Method for Producing a Composition Containing Archaeal Lipids from Sulfolobus Cell Cultures - Patent application

Abstract

The present invention provides (1) growing a S. sulfolobus cell culture at a temperature of 75°C to 85°C and a growth rate of 0.025 to 0.035 per hour, and extracting archaeal lipids from the archaeal culture to obtain a composition for use in oral delayed treatment; or (2) growing a Sulfolobus cell culture at a temperature of 75°C to 85°C and a growth rate of 0.005 to 0.015 per hour, and extracting archaeal lipids from the archaeal culture to obtain a composition for use in oral rapid treatment; The present invention also provides a method for producing a composition containing archaeal lipids from a Sulfolobus cell culture, the method comprising:

Description

[Technical field]

[0001] The present invention relates to the preparation of compositions comprising archaeal lipids and to compositions comprising archaeal lipids.

[0002] Liposomes are spherical lipid bilayer vesicles (lipid esters) that enclose an aqueous space. They are important carriers for the administration of drugs, vaccines, genes, proteins, small molecules, antibiotics, and nutrients. Liposomes are composed of phospholipids (mainly phosphatidylcholine) and cholesterol, but may also contain other lipids such as phosphatidylethanolamine. Liposomes are produced by a variety of methods (reviewed, for example, in van Hoogevest, Eur. J. Pharm. Sci. 108 (2017), 1-12; Szoka et al., Ann. Rev. Biophys. Bioeng. 9 (1980), 467-508), for example, by dispersing phospholipids in an aqueous medium, for example using mechanical treatment (e.g., with a homogenizer, preferably high-pressure homogenization) or ultrasonic treatment. Liposomes vary in diameter between 0.02 and 10 μm. Liposome size and composition greatly affect the properties of the liposomes.

[0003] Archaeosomes (i.e. liquid vesicles composed partly or entirely of archaeal lipids) represent a special class of liposomes derived from membrane lipids isolated from archaea. Archaeosomes are composed of lipid ethers, i.e. diether structures (archaeols) or tetraether structures (caldarchaeols) (e.g. Kaur et al., Drug delivery 23 (2016), 2497; Patel et al., Critical Reviews in Biotechnology 19 (1999), 317; Fig. 4). Diether structures consist of a glycerol moiety with two phytanyl chains (20-40 carbons long) at the sn-2,3 positions. Tetraether structures have two diphytanyl chains linked antiparallel (caldarchaeols) or parallel (isocaldarchaeols) to two glycerol residues. Additionally, one or several cyclopentane rings may be present. In general, archaeosomes are composed of a wide range of heterogenous lipid structures that greatly affect their properties. Depending on the composition (amount of archaeols vs. cardoarchaeols), the lipid layers are monolayers or bilayers or a mixture of both. Total archaeal lipids obtained by solvent extraction represent about 5% of the cell dry weight. As shown in several studies, the type and structure of archaeal lipids vary depending on the organism from which they were extracted and the respective living environment (e.g. temperature) as well as the growth phase (Jensen et al., Life 5 (2015), 1539). For example, increasing the growth temperature for M. jannaschii from 50 to 65 °C changed the % core lipid distribution of archaeols, cardoarchaeols, and macrocyclic archaeols from 60%, 21%, and 19% to 15%, 42%, and 43%, respectively (Sprott et al., J. Bact. 173 (1991), 3907). For the archaeon Sulfolobus, membrane fluidity is also speculated to be controlled by the number of cyclopentane rings in the phytanyl chains of the cardoarchaeolipids, which increases with increasing growth temperature (Jensen et al., 2015).

[0004] US 5,098,588A discloses the use of archaeal lipids as a new class of lubricants for metal surfaces. The document does not disclose the process of culturing archaea, nor does it describe compositions in which specific quantitative amounts of various archaeal lipids are provided. WO 2007 / 112567A1 discloses polar synthetic lipids that contain at least one carbohydrate or anionic group covalently linked to at least one free hydroxyl group of an archaeal core lipid, where Thermoplasma is used as a source of cardoarchaeols. The document discloses that the use of these lipids obtained from Thermoplasma is particularly preferred compared to the use of Sulfolobus as a source, in order to avoid the mixture of cardoarchaeols and nonitol-caldoarchaeols found in Sulfolobus. DE 10 2012 216 378 A1 describes an immobilization matrix comprising a tetraether lipid layer onto which ligands (capture molecules) capable of binding to targets can be covalently immobilized, as well as a biosensor chip comprising said matrix for chip-based analytical methods.

[0005] Currently, archaeosomes have received more and more attention because they have been proven to be highly stable against heat, pH, bile salts, lipase, and oxidation, which makes them more attractive than traditional liposomes. Several in vitro studies have shown that archaeosomes can be used as a versatile nanocarrier system for various cargoes, which has great promise in fighting various diseases. However, a breakthrough in the application of archaeosomes has yet to be seen due to the following limitations: 1) Archaeosomes are produced in complex environments (complex media and uncontrolled shake flask cultures) and are therefore highly heterogeneous; 2) Archaeosomes cannot be manufactured under defined conditions, in contradiction to regulatory guidelines (Quality by Design); 3) the inability to control the lipid composition of archaea; 4) Archaeosomes cannot be produced in large quantities cost-efficiently. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, one object of the present invention is to reduce the heterogeneity of archaeosomes and to allow the production of tailor-made archaeolipids by controlling process parameters under controlled culture conditions for archaea. More specifically, the object of the present invention is to provide an archaeosome composition that contains a variety of cyclopentane ring lipids, but at the same time also contains phosphatidylarchaeols. It is a further object to provide a composition of archaeolipids suitable for a particular delivery route and / or administration (e.g. oral delayed delivery and / or oral rapid delivery). It is a further object to provide an archaeolipid composition with adequate archaeosome stability (e.g. defined as the ability to stably encapsulate a drug of interest and allow passage through the stomach or skin or mucous membranes) and / or sufficient functionality (e.g. defined as the ratio between cardoarchaeols and archaeols). Another object of the present invention is to prepare lipid mixtures with the maximum possible number of rings in the phytanyl chain of cardoarchaeols, while maintaining the adjustability (within physiological limits) of the ratio of cardoarchaeols:archaeols in archaeobic liposomes.

[0007] Thus, the present invention provides a method for culturing Sulfolobus in a defined medium and controlled process conditions for a specific, more uniform lipid composition to produce tailored archaeosomes with reduced heterogeneity as vesicles for the biopharmaceutical industry. [Means for solving the problem]

[0008] More specifically, the present invention relates to a method for producing a medicament for use in a method for producing a medicament comprising the steps of: (1) growing a Sulfolobus cell culture at a temperature between 75°C and 85°C and a growth rate between 0.025 and 0.035 per hour, and extracting archaeal-type lipids from the archaeal culture to obtain a composition for use in oral delayed therapy; or (2) growing a Sulfolobus cell culture at a temperature between 75°C and 85°C and a growth rate between 0.005 and 0.015 per hour, and extracting archaeal-type lipids from the archaeal culture to obtain a composition for use in oral rapid therapy; The present invention provides a method for producing a composition comprising archaeal lipids from a Sulfolobus cell culture, the method comprising: [Brief description of the drawings]

[0009] [Figure 1] The composition of lipid patterns as a function of growth temperature is shown. [Diagram 2] The composition of lipid patterns as a function of proliferation rate is shown. [Diagram 3] The composition of lipid patterns according to process mode is shown. [Figure 4] The structures of the major lipids of Sulfolobus acidocaldarius are shown. [Diagram 5] The effect of growth temperature on the number of cyclopentane rings is shown. [Figure 6] Spectral data for biomass samples grown at 68° C. are shown. [Figure 7] Spectral data for biomass samples grown at 75° C. are shown. [Figure 8] Spectral data for biomass samples grown at 82° C. are shown. [Figure 9] Spectral data for biomass samples grown at a growth rate of 0.010 h-1 are shown. [Figure 10] Spectral data for biomass samples grown at a growth rate of 0.020 h −1 are shown. [Figure 11] 4 shows the spectral data for biomass samples grown at a growth rate of 0.034 h −1 . [Figure 12] 1 shows spectral data for biomass samples grown in fed-batch mode. [Figure 13] 1 shows spectral data for biomass samples grown in continuous mode. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Archaeosomes have advantageous properties in the prior art that make them promising candidates for future medicines. However, until now, the major obstacles to making archaeosomes a practically suitable candidate were mainly the lack of control over the manufacturing process and the high variability regarding their lipid ether composition. The present invention now enables such an industrial approach, making archaeosomes a realistic candidate for drug cargo intended to be delivered to human patients.

[0011] For therapeutic use in humans, formulations are generally required to have a minimum shelf life of 1-2 years. This means physical stability (aggregation, cargo loss) and chemical stability (oxidation, hydrolysis) during storage. Recently, the use of liposomes in drug / vaccine delivery has been extended to oral delivery systems. Conventional liposomes cannot be used for this purpose due to their poor stability in the harsh environment of the gastrointestinal (GI) tract (pH as low as pH 1 in the stomach and attack by lipases and bile salts). The main advantage of archaeosomes over conventional liposomes is their high stability. Different stability aspects have been tested so far, showing the advantages of archaeosomes over liposomes. Storage stability: Archaeosomes contain saturated phytanyl chains and therefore are not oxidized by air. As a result, archaeosomes can be stored in chloroform / methanol at room temperature for years without any changes. ● Thermostability: Thermostability of archaeosomes, for example those made from Sulfolobus acidocaldarius, compared to conventional liposomes showed that unlike conventional liposomes that become permeable and lose their cargo at 35°C to 40°C, archaeosomes did not become leaky at much higher temperatures. The ring structures in archaeotype lipids deposited as a thermoadaptive response result in tight membrane packing and reduced membrane fluidity. This high thermostability allows sterilization by autoclaving, a crucial requirement for in vivo applications of lipid vesicles. In contrast, heat sterilization of conventional liposomes results in aggregation, hydrolysis, and leakage, making the preparation of sterile liposomes a challenging endeavor. ● pH stability: In vitro assays revealed that archaeosomes are more stable at acidic pH than conventional liposomes: after 21 days of storage at pH 3.0, liposomes lost up to 60% of their cargo, whereas archaeosomes only lost 30%. Stability against lipases: Lipases in the gastrointestinal tract cause severe hydrolysis of liposomes, resulting in rapid release of the encapsulated cargo. Previous studies have shown that conventional liposomes lost up to 100% of their cargo when exposed to phospholipase A2 for 4 hours, whereas archaeosomes lost less than 20%. Stability in the presence of bile salts: Archaeosomes were shown to retain all of their cargo upon incubation in simulated human bile at 37°C for 90 min, a time longer than the average residence time of lipid vesicles in the stomach. Stability against the combined effects of lipases and bile salts: In the gastrointestinal tract, lipid vesicles encounter both lipases and bile salts, which affect the stability of lipid vesicles. Conventional liposomes lose 100% of their cargo already after 30 min when incubated in the presence of pancreatic lipase in simulated human bile, whereas lipases and bile salts show no synergistic effect on archaeosomes.

[0012] In summary, archaeosomes already have the ability to surpass currently used liposomes in terms of their stability (more stable over time, more resistant to heat, pH, lipase, bile acids, autoclavable). According to the present invention, a reliable method is provided to allow suitable and reproducible compositions for specific delivery of drugs via archaeosomes. Thus, the present invention allows the production of archaeosomes of sufficient quality and sufficient quantities.

[0013] Archaeosomes have been recognized as very useful carriers for the delivery of various cargoes. The most promising drug carrier application to date is the use of archaeosomes as microcapsules for the oral delivery of therapeutic proteins or peptides (such as insulin). Archaeosomes have also been considered as highly interesting adjuvants in vaccines, since the uptake of archaeosomes in phagocytes is more than 50 times greater than conventional liposomes. Studies in mice have shown that systemic administration of several test antigens entrapped in archaeosomes conferred a humoral immune response comparable to that obtained with the potent but toxic Freund's adjuvant. All in vitro and in vivo studies performed to date have shown that archaeosomes are safe and do not cause any appreciable toxicity. In general, the requirements for lipid carriers in medical applications are: ●Safety, stability, bioavailability, cost-effectiveness ●No toxicological effects on the human body Induction of non-pathological inflammatory responses Promoting crosstalk between the innate and adaptive immune systems Enhancement of humoral antibody responses and induction of T cell-mediated responses

[0014] The archaeosome meets all of these requirements and has therefore been successfully tested as a drug delivery system for cancer vaccines, immune adjuvants against Chagas disease, novel gene delivery systems, carriers for the oral delivery of proteins and peptides, novel antigen delivery systems, etc.

[0015] A wide range of potential medically relevant applications of the archaeosome have been demonstrated in vitro and in animal models, but clinical application has yet to be realized due to insufficient uniformity of the lipid composition.

[0016] Archaeosomes have been successfully prepared from various archaeal species. Usually, total archaeal lipids are extracted from frozen biomass by organic solvent extraction with chloroform / methanol / water. Polar and neutral lipids are then separated by precipitation with acetone. The resulting liquid extract can be used directly for the preparation of archaeosomes as a complex mixture of different lipid classes or can be further purified by chromatography to isolate pure polar lipids of a specific class. Starting from these special, naturally occurring archaeal lipids, it is possible to make preparations of archaeosomes and encapsulate or associate hydrophilic or hydrophobic compounds using available methods developed for the preparation of conventional liposomes (mechanical dispersion, lipid hydration, membrane extrusion, sonication, etc.). Methods for preparing lipids from archaea or culturing archaea are disclosed, for example, in US2017 / 0152533A1, US6,316,260B1, EP1999137B1, EP0883624B1, EP2109459B1, Siliakus et al. (Extremophiles 21 (2017), 651-670), Jain et al. (Frontiers in Microbiol. 5 (2014), article 641).

[0017] Although archaea are known as a useful source of these special lipids, they have not yet been widely used for this purpose. This is because, currently, the composition of liposomes cannot be controlled and is highly heterogeneous. Most archaea are cultivated on complex media in shake flasks under uncontrolled conditions, leading to batch-to-batch variations in terms of lipid class composition, lipid core structure and branching. Furthermore, the composition and properties of the lipodome have not been controllable so far. However, this is essential for reproducible high-quality archaeosome formation with reduced heterogeneity and specific properties required for pharmaceutical applications. It has been observed that the lipid composition of S. islandicus and S. tokodaii was not constant between different processes, especially when the temperature was changed or samples were taken at different growth phases (Jensen et al., Life 5 (2015), 1539). By culturing archaea in a defined medium in the controlled environment of a bioreactor, the present invention provides efficient control of the quality (heterogeneity and composition) of archaeal lipid ethers that prevent the preparation of well-defined archaeosomes by varying key process parameters (e.g. temperature, growth rate, etc.).

[0018] Archaeosomes can be used for a similar task as liposomes (delivering drugs to patients), but they show much better properties than liposomes (they are more stable over time, more resistant to heat, pH, bile acids, and are autoclavable). Archaeosomes can be produced in sufficient quality and quantity, making them promising as a viable alternative to conventional liposomes.

[0019] The global liposome drug delivery market was valued at US$ 2.4 billion in 2017 and is projected to reach US$ 5.2 billion by the end of 2025, growing at a CAGR of 10.1% from 2018 to 2025.

[0020] A recent review of the clinical use of liposomal formulations listed a total of 15 approved clinical products worldwide, covering the areas of anticancer drugs, anti-fungal drugs, vaccines, anti-inflammatory drugs, and therapeutic genes.

[0021] For the use of archaeasomes as carriers of pharma- ceutical active ingredients, different release kinetics must be ensured depending on the desired site of action and pharmacokinetics, which can essentially be influenced by the stability of the archaeasomes.

[0022] For stability, in addition to the basic type of lipid, in particular the ratio of short-chain lipids to long-chain lipids is important; also the presence of ring structures in the lipid chains that restrict the mobility of the chains, resulting in a tighter packing and a reduction in membrane permeability. The combination of archaeal (Arc, 40 total carbon atoms in the phytanyl chain) and caldarchaeal (Cal, 80 total carbon atoms in the phytanyl chain) represents a suitable approach here. Both substances are natural membrane constituents of Sulfolobus acidocaldarius and other archaeal species. Caldarchaeal variants exist with different modifications of the terminal glycerol residue (no modification, phosphatidylinositol (PI), dihexose (2Hex), and combinations thereof; Cal, PI-Cal, 2Hex-Cal, PI-Cal-2Hex). Furthermore, Cal variants exist with different numbers of rings in the phytanyl chain (preferably 4, 5 or 6 rings in the case of S. acidocaldarius). Archaeoles exist primarily as PI-Arc.

[0023] For a number of reasons, a mixture of 2Hex-Cal and PI-Arc is a particularly interesting combination for the preparation of archaeosomes according to the invention, especially for oral use. • Cardoarchaeoles (multiple) with different numbers of rings can be used. • The mixture of short and long chain lipids ensures an effect on the stability of the archaeosomes formed. • Both lipids have a sufficient number of functional groups in their head groups for efficient modification with linkers (e.g., attachment of antibody fragments, aptamers, etc.) for use in targeted drug delivery. • This modification can be selective for one of the two lipid classes since the head groups differ in structure and chemistry.

[0024] As experimentally shown by the present invention, it is possible to influence the ratio of Cal to Arc via the process parameters temperature and growth rate (at optimal growth temperature and highest growth rate the Cal:Arc ratio is highest and vice versa). However, the proportion of relatively high ring numbers (6 rings vs. 5 or 4 rings) surprisingly depends only on temperature and does not correlate with growth rate. The effect of temperature on ring number has been observed in Sulfolobus spp., but no literature is known about the effect of growth rate.

[0025] Essentially, the maximum possible number of rings is necessary for the lowest possible membrane permeability and thus gastric resistance, which the present invention has shown requires incubation at the highest possible temperature. This leads to the loss of temperature, a suitable tool for establishing the Cal:Arc ratio, which according to the present invention can be controlled by the growth rate. For drug release in the intestine, the archaeosomes must not be too stable, because otherwise the drug cannot be delivered. This maintains the tunability of the release kinetics of the archaeosome preparations according to the present invention, and medicines produced on the basis of archaeosomes from tailored lipid mixtures provided by the present invention have different durations of action in the intestine, for example after oral administration.

[0026] The present invention is based on at least two remarkable results obtained in the course of the invention. First, the present invention provides a controlled production method that allows a reproducible and defined composition of archaeal lipids. Second, this knowledge of applying and using the controlled production method of archaeal lipids according to the present invention (by specific and different culture conditions) allows to provide also in a reproducible scaled-up format two specific types of archaeal compositions by applying different culture conditions, where said two specific types of archaeal compositions can be used in particular (a) for oral delayed treatment (i.e. for pharmaceutical compositions that show a delayed effect) or (b) for oral rapid treatment (i.e. for pharmaceutical compositions that show an immediate release to the patient when delivered orally).

[0027] Bioreactor cultivation methods have been developed in recent years with new analytical tools, computer-directed process monitoring and software-guided process control, achieving a completely different level of breakthrough technology in the cultivation of microorganisms. This has had a particular impact on cultivation techniques for microorganisms that require complex cultivation conditions, such as archaea, which require cultivation temperatures that are significantly higher than the usual cultivation temperatures of 30° C. or 37° C. or even room temperature. Modern methods of observing process parameters, which were not available 10 or 15 years ago, allow the method according to the invention to be the necessary basis for a controllable and reproducible production method for the cultivation of archaeal cell cultures according to the invention, in particular for an effective control of the growth rate.

[0028] For example, Patel et al. (1999, supra) report in Table 1 the relative abundance (expressed in %) of the main core lipids in representative archaea. Due to the lack of suitable analytical tools at that time, only the core lipids were analyzed semi-quantitatively, i.e., as relative amounts. Apart from the low growth temperature (70° C.; compared with the optimum growth temperature of 75° C. or higher), lipids were obtained from the late exponential growth phase. This was because, at that time, more specific growth phases were simply not controllable and varied greatly during the culture (Sprott et al., Can. J. Microbiol. 43 (1997), 467-476). Until the introduction of the new culture methods now available, it was not possible to handle archaeal cell cultures in such a way that culture parameters such as specific growth rate could be controlled. Thus, the prior art could not even provide archaeal-type lipid compositions reproducibly provided with different - but always and reproducibly defined - qualitative and quantitative lipid body compositions due to different process parameters and / or different culture conditions. This has been done for the first time for archaeal lipids derived from archaeal cell cultures provided by the present invention, and in particular for the first time for Sulfolobus cell cultures.

[0029] According to the invention, it is possible to provide lipid mixtures with the maximum possible number of rings in the phytanyl chain of the cardoarchaeologous species, while maintaining the possibility of adjusting the Cal:Arc ratio (within physiological limits). 2Hex-Cal and PI-Arc are particularly preferred target species according to the invention due to their particular chemical suitability. Therefore, according to the invention, compositions can be provided that contain archaeosomes with a high number of rings due to the elevated growth temperature, and at the same time, the Cal:Arc ratio can be adjusted due to the growth rate. Due to the adjusted process conditions, the invention makes it possible to obtain lipid extracts that can be used directly or with minimal purification efforts to produce archaeosomes with high gastric juice resistance and tailor-made release kinetics for various pharmacoactive ingredients.

[0030] In accordance with the present invention, two specific compositions are provided that include archaeal lipids from Sulfolobus cell cultures, one of which is particularly suitable for use in oral delayed therapy and the other of which is particularly suitable for oral rapid therapy. In accordance with the present invention, the terms "oral delayed therapy" and "oral rapid therapy" are used in their ordinary sense. "Delayed" (or "time-release", "sustained release", or "extended release") oral treatments are those that exhibit a "retard effect" allowing for continuous release after some time has passed since delivery, e.g., 12 hours after (oral) delivery, while "rapid" (or "immediate release") oral treatments are released immediately after administration, with maximum release shortly after delivery, e.g., within 3 hours of oral administration. (Pschyrembel, "retard effect": protracted release of an active substance due to galenic formulation; Andrade, J. Clin. Psychiatry 76 (2015), e995-e999; Perrie et al. "Controlling drug delivery" in Pharmaceutics: drug Delivery and Targeting" (2012), Pharmaceutical Press).

[0031] The lipids produced for the production of oral archaeosomes with sustained release effect are particularly suitable for the needs of oral administration and therefore show high gastric juice resistance. This resistance is protected by the presence of a large number of cyclopentane rings. According to the invention, it has been found that this can be achieved by applying high process temperatures, in particular by applying process temperatures in the range of 75-85°C. For the retardation effect necessary for sustained release, a sustained release of the active substance over a longer period is required, which therefore requires temporal stability. According to the invention, this can be achieved by the presence of PI-Arc, which is less destabilizing. The invention provides the teaching that in order to obtain this particular lipid composition, a high growth rate is necessary to obtain the particular lipid composition, i.e. a growth rate of preferably 0.025-0.035 per hour, in particular 0.027-0.033 per hour. In a preferred embodiment of the invention, the method provides a composition for use for oral retardation therapy, wherein the ratio of dihexose-caldoarchaeal (2Hex-Cal) to phosphatidylinositol-Archaeal (PI-Arc) is 1:5 to 1:7, particularly 1:5.5 to 1:6.5.

[0032] The lipids produced for the production of oral archaeosomes for rapid treatment are on the one hand also particularly suited to the need for oral administration and therefore exhibit high gastric juice resistance. This resistance, as in the case of delay, is protected by the presence of a large number of cyclopentane rings. According to the invention, it has been found that this can be achieved by applying high process temperatures, in particular by applying process temperatures in the range of 75-85°C. On the other hand, compositions for rapid treatment require a rapid release of the drug in the intestine. Therefore, a lower stability of said composition is also required. According to the invention, this can be achieved by the presence of PI-Arc, which is more prone to destabilization. The invention provides the teaching that in order to obtain this particular lipid composition, a low growth rate is necessary to obtain the particular lipid composition, i.e. a growth rate of preferably 0.005-0.015 per hour, in particular 0.007-0.013 per hour. In a preferred embodiment of the invention, the method provides a composition for use for oral rapid therapy, wherein the ratio of dihexose-caldoarchaeal (2Hex-Cal) to phosphatidylinositol-Archaeal (PI-Arc) is 1:2 to 1:4, particularly 1:2.5 to 1:3.5.

[0033] These compositions obtainable by this method can provide tailor-made archaeosomes in which the drug to be delivered can be packaged for administration, preferably oral administration, to a human patient in need of an effective amount of the drug.

[0034] Cultivating Sulfolobus cell cultures is well available to the skilled artisan and is, in principle, a long-established technique (as early as Brock et al., Arch. Mikrobiol. 84 (1972), 54-68). A preferred Sulfolobus strain is Sulfolobus acidocaldarius; this strain is one of the established strains in the art. A repository for this Sulfolobus organism used may be, for example, the German strain collection for microorganisms (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ), catalogue number DSM 639. An alternative source of supply is, for example, the American Type Culture Collection (ATCC), catalogue number ATCC 33909. Other archaea are also available, for example, at the DMSZ or ATCC.

[0035] After culturing the Sulfolobus cell culture under defined culture conditions, the Sulfolobus cell lipids are extracted. This can be done by methods disclosed in the art. Preferably, the cells are homogenised after culturing, preferably by homogenisation, particularly high pressure homogenisation, or by sonication, so that lipids can be extracted from the aqueous culture. One preferred method for purifying lipids according to the present invention is supercritical fluid extraction, particularly extraction with supercritical CO2 (see, for example, Hanif et al., Int. J. Mol. Sci. 13 (2012), 3022-3037).

[0036] Prior to extraction, the homogenised cells are preferably mixed with a buffer, such as a buffer known and described in the art as suitable for the extraction of Sulfolobus cell lipids, for example an ammonium acetate buffer, particularly an ammonium acetate buffer containing 100-200 mM ammonium acetate.

[0037] According to a preferred embodiment of the present invention, the archaeal lipids are obtained by extracting an aqueous phase containing cells and / or homogenised cells into an organic phase, preferably a chloroform / methanol organic phase, and phase separating said organic phase from said aqueous phase. The organic phase then contains the lipid groups in the desired "tailor-made" ratio. The archaeal lipids can then be obtained by evaporating the organic phase, preferably by evaporating the organic phase to a dry state.

[0038] Preferably, the obtained archaeal lipid is combined with a pharma- ceutical active drug, also preferably with a pharma- ceutical acceptable carrier, to obtain a composition comprising the archaeal lipid and said pharma- ceutical active drug. This is preferably done after a purified composition (archaeosome) comprising the archaeal lipid is provided; however, it is also possible that said drug and / or further components have already been added in the purification process and are co-purified with said lipid.

[0039] According to another preferred embodiment of the method, the archaeal lipids are obtained by supercritical fluid extraction, preferably by extraction with supercritical CO2. In another aspect, the present invention also relates to compositions comprising archaeal lipids obtainable by the method according to the invention. These "tailor-made" archaeosome compositions are novel and highly useful for packaging drugs to be delivered to patients, in particular for delivering drugs known to be suitable for therapeutic or prophylactic use in humans.

[0040] The present invention also provides two specially designed compositions, one for oral delayed use and the other for oral active use. Thus, the preferred compositions according to the present invention have a ratio of dihexose-caldoarchaeoI (2Hex-Cal) to phosphatidylinositol-ArchaeoI (PI-Arc) of 1:5 to 1:7 (delayed oral use) or a ratio of dihexose-caldoarchaeoI (2Hex-Cal) to phosphatidylinositol-ArchaeoI (PI-Arc) of 1:2 to 1:4 (rapid oral use). Both of these compositions defined by this specific 2Hex-Cal / PI-Arc ratio have high resistance to gastric juices, which allows them to pass through the stomach efficiently after oral administration. The delayed oral composition further contains PI-Arc which is less destabilized and therefore has delayed release properties in the intestine, which allows a timely delayed release. Rapid oral compositions are less stable in the intestine, and therefore the packaged drug can be released immediately from the liposomes, allowing for rapid delivery of the drug for intestinal uptake. Unexpectedly, culturing Sulfolobus cell cultures at a growth rate of 0.020 per hour did not yield delayed or immediate release compositions with the specific 2Hex-Cal / PI-Arc ratios defined for delayed / rapid compositions.

[0041] This is all the more unexpected in light of the data obtained on lipid composition, e.g., Table 1, by Patel et al. (1999, supra), which was obtained without controlling growth conditions (see, e.g., Sprott et al., 1997, supra).

[0042] The composition according to the invention is suitable for packaging any drug suitable for packaging in liposomes. The composition according to the invention is therefore particularly suitable for further comprising a drug, preferably a drug for pharmaceutical use in humans, in particular a drug that is the subject of an approval issued in the European Union or the United States for marketing the product as a medicinal product. However, since there are practically no physicochemical limitations on the drug to be combined in the archaeosome preparation, any drug can be packaged in the lipid composition according to the invention. It is therefore preferred to include a drug in the lipid composition according to the invention, which is specified for human (or animal) use, whether the drug is the subject of a national or regional marketing authorization or the drug is to be administered to humans for other reasons.

[0043] A preferred composition according to the present invention comprises 5% w / w or more, preferably 10% w / w or more, of PI-Arc, and 50% w / w or more of 2Hex-Cal (% w / w is % w / w of total lipids).

[0044] According to a more general aspect, the present invention provides a method for producing a composition comprising archaeal lipids from an archaeal culture, the method comprising growing an archaeal cell culture under controlled conditions and extracting archaeal lipids from the archaeal culture to obtain a composition comprising archaeal lipids. The present invention provides a controlled production method allowing a reproducible and defined composition of archaeal lipids. Preferably, the controlled conditions are selected from pH, growth rate, temperature and process mode. The method according to the invention is in principle applicable to all archaeal cells. Thus, the archaeal cell culture of the present invention is a culture of S. acidocaldarius, M. hungatei, M. voltae, M. concilii, M. smithii, M. espanolae, T. acidophilum, M. mazei, M. espanole, T. acidophilum, H. salinarum, M. smithii, M. stadtmanae, H. halobium, H. morrhuae, M. jannaschii, S. islandicus, S. solfataricus, S. shibatae, S. tokodaii, S. metallicus, M. sedula, H. hispanica, and H. volcanii, preferably a culture of S. acidocaldarius.

[0045] Preferred temperatures for growing archaeal cultures can be optimized using the teachings of the present invention and are preferably in the range of temperatures from 55 to 90°C, preferably in the range of 70 to 85°C, especially in the range of 75 to 85°C.

[0046] Particularly preferred embodiments of the invention include growing the archaeal culture at a growth rate of 0.025 to 0.035 per hour, in particular 0.027 to 0.033 per hour, or at a growth rate of 0.005 to 0.015 per hour, in particular 0.007 to 0.013 per hour.

[0047] A particularly preferred embodiment of the invention involves growing the archaeal culture at a pH between 2 and 4, preferably between 2.5 and 3.5.

[0048] The invention can be applied by growing archaeal cultures in continuous mode and by growing archaeal cultures in batch mode, in particular fed-batch mode.

[0049] The method according to the invention preferably comprises extracting archaeal lipids by two-phase extraction, organic extraction, in particular Soxhlet, high pressure or high temperature extraction, ionic liquid extraction or supercritical fluid extraction.

[0050] According to a preferred embodiment, the method comprises extracting archaeal lipids with a solvent selected from the group consisting of water, hexane, chloroform, acetone, diethyl ether, methanol, ethanol, ethyl acetate, dichloromethane, MTBE, CO2, and mixtures thereof. The present invention is further illustrated by the following examples and figures, but is not limited thereto.

[0051] FIG. 1 shows the composition of lipid patterns as a function of growth temperature. FIG. 2 shows the composition of lipid patterns as a function of proliferation rate. FIG. 3 shows the composition of lipid patterns depending on the process mode. FIG. 4 shows the structures of the major lipids of Sulfolobus acidocaldarius. FIG. 5 shows the effect of growth temperature on the number of cyclopentane rings. FIG. 6 shows the spectral data for biomass samples grown at 68° C. FIG. 7 shows the spectral data for biomass samples grown at 75° C. FIG. 8 shows the spectral data for biomass samples grown at 82° C. Figure 9 shows the time t0.010h -1 4 shows spectral data for biomass samples grown at growth rates of 100-200 μg / ml. Figure 10 shows the time -14 shows spectral data for biomass samples grown at growth rates of 100-200 μg / ml. Figure 11 shows the time 0.034h -1 4 shows spectral data for biomass samples grown at growth rates of 100-200 μg / ml. FIG. 12 shows the spectral data of biomass samples grown in fed-batch mode. FIG. 13 shows the spectral data of biomass samples grown in continuous mode. EXAMPLES

[0052] 1. Production of Tailor-Made Archaeal Lipids The present invention describes techniques for the production of unique lipid patterns in Sulfolobus acidocaldarius with defined and tunable composition by varying process parameters in the upstream process. This example shows that by varying key process parameters during upstream processing, such as temperature, growth rate, process mode, the organism Sulfolobus acidocaldarius produces different lipid patterns (ratios between distinct lipid fractions). Thus, as a result, different lipid compositions can be harvested after downstream processing. Since the lipid pattern can be influenced by a wide number of key process parameters (temperature, growth rate, process mode, pressure, osmolality, medium composition, dissolved oxygen content, etc.), very specific tailoring of the pattern is possible.

[0053] The following paragraphs provide a showcase of the impact of (A) temperature, (B) growth rate, and (C) process mode on the ratio of lipid fractions. Depending on the application of the lipid mix by the user / customer, the desired lipid pattern can be tailored by careful selection of the required process parameters. Cultures were grown on defined media in a controlled bioreactor environment.

[0054] 1.1 Temperature effects The temperatures were set at 68 / 75 / 82 °C for a period of three generation times for each temperature, after which biomass samples were taken. The relative abundance of the five major lipid fractions was analyzed by MALDI-MS (positive ion mode; Na + The results are shown in Figure 1 and Figures 5 to 8.

[0055] 1.2 Effect of growth rate The growth rates are 0.010 / 0.020 / 0.034 h for three generation times for each growth rate. -1 The relative abundance of the five major lipid fractions was determined by MALDI-MS (positive ion mode; Na + The results are shown in Figures 2, 9, 10 and 11.

[0056] 1.3 Influence of process mode The cultivations were run in different process modes (fed-batch and continuous) for a period of three generation times for each process mode, after which biomass samples were taken. The relative abundance of the five main lipid fractions was analyzed by MALD-MS (positive ion mode; Na + The results are shown in Figures 3, 12, and 13.

[0057] 2. Materials and Methods 2.1 Upstream: Cultivation of Sulfolobus acidocaldarius DSM639 in a bioreactor To produce tailor-made archaeal lipids, S. acidocaldarius was cultivated in a continuous process mode to ensure constant process conditions for more than three generation times. Prior to the continuous phase, batch and fed-batch phases were performed to increase biomass. The volumes in the following paragraphs are exemplary and can be scaled up or down as needed.

[0058] Precultures of Sulfolobus acidocaldarius DSM639 were grown for 100 h in a defined medium according to Quehenberger et al. (Journal of Biotechnology (2019) Submission JBIOTEC-D-18-01483; “VD medium”; see table below) in 100 mL shake flasks in a rotating oil bath at 75 °C and 100 rpm. [Table 1]

[0059] The preculture was aseptically transferred to a culture vessel containing a defined medium according to Quehenberger et al. (2019) with a total starting volume of 1.5 L for the batch phase. The batch phase was performed at 75 °C until a decrease in the CO2 signal was observed, followed by a fed-batch phase (medium containing 10 g / L monosodium glutamate and 3 g / L D-glucose, exponential feed, μ = 0.03 h) at 75 °C. -1 ) until a total volume of 2.5 L was reached. The fed-batch phase was followed by continuous cultivation at the desired growth temperature and growth rate. Biomass was harvested after at least 3 generation times to allow sufficient time for the organism to adapt to the specific growth conditions during the continuous phase.

[0060] The calculation of the initial flow rate of the feed (F0) for the exponential phase is based on the mass balance of the carbon source. Equation 1: General mass balance

number

number

[0061] Equation 2: Simplified Mass Balance for Fed-Batch After simplification

number

number

[0062] Equation 3: Initial feed rate during fed-batch phase (assuming broth density is 1 kg / L) Set the right-hand side (constant terms) to 0 and rewrite it as follows:

number

[0063] For calculation of the initial feed rate (F0, Eq. 3), x0 was determined by OD600 measurements and a calibration curve for conversion of OD600 to dry cell weight. S0 was calculated as the sum of the concentrations of monosodium glutamate and D-glucose, assuming an experimentally determined biomass yield of 0.33. Then, F0 was exponentially increased during the fed-batch phase according to Eq. 4. Equation 4: Calculation of the feed rate during the fed-batch phase

number

[0064] The feed was controlled by the weight of the feed vessel and used a PID controller to compensate for deviations in the feed pump. During the continuous phase, the value of the dilution rate (D) was set equal to the desired growth rate (D=μ), whereby the dilution rate was calculated as the sum of the added acid stream and feed. R ) to determine the feed rate (F conti ) to F conti =D×V R (The density of the broth was assumed to be 1 kg / L.)

[0065] Dissolved oxygen levels were monitored online and maintained above 15% by aeration with pressurized air at 0.25–0.5 vvm (volume of gas / culture volume / min). If necessary, air was replaced with pure oxygen. pH was monitored online and maintained at 3.0 ± 0.1 by addition of H2SO4 (9.6% v / v). The feed medium was delivered by a peristaltic pump module according to a feed-forward controlled exponential feed strategy. Mixing was performed at 300–500 rpm. The CO2 content in the off-gas was monitored.

[0066] 2.2 Downstream: cell harvest, biomass homogenisation and lipid extraction Cells were harvested by centrifugation at 14,000g for 15 min. After discarding the supernatant, the cell pellets were stored at -20°C for further processing. Cell pellets from 100 mL culture broth per sample (dry cell weight at harvest is shown in Table 1) were thawed and resuspended in 10 mL ice-cold ammonium acetate buffer (155 mM). The suspension was homogenised by sonication (6 times for 1 min with 30 s rest between pulses) in a glass beaker in an ice / water bath. After homogenisation, the samples were diluted with ice-cold ammonium acetate buffer (155 mM) to an OD600 of 1.5. 3.3 parts of ice-cold 2:1 CHCl3 / methanol mixture were added to 1 part of the diluted homogenate and the resulting mixture was vortexed at 1,400 rpm for 30 min at 4°C. The samples were centrifuged for phase separation (2 min, 1,000g, 4°C). The organic phase was collected in a glass vial and the sample was evaporated to dryness with N2 at room temperature. The lipids were stored at -20 °C for further analysis.

[0067] 2.3 Analysis: Identification of lipid fractions by MALDI mass spectrometry The dried lipid fractions were reconstituted in a 1:3 CHCl3 / methanol mixture. These fractions were immediately used for MALDI sample preparation using 2-4-,6-trihydroxyacetophenone (THAP) as the MALDI matrix (approximately 15 mg THAP / mL, with or without added sodium chloride to promote sodium adduct ions). Finally, the matrix and analyte solutions were mixed 1:1 for deposition onto a 384-well plate stainless steel MALDI target. The mass spectrometer used was a Shimadzu MALDI 7090 TOF / RTOF tandem time-of-flight mass spectrometer equipped with a grounded gas collision cell for high-energy CID experiments (E LAB= 20 keV, collision gas: helium). Prior to the tandem mass spectrometry experiments, samples were measured by positive and negative ion reflectance TOF-MS. Mass calibration was performed using a commercial peptide mixture provided by Shimadzu, which allows mass calibration up to m / z 3600. In the positive ion mode, depending on the salt doping, mainly the [M+Na] of the analyte molecule was observed. + and [M+2Na-H] + Adduct ions were detected. Most lipids contained phosphoinositol head groups. All analytes were characterized by high-energy CID TOF / RTOF-MS. In contrast, in the negative ion mode, only phosphoinositol-containing lipids (and not neutral lipids) were detected, which was [MH] - The ions were also characterized by high energy CID TOF / RTOF-MS. To date, all charged lipid analytes contain phosphoinositol polar head groups. Minor lipids that may further be present in this mixture can be analyzed by HPTLC analysis and micropreparative extraction followed by MALDI analysis.

[0068] Therefore, the present invention provides the following preferred embodiments. 1. A method for producing a composition comprising archaeal lipids from an archaeal cell culture, comprising growing an archaeal cell culture under controlled conditions and extracting archaeal lipids from the archaeal culture to obtain a composition comprising archaeal lipids. 2. The method of embodiment 1, wherein the controlled conditions are selected from pH, growth rate, temperature, and process mode. 3. The archaeal cell culture contains S. acidocaldarius, M. hungatei, M. voltae, M. concilii, M. smithii, M. espanolae, T. acidophilum, M. mazei, M. espanole, T. acidophilum, H. salinarum, M. smithii, M. stadtmanae, H. halobium, H. morrhuae, M. jannaschii, S. islandicus, S. solfataricus, S. shibatae, S. tokodaii, S. metallicus, M. sedula, H. hispanica, and H. volcanii. 4. The method according to any one of embodiments 1 to 3, comprising growing the archaeal culture at a temperature between 55°C and 90°C, preferably between 70°C and 85°C, in particular between 75°C and 85°C. 5. The method according to any one of the preceding claims, comprising growing the archaeal culture at a growth rate of 0.025 to 0.035 per hour, in particular 0.027 to 0.033 per hour, or at a growth rate of 0.005 to 0.015 per hour, in particular 0.007 to 0.013 per hour. 6. The method of any one of embodiments 1 to 5, comprising growing the archaeal culture at a pH of 2 to 4, preferably at a pH of 2.5 to 3.5. 7. The method of any one of embodiments 1 to 6, comprising growing the archaeal culture in continuous mode. 8. The method of any one of embodiments 1 to 6, comprising growing the archaeal culture in batch mode, in particular in fed-batch mode. 9. The method according to any one of the preceding embodiments, wherein the archaeal lipids are obtained by extraction of an aqueous phase comprising cells and / or homogenised cells into an organic phase, preferably a chloroform / methanol organic phase, and phase separation of the organic phase from the aqueous phase. 10. The method according to any one of the preceding embodiments, wherein the cells are homogenised after cultivation, preferably by homogenisation, in particular high pressure homogenisation, or by sonication. 11. (1) growing a Sulfolobus cell culture at a temperature of 75°C to 85°C and a growth rate of 0.025 to 0.035 per hour, and extracting archaeal-type lipids from the archaeal culture to obtain a composition for use in oral delayed therapy; or (2) growing a Sulfolobus cell culture at a temperature between 75°C and 85°C and a growth rate between 0.005 and 0.015 per hour, and extracting archaeal-type lipids from the archaeal culture to obtain a composition for use in oral rapid therapy; A method for producing a composition comprising archaeal lipids from a Sulfolobus cell culture, comprising: 12. The method of any one of Examples 1 to 11, wherein the Sulfolobus cell culture is a culture of Sulfolobus acidocaldarius cells. 13. The method according to any one of the preceding embodiments, wherein the composition for use in oral retardation therapy has a ratio of dihexose-caldoarchaeal (2Hex-Cal) to phosphatidylinositol-archial (PI-Arc) of 1:5 to 1:7, in particular 1:5.5 to 1:6.5. 14. The method according to any one of the preceding embodiments 1 to 12, wherein the composition for use in oral rapid therapy has a ratio of dihexose-caldoarchaeal (2Hex-Cal) to phosphatidylinositol-archial (PI-Arc) of 1:2 to 1:4, in particular 1:2.5 to 1:3.5. 15. The method according to any one of the preceding embodiments, wherein the cells are homogenised after cultivation, preferably by homogenisation, in particular high pressure homogenisation, or by sonication. 16. The method according to embodiment 15, wherein the homogenised cells are mixed with a buffer, preferably with an ammonium acetate buffer, in particular with an ammonium acetate buffer comprising 100 to 200 mM ammonium acetate. 17. The method according to any one of the preceding embodiments, wherein the archaeal lipids are obtained by extraction of an aqueous phase comprising cells and / or homogenised cells into an organic phase, preferably a chloroform / methanol organic phase, and phase separation of the organic phase from the aqueous phase. 18. The method of embodiment 17, wherein the archaeal lipids are obtained by evaporating the organic phase, preferably by evaporating the organic phase to a dry state. 19. A method according to any one of embodiments 1 to 18, wherein the obtained archaeal lipid is combined with a pharma- ceutical active agent, and preferably combined with a pharma- ceutical acceptable carrier, to obtain a composition comprising the archaeal lipid and the pharma- ceutical active agent. 20. A method according to any one of the preceding embodiments, wherein the archaeal lipids are obtained by supercritical fluid extraction, in particular by extraction with supercritical CO2. 21. A composition comprising archaeal lipids, obtainable by a method according to any one of embodiments 1 to 20. 22. A composition comprising archaeal lipids, preferably as described in embodiment 21, in which the ratio of dihexose-caldoarchaeal (2Hex-Cal) to phosphatidylinositol-archaeal (PI-Arc) is 1:5 to 1:7 or 1:2 to 1:4. 23. The composition of embodiment 21 or 22, further comprising a drug, preferably a drug for pharmaceutical use in humans, in particular a drug that is the subject of approval issued in the European Union or the United States to market the product as a medicinal product. 24. A composition according to any one of embodiments 21 to 23, wherein the archaeosome comprises 5% w / w or more, preferably 10% w / w or more, of PI-Arc, and 50% w / w or more of 2Hex-Cal (% w / w is % w / w of total lipids). 25. A composition according to any one of embodiments 21 to 24, wherein the archaeosome comprises an antigen, an oral drug, a vaccine, an antioxidant, a virus-like particle, a nucleic acid, in particular DNA, RNA, pDNA, an antibiotic, a polypeptide, or a protein, as a drug to be administered to a patient. 26. A composition described in any one of embodiments 21 to 25, wherein the archaeosome is for oral, transmucosal, or transdermal administration. 27. A liposomal composition that is the subject of approval issued in the European Union or the United States for marketing the product as a pharmaceutical, wherein the liposome is replaced by an archaeosome provided by the method described in any one of embodiments 1 to 20.

Claims

1. (1) growing a Sulfolobus cell culture at a temperature between 75°C and 85°C and a growth rate between 0.025 and 0.035 per hour and extracting archaeal lipids from the archaeal culture; or (2) growing a Sulfolobus cell culture at a temperature between 75°C and 85°C and a growth rate between 0.005 and 0.015 per hour, and extracting archaeal lipids from the archaeal culture; wherein the Sulfolobus cell is a Sulfolobus acidocaldarius DSM639 cell. A method for producing an extract containing archaeal lipids from a culture of Bacillus sulfolobus cells.

2. The method described in claim 1, wherein the extract (1) has a ratio of dihexose-caldoarchaeal (2Hex-Cal) to phosphatidylinositol-archeal (PI-Arc) of 1:5 to 1:

7.

3. The method described in claim 2, wherein the extract of (1) has a ratio of dihexose-caldoarchaeal (2Hex-Cal) to phosphatidylinositol-archeal (PI-Arc) of 1:5.5 to 1:6.

5.

4. The method according to claim 1, wherein the extract (2) has a ratio of dihexose-caldoarchaeal (2Hex-Cal) to phosphatidylinositol-archeal (PI-Arc) of 1:2 to 1:

4.

5. The method described in claim 4, wherein the extract (2) has a ratio of dihexose-caldoarchaeal (2Hex-Cal) to phosphatidylinositol-archeal (PI-Arc) of 1:2.5 to 1:3.

5.

6. The method according to any one of claims 1 to 5, wherein the Sulfolobus cells are homogenised after cultivation.

7. The method according to claim 6, wherein the disruption is by high pressure homogenization or ultrasonic treatment.

8. 8. The method of claim 6 or 7, wherein the homogenised Sulfolobus cells are mixed with a buffer.

9. The method of claim 8, wherein the buffer is an ammonium acetate buffer.

10. The method of claim 9, wherein the ammonium acetate buffer is an ammonium acetate buffer containing 100 to 200 mM ammonium acetate.

11. 11. The method according to claim 1, wherein the archaeal lipids are obtained by extracting an aqueous phase comprising Sulfolobus cells and / or homogenised Sulfolobus cells into an organic phase and phase separating the organic phase from the aqueous phase.

12. The method of claim 11, wherein the organic phase is a chloroform / methanol organic phase.

13. The method according to claim 11 or 12, wherein the archaeal lipids are obtained by evaporating the organic phase.

14. A method according to claim 11 or 12, wherein the archaeal lipids are obtained by evaporating the organic phase to a dry state.

15. The method according to any one of claims 1 to 14, wherein the archaeal lipids are obtained by supercritical fluid extraction.

16. The method according to claim 15, wherein the supercritical fluid extraction is extraction using supercritical CO2.

17. An extract containing archaeal lipids from a Sulfolobus acidocaldarius DSM639 cell culture, the ratio of dihexose-caldoarcheols (2Hex-Cal) to phosphatidylinositol-archeols (PI-Arc) being 1:5 to 1:7 or 1:2 to 1:4.

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