a "cell culture density" of 1000 g / L):

[0492]

number

[0493] result As shown in FIG. 10, in the absence of nitrogen depletion and / or induction, no production of QS-21 saponin was observed, whereas a nitrogen depletion step followed by induction results in the production of QS-21 saponin.

[0494] 3.7 Experiment 7 – QS-21 volumetric productivity (no nitrogen source during nitrogen starvation) A fourth suspension cell line (CMC5B-1) (established as described in Examples 1 and 2) was tested at passage P11. Parental cultures of CMC5B-1 grown in medium 4 were centrifuged and resuspended in medium 1 (without nitrogen source) at a PCV between 20-25% (this is day 0). After 5 days, the PCV% of each culture was measured at day 5, and 3.3 μM / PCV% MeJa was added directly to the culture medium (i.e., after adding MeJa) for 5 days. Cells were then harvested, disrupted, and the saponin content in plant cell extracts was measured (as described in Example 6), as described below. QS-21 volumetric productivity was investigated. The data are shown in Table 6, and the results are presented in graphical form in Figure 11.

[0495] Saponin Extraction Before harvesting the cells, the PCV of the cell culture was measured. 1 ml of cell culture ("Vol. PCC") was centrifuged at 4000 rpm for 10 minutes. The supernatant was discarded, while the cell pellet was frozen at -70°C for 24 hours to induce cell lysis. The thawed sample cell pellet was then diluted with 4 ml of methanol, vortexed for 30 seconds, and then centrifuged. The supernatant was collected and diluted. 1 μL of the diluted sample was then used to analyze the saponin content according to the method described in Example 6.

[0496] [Table 6]

[0497] The calculation is as follows:

[0498]

number

[0499] result As for experiments 5 and 6, after the nitrogen depletion step and subsequent induction, production of QS-21 saponin was observed (as shown in FIG. 11).

[0500] 3.8 Experiment 8 – QS-7 production (no nitrogen source during nitrogen starvation) Samples #2 and #12 from experiment 6 were also tested for the presence of QS-7 saponin. As detailed in experiment 6, samples #2 and #12 represent the "no nitrogen depletion" / "no induction" and "nitrogen depletion / induction" conditions, respectively.

[0501] The presence of QS-7 was detected by UPLC / MS using the following parameters:

[0502] TIFF2024538802000068.tif239169

[0503] The presence of QS-7 saponin in plant cell extracts was confirmed by doubly charged [M-2H] 2- The singly charged [MH] corresponding to QS-7 1862 was determined by extracting (1861.78-1) / 2=930.39. - 1861.78 ions. Chromatograms representing the "no nitrogen depletion" / "no trigger" and "nitrogen depletion / trigger" conditions are provided in FIG.

[0504] result In the absence of both nitrogen starvation and induction, QS-7 1862 was not detectable, but after both nitrogen starvation and induction, QS-7 1862 becomes detectable (see Figure 14, Panels A and B, respectively), indicating that QS-7 saponin is produced as well when the methods of the present invention are used.

[0505] 3.9 Experiment 9 – QS-18 volumetric productivity (reduced concentrations of nitrogen source during nitrogen depletion) The same suspension cell line (CMC16B) used in experiments 4, 5 and 6 above was tested at passage P47. A parental culture of CMC16B grown in medium 6 was split into 13 separate cultures (this is day 0) by centrifuging the cells of the parental culture and resuspending them in medium 1 (i.e., no nitrogen source) at approximately 20% PCV, or medium 1 supplemented with 1.25 mM, 2.5 mM or 5 mM NH4Cl, NH4NO3 or KNO3 (as shown in Figure 13). After 5 days, cultures were left untreated (i.e., no Meja addition) or 8 μM / PCV% MeJa was added directly to the culture medium (as shown in Figure 13). At 4, 7, 10 and 14 days after induction, cells were harvested and disrupted (as described in Example 4) and saponin content in plant cell extracts was measured (as described in Example 5). QS-18 volumetric productivity was investigated, and the data is shown in Tables 7, 8, and 9, with the results presented in FIG.

[0506] [Table 7]

[0507] The calculation is as follows (assuming a "cell culture density" of 1000 g / L):

[0508]

number

[0509] [Table 8]

[0510] The calculation is as follows (assuming a "cell culture density" of 1000 g / L):

[0511]

number

[0512] [Table 9]

[0513] The calculation is as follows (assuming a "cell culture density" of 1000 g / L):

[0514]

number

[0515] [Table 10]

[0516] The calculation is as follows (assuming a "cell culture density" of 1000 g / L):

[0517]

number

[0518] result Apart from the condition “NH4Cl 5 mM”, all other reduced concentrations of nitrogen sources tested during the nitrogen depletion phase (i.e., 1.25 mM NH4Cl, KNO3 or NH4NO3, 2.5 mM NH4Cl, KNO3 or NH4NO3) were 3、 and 5 mM KNO3 or NH4NO3) resulted in saponin production (after induction). These results indicate that saponin production can be successfully induced by induction in the presence of residual concentrations of the nitrogen source. Complete elimination of the nitrogen source from the culture medium is not required prior to induction by the method of the present invention.

[0519] 3.10 Experiment 10 – QS-21 volumetric productivity (natural nitrogen depletion) The following figure provides a schematic diagram illustrating an embodiment of the method of the present invention that was implemented in this experiment.

[0520] TIFF2024538802000078.tif58161

[0521] The same suspension cell line (CMC40B6) used in experiments 1, 2, 4 and 5 above was tested at passage P29. A 10 L bioreactor was seeded with cells at a PCV of about 10% in 8 L of medium 4 (containing 15 mM KNO3 and 10 mM NH4NO3) and the cells were grown until they reached a PCV of about 20%. At this point, to perform the sub-experiment* (see below), 1 L was harvested from the bioreactor and 3 L of medium 4 (containing 15 mM KNO3 and 10 mM NH4NO3) was further added to the remaining 7 L of culture in the bioreactor (i.e., the cells were diluted 1.43 times in the bioreactor, resulting in a PCV of about 14%). This represents the "last supplementation with nitrogen source" (=D0) in the bioreactor experiment, where the culture medium is not further supplemented with nitrogen source. The cells were maintained in the same medium, thus allowing the cells to naturally consume the nitrogen source contained in medium 4 to residual levels. Ammonium (NH4 + ions) and nitrates (NO3 - The levels of ammonium, nitrate and nitrate (ions) were monitored regularly by measuring their respective levels every few days. At D9, the levels of ammonium were undetectable and the levels of nitrate were about 5 mM (see panels B and C of FIG. 15, respectively). The cells were further maintained in the same medium for another 5 days. Then, 2 μM / PCV% MeJa was added (D14). Seven days after induction (i.e., D21), the cells were harvested and disrupted (as described in Experiment 5) and the saponin content in the plant cell extract was measured (as described in Example 6). The QS-21 volumetric productivity was examined. The data are presented in Table 11 below and the results are presented in graphical form in FIG. 15 (Panel A).

[0522] Glucose and phosphate (PO4) supply conditions during the nitrogen depletion and induction phases: The minimum targets for glucose and phosphate (PO4) levels during both the nitrogen depletion and induction phases were 15 mM and 0.6 mM, respectively. Glucose and phosphate (PO4) levels were measured every few days. If the measured level of glucose was lower than 15 mM, the culture medium was fed with a 60 mM glucose solution. If the measured level of phosphate (PO4) was lower than 0.6 mM, the culture medium was fed with a 2.5 mM phosphate solution.

[0523] *Side experiment 1 L harvested from the bioreactor as mentioned above was centrifuged and resuspended in medium 1 (without nitrogen source) at a PCV between 20-25% (this is day 0). After 5 days, the PCV% of each culture was measured at day 5, and 2 μM / PCV% MeJa was added directly to the culture medium (i.e., after adding MeJa) for 7 days. Cells were then harvested and disrupted (as described in experiment 6), and saponin content in plant cell extracts was measured (as described in example 6). QS-21 volumetric productivity was investigated. The results are presented in graphical form in panel D of FIG. 15 (data not shown).

[0524] [Table 11]

[0525] The calculation is as follows (assuming a "cell culture density" of 1000 g / L):

[0526]

number

[0527] result FIG. 15A shows that natural nitrogen depletion prior to induction (i.e., allowing the cells to naturally consume the nitrogen source present in the culture medium) also results in saponin production (as demonstrated herein by examining QS-21 saponin).

[0528] FIG. 15D shows that the QS-21 volumetric productivity achieved by natural depletion is within the same range as the volumetric productivity achieved when nitrogen depletion is performed by removing the culture medium and replacing it with culture medium that does not contain a nitrogen source.

[0529] 3.11 Conclusion Saponin production (e.g., QS-7, QS-21 and QS-18 saponins) was reproducibly observed and obtained using at least four different suspension cell lines used at various passages when using the methods according to the invention. Different nitrogen depletion conditions and different induction conditions using different concentrations of inducer resulted in similar saponin production, regardless of the extraction process used, as confirmed by different analytical methods.

[0530] Example 4 – Extraction of Saponins At the end of the induction, the PCV of the cultures to be extracted was measured. Approximately 3 ml of each suspension culture was transferred to a 7 ml tube pre-filled with Precellys® ceramic beads. The tubes were centrifuged at 1000 g for 5 minutes. After centrifugation, the supernatant was discarded. After weighing the cell pellets ("cell fresh weight" or "cell FW"), a volume of sodium acetate buffer (30 mM, pH 6) equivalent to the volume of the cell pellet was added to the cell pellet. Based on the assumption that 1 g of cell pellet is equal to 1 mL of cell pellet, the "total extraction volume" in Tables 2, 3, 7, 8, 9 and 10 represents the sum of the sodium acetate buffer volume added and the cell FW. The cell pellets were then homogenized using a Precellys® with Cryolis using the following conditions maintained at 4°C: 3 cycles of 30 seconds at 8000 rpm with a rest interval of 60 seconds between each cycle. The tubes were then centrifuged at 5000 g for 10 minutes at 4°C. The supernatant was then filtered over a series of filters made of Millex-HV PVDF 0.45 μm and Millex-HV PVDF 022 μm filters. After filtration, the extract is ready to give its saponin content and may be stored at 4° C. prior to analysis. The saponin content was given in 20 μl samples of plant cell extracts to be analyzed according to the method described in Example 5.

[0531] Example 5 – Analysis of saponin content by HPLC / ELSD The saponin content in the plant cell extracts was measured by HPLC / ELSD using the following parameters:

[0532] Chromatography system: Agilent 1290LC Column: Waters Acquity BEH C18, 2.1 mm x 100 mm; 1.7 μm 130A Oven temperature: 55°C Autosampler temperature: 10℃ Flow rate: 0.6ml / min Runtime: 11.00 min Injection volume: 20 μl Mobile phase A: H2O / ACN / IPA (75 / 20 / 5) v / v / v 0.025% FA Mobile phase B: H2O / ACN / IPA (10 / 72 / 18) v / v / v 0.025% FA

[0533] TIFF2024538802000081.tif107161

[0534] ELSD detection parameters: Evaporator: 80℃ Nebulizer: 90℃ Nitrogen flow rate: 1.5SLM

[0535] retention time - QS-17 A standard (50 μg / ml) corresponding to the QS-17 fraction isolated and purified from the crude bark extract of Quillaja saponaria tree ("QS-17 standard") was used to establish a calibration curve, which allowed for the subsequent quantification of the QS-17 saponin family present in the plant cell extract. The retention time at which the QS-17 standard peaked by HPLC / ELSD is approximately 4.32 min (data not shown).

[0536] - QS-21 A standard (50 μg / ml) corresponding to the QS-21 fraction isolated and purified from the crude bark extract of Quillaja saponaria tree ("QS-21 standard") was used to establish a calibration curve, which allowed for the subsequent quantification of the QS-21 saponin family present in the plant cell extract. The QS-21 standard was obtained using the purification method described in Example 3 of WO 19 / 10692. The retention time at which the QS-21 standard peaked by HPLC / ELD is approximately 4.80 min (see FIG. 6).

[0537] - QS-18 A standard (50 μg / ml) corresponding to the QS-18 fraction isolated and purified from a crude bark extract of the Quillaja saponaria tree ("QS-18 standard") was used to establish a calibration curve, which allowed the subsequent quantification of the QS-18 saponin family present in the plant cell extracts.

[0538] QS-18 standard was obtained using the purification method described in Example 3 of WO 19 / 10692 as follows: after reverse phase chromatography using phenyl resin (EPDM), the phenyl fraction containing QS-18 was collected (the presence of m / z corresponding to the major component was confirmed by MS - data not shown). The retention time at which the QS-18 standard peaked by HPLC / ELSD is approximately 4.54 min (see Figure 5A).

[0539] The amount of QS-18 saponin in a given plant cell extract was determined by comparing the peak area obtained for the plant cell extract with the peak area obtained for the QS-18 standard. Taking into account the PCV% measured before harvesting and extraction, the fresh cell weight in the plant cell extract, and the total extraction volume, the amount of QS-18 saponin in a given plant cell extract was converted to a QS-18 volumetric productivity expressed in μg / L (assuming that 1 g fresh cell weight is equal to 1 ml). Details of the calculation are provided in the above table reporting the data used in the calculation.

[0540] QS-18 Standard Composition The identification of saponin species contained in the QS-18 standard has been analyzed in parallel using a 120 min high-resolution LCMS mass spectrometry method using a Qtof mass spectrometer, which interrogates all saponin species with monoisotopic molecular weights (m / z) ranging from 300 to 4000. As provided in the chromatogram shown in Figure 5B, the major saponin species in the QS-18 standard is QS-18 2150 A (V1 and V2). It also contains minor saponin species such as QS-18 2150 B (V1 and V2), QS-18 2032, QS-18 2164, QS-18 2018, QS-17 2134, and QS-21 1988.

[0541] Example 6 – Analysis of saponin content by LC-MS / MS Alternatively, saponin content in plant cell extracts was measured by LCMS / MS using the following parameters:

[0542] TIFF2024538802000082.tif238166

[0543] A QS-21 fraction isolated and purified from a crude bark extract of the Quillaja saponaria tree using the purification method described in Example 3 of WO 19 / 10692 is used as a standard ("QS-21 standard" - same as in Example 5).

[0544] The amount of QS-21 saponin in a given plant cell extract was determined by comparing the peak area obtained for the plant cell extract with that obtained for the QS-21 standard (10 μg / ml) at the above MRM 993.46>755.55 transition (corresponding to QS-21 1988). A representative chromatogram is provided in FIG. 12B. Taking into account the PCV% measured before harvesting and extraction, the cell fresh weight in the plant cell extract (or the volume of the sample to be analyzed - "Vol. PCC"), and the total extraction volume, the amount of QS-21 saponin in a given plant cell extract was converted to QS-21 volumetric productivity expressed in μg / L (assuming that 1 g fresh cell weight is equal to 1 ml). Details of the calculations are provided in the above tables reporting the data used in the calculations.

[0545] QS-21 Standard Composition The identification of the saponin species contained in the QS-21 standard has been analyzed in parallel using a 120 min high resolution LCMS mass spectrometry method using a Qtof mass spectrometer that interrogates all saponin species with monoisotopic molecular weights (m / z) ranging from 300 to 4000. As provided in the chromatogram shown in Figure 12A, the major saponin species in the QS-21 standard are QS-21 1988 A V1 and QS-21 1988 A V2. It also contains minor saponin species such as QS-21 2002 A V1 and QS-21 2002 A V1.

[0546] References Reichert et al. in 2019 (“Quillaja Saponin Characteristics and Functional Properties”; Annu Rev Food Sci Technol. Mar 25;10, p43-73) Dalsgaard et al. in 1974 (“Saponin adjuvants”; Archiv. Fur die gesamte Virusforschung, Vol. 44, Springer Verlag, Berlin, p243-254) Kensil et al. in 1991 (“Separation and characterization of saponins with adjuvant activity from Quillaja aponaria Molina cortex”; Journal of immunology, Vol. 146: p431-437) Ragupathi, G. et al. 2011 (“Natural and synthetic saponin adjuvant QS-21 for vaccines against cancer”; Expert Review of Vaccines, Vol. 10: p463-470) Garcon, N. et al. in 2011 (“Recent clinical experience with vaccines using MPL and QS-21-containing adjuvant systems”; Expert Review of Vaccines, Vol. 10(4): p71-486) Didierlaurent, A. et al. in 2017 (“Adjuvant system AS01: helping to overcome the challenges of modern vaccines”; Expert Review of Vaccines, Vol. 16(1): p55-63) Didierlaurent A., et al. in 2014 (“Enhancement of Adaptive Immunity by the Human Vaccine Adjuvant AS01 Depends on Activated Dendritic Cells”; Journal of Immunology, Vol. 193(4): p1920-1930). De Becker, G. et al. in 2000 (“The adjuvant monophosphoryl lipid A increases the function of antigen-presenting cells”; International immunology, Vol. 12: p807-815) lsmaili, et al. in 2002 (“Monophosphoryl lipid A activates both human dendritic cells and T cells”; Journal of immunology, Vol. 168(9): p26-932) Martin, M. et al. in 2003 (“Role of innate immune factors in the adjuvant activity of monophosphoryl lipid A”; Infection and immunity, Vol (71): p2498-2507) Mata-Haro, V. et al. in 2007 (“The vaccine adjuvant monophosphoryl lipid A as a TRIF-biased agonist of TLR4”; Science, Vol. (316): p1628-1632) Kensil, C. et al. in 1998 (“QS-21: a water-soluble triterpene glycoside adjuvant”; Expert Opinion on Investigational Drugs, Vol. 7: p1475-1482) Newman, M.J. et al. in 1992 (“Saponin adjuvant induction of ovalbumin-specific CDS+ cytotoxic T lymphocyte responses”; Journal of immunology, Vol. 148: p2357-2362) Soltysik, S. et al. in 1995 (“Structure / function studies of QS-21 adjuvant: assessment of triterpene aldehyde and glucuronic acid roles in adjuvant function”; Vaccine, Vol. 13: p1403-1410) Lambrecht, B.N. et al. in 2009 (“Mechanism of action of clinically approved adjuvants”; Current Opinion in Immunology, Vol. 21 : p23-29) Li, H., S.B. et al. in 2008 (“Cutting edge: inflammasome activation by alum and alum’s adjuvant effect are mediated by NLRP3”; Journal of Immunology, Vol. 181: p17-21) Marty-Roix, R. et al. in 2016 (“Identification of QS-21 as an lnflammasome-activating Molecular Component of Saponin Adjuvants”; J. Biol. Chem. Vol. 291: p1123-36) Yendo, A et al. in 2010 (“Production of Plant Bioactive Triterpenoid Saponins: Elicitation Strategies and Target Genes to Improve Yields”; Mol. Biotech. Vol. 46: p94-104)

[0547] US 2019 / 0134128 WO 2011 / 161151 WO 2015 / 082978 WO 2019 / 106192 WO 2013 / 041572

Claims

1. A method for converting a non-producing plant cell capable of naturally synthesizing a saponin containing a quillaic acid triterpenoid aglycone into a saponin-producing plant cell, the method comprising at least the following steps: i) culturing non-producing plant cells in a culture medium containing a nitrogen source; ii) depleting the culture medium of all nitrogen sources; and iii) inducing the production of saponins using at least one inducer. A method comprising:

2. 1. A method for producing saponins containing quillaic acid triterpenoid aglycones, comprising at least the following steps: i) culturing plant cells capable of naturally synthesizing saponins containing quillaic acid triterpenoid aglycones in a culture medium containing a nitrogen source; ii) depleting the culture medium of all nitrogen sources; iii) inducing the production of saponins using at least one inducer; and iv) Recovering the saponin produced A method comprising:

3. The method of claim 1 or 2, wherein the plant cells are suspension cell lines.

4. 3. The method of claim 1, wherein the total concentration of nitrogen sources in the culture medium in step i) is between 10 mM and 50 mM.

5. 3. The method according to claim 1 or 2, wherein the culture medium in step i) comprises one or more of sucrose, glucose and fructose as carbon sources.

6. 3. The method of claim 1 or 2, wherein step ii) is carried out by replacing the culture medium at the end of step i) with a culture medium containing no nitrogen source or between 0.5 mM and 5 mM of a nitrogen source, and maintaining the cells in the replaced culture medium.

7. 3. The method of claim 1 or 2, wherein step ii) is 1 to 9 days.

8. 3. The method according to claim 1 or 2, wherein step ii) is carried out by allowing the cells to naturally consume the nitrogen source contained in the culture medium in step i) to a residual level without further supplementing the culture medium with a nitrogen source, and maintaining the cells in the consumed culture medium.

9. 9. The method of claim 8, wherein the residual level of the nitrogen source is less than 10 mM.

10. 9. The method of claim 8, wherein step ii) is performed for 5 to 20 days.

11. 9. The method of claim 8, wherein the cells are maintained in the spent culture medium for 1 to 9 days.

12. 3. The method according to claim 1 or 2, wherein at least one inducer in step iii) is a monocarboxylic acid compound type inducer.

13. 13. The method of claim 12, wherein at least one elicitor is methyl jasmonate (MeJa).

14. 3. The method according to claim 1 or 2, wherein the at least one inducer in step iii) is added directly to the cells at the end of step ii).

15. 3. The method of claim 1 or 2, wherein step iii) is between 1 and 14 days.

16. A suspension cell line of plant cells capable of naturally synthesizing quillaic acid-based triterpenoid saponins and producing said saponins at a volumetric productivity of at least 5 mg / L.

17. The method of claim 1 or 2, or the suspension cell line of claim 16, wherein the plant cells are derived from the genus Quillaja.

18. 18. The method or suspension cell line of claim 17, wherein the plant cells are from the species Quillaja Saponaria.

19. 18. The method or suspension cell line of claim 17, wherein the saponin is one or more saponin species from the QS-7 saponin family, the QS-17 saponin family, the QS-18 saponin family and / or the QS-21 saponin family.

20. 20. The method or suspension cell line of claim 19, wherein the saponin is one or more saponin species from the QS-21 saponin family.

21. 21. The method of claim 20, or the suspension cell line, wherein the saponin is one or more of QS-21 1988 A V1, QS-21 1988 A V2, QS-21 1988 B V1 and QS-21 1988 B V2.

22. A plant cell obtainable by the method of claim 1.

23. 1. A method for preparing an adjuvant comprising saponin, the method comprising the steps of: a) preparing the saponin according to the method of claim 2, and b) formulating the recovered saponin as an adjuvant. A method comprising:

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