WO2020187526A1 - Method for producing a composition comprising archaeal lipids from a sulfolobus cell culture - Google Patents
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- WO2020187526A1 WO2020187526A1 PCT/EP2020/054579 EP2020054579W WO2020187526A1 WO 2020187526 A1 WO2020187526 A1 WO 2020187526A1 EP 2020054579 W EP2020054579 W EP 2020054579W WO 2020187526 A1 WO2020187526 A1 WO 2020187526A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
- C12P7/6436—Fatty acid esters
- C12P7/6445—Glycerides
- C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5063—Compounds of unknown constitution, e.g. material from plants or animals
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Definitions
- the present invention relates to the production of compo sitions comprising archaeal lipids and compositions comprising archaeal lipids.
- Liposomes are spherical lipid bilayer vesicles (lipid es ters) surrounding an aqueous space. They are important carriers for the administration of drugs, vaccines, genes, proteins, small molecules, antibiotics and nutrients. Liposomes are made of phospholipids, mainly phosphatidylcholine, and cholesterol, but might also include other lipids, like phosphatidylethanola- mine . Liposomes are generated by a large number of different methods (reviewed e.g. by van Hoogevest, Eur . J. Pharm. Sci. 108 (2017), 1-12; Szoka et al . , Ann. Rev. Biophys . Bioeng.
- phospholipids for example by dispersing phospholipids in aqueous medium, e.g. using mechanical treatment (e.g. in a homogenizer, preferably by high pressure homogenization) or sonication. They vary between 0.02 to 10 pm in diameter. Their size and composi tion significantly affect their properties.
- Archaeosomes i.e. lipid vesicles which are composed partly or completely of archaeal lipids
- Archaeosomes represent a special class of liposomes originating from membrane lipids isolated from Ar- chaea.
- Archaeosomes are made of lipid ethers, namely 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) .
- Di ether structures are composed of a glycerol moiety carrying two phytanyl chains (20-40 carbons in length) on the sn-2,3 posi tions. Tetraether structures carry two di-phytanyl chains linked to two glycerol residues in either an antiparallel manner (cal- darchaeol) or a parallel manner ( isocaldarchaeol ) . Furthermore, one or several cyclopentane rings may occur. In general, archae osomes are made of a large range of heterogeneous lipid struc tures, which significantly affects their characteristics. De pending on the composition (amount of archaeols vs.
- the lipid layer is a mono- or a bi-layer or a mixture thereof.
- the total lipids of Archaea obtained by solvent ex traction, account for about 5% of the cell dry weight.
- the type and structure of archaeal lipids depend on the organism they are extracted from and their respec tive living environments (e.g. temperature) as well as growth phases (Jensen et al . , Life 5 (2015), 1539) . For example, an increase in growth temperature for M.
- US 5,098,588 A discloses the use of archaebacterial lipids as a new class of lubricants for metal surfaces. Neither a cul tivation process for the archaebacteria is disclosed in this document not are compositions with specific quantitative amounts of different archaeal lipids provided.
- WO 2007/112567 Al dis closes a polar synthetic lipid comprising at least one carbohy drate or anionic group linked by covalent bonding to at least one free hydroxyl group of an archaeal core lipid, wherein Ther moplasma is used as a source of caldarchaeol lipids.
- lipids from Thermoplasma is disclosed in this document to be specifically preferred compared to using Sulfolobus as a source, to avoid mixtures of caldarchaeols and nonitol-caldarchaeols found in Sulfolobus .
- DE 10 2012 216 378 Al describes an immobi lisation matrix comprising a tetraether lipid layer, on which ligands (capture molecules) that can bond to a target can be covalently immobilised and biosensor chips, for chip-based anal ysis methods, which comprise such matrix.
- archaeosomes are receiving increased attention as they have proven to be highly stable against heat, pH, bile salts, lipases and oxidation, making them more attractive than conventional liposomes.
- archaeosomes can be used as versatile nano carrier systems for different cargo with a huge prospect to fight various diseases.
- archaeosomes have not seen their breakthrough in application yet due to the following limita tions : 1) They are produced in a complex environment (complex cul tivation media and uncontrolled shake flask cultivations) and are thus very heterogeneous,
- the ob ject of the present invention is to provide an archaeosome com position which comprises many cyclopentane ring lipids but at the same time also phosphatidyl archaeol. It is a further object to provide compositions of archaeal lipids which are suitable for specific delivery route and/or administration, e.g. for oral retard delivery and/or for oral acute delivery. It is a further object to provide archaeal liposome compositions which have an appropriate stability (defined e.g.
- Another aim of the present invention is the preparation of a lipid mixture with the highest possible number of rings in the phytanyl chains of the caldarchaeol species while maintaining the adjustability of the ratio caldarchaeol: Ar chaeol in the liposomes derived from Archaea (within the physi ological limits) .
- the present invention provides cultivation methods for Sulfolobus on a defined medium und controlled process conditions for a certain, more homogeneous lipid composition to generate tailor-made archaeosomes with reduced heterogeneity as vesicles for the biopharmaceutical industry.
- the present invention provides a method for producing a composition comprising archaeal lipids from a Sulfolobus cell culture, comprising
- Archaeosomes have advantageous properties which have made them as promising candidates for future pharmaceutical in the prior art.
- the major obstacles from making them practically relevant candidates were mainly the lack of controllability of the production processes and their high var iability concerning their lipid ether composition.
- the present invention now enables such industrial approach making archaeo somes realistic candidates for drug cargo intended to be deliv ered to human patients:
- archaeosomes already had the potential to out- compete currently used liposomes regarding their stability (more stable over time and more resistant against heat, pH, lipases, bile acid, autoclavable ) .
- a reliable method is provided to enable suitable and reproducible composi tions for specific delivery of drugs via archaeosomes.
- the pre sent invention therefore enables production of archaeosomes in sufficient quantities at sufficient quality.
- Archaeosomes have been recognized as tremendously useful carriers for the delivery of a variety of cargo.
- the drug-carrier application which currently is most promising, is the use of archaeosomes as microcapsules for oral delivery of therapeutic proteins or peptides (like insulin) .
- they are also regarded as highly interesting adjuvants in vaccines since the uptake of archaeosomes in phagocytic cells is more than 50-fold greater than of conventional liposomes.
- Studies in mice show that systemic administration of several test antigens entrapped in archaeosomes gave humoral immune responses that were compa rable to those obtained with the potent, but toxic Freund's adjuvant. All in vitro and in vivo studies performed to date indicate that archaeosomes are safe and do not invoke any no ticeable toxicity.
- requirements for lipid carriers in medical applications are:
- Archaeosomes fulfill all of these requirements and thus have been successfully tested as drug delivery systems for cancer vaccines, immunoadj uvants against Chagas disease, novel gene de livery systems, carriers for oral delivery of proteins and pep tides, novel antigen delivery systems and others.
- Archaeosomes have been successfully prepared from different archaeal species. Usually, total archaeal lipids are extracted from frozen biomass by organic solvent extraction using chloro form/methanol/water. Then, the polar and the neutral lipids are separated by precipitation using acetone. The resulting lipid extracts can either be used directly for the preparation of archaeosomes as complex mixtures of different lipid classes or can be further purified by chromatography to isolate pure polar lipids of a particular class.
- tokodaii was not constant in dif ferent processes, especially when the temperature was changed or samples were taken in different growth phases (Jensen et al . , Life 5 (2015) , 1539) .
- the present in vention provides an efficient control of the quality (heteroge neity and composition) of the lipid ethers of the Archaea by changing critical process parameters (e.g. temperature, growth rate, etc.), which impedes the preparation of well-defined ar- chaeosomes .
- Archaeosomes can be used for similar tasks as liposomes (administration of drugs to patients), but show much better properties than liposomes (more stable over time and more re sistant against heat, pH, bile acid, autoclavable ) and are ex pected to be a viable alternative to conventional liposomes as they can be produced in sufficient quantities at sufficient quality .
- the global liposome drug delivery market is valued at 2.4 billion USD in 2017 and expected to reach 5.2 billion USD by the end of 2025, growing at an annual growth rate of 10.1% during 2018-2025.
- archaeosomes As a carrier of pharmaceutical active ingredients, it is necessary to ensure a different release kinetics depending on the desired site of action and pharmaco dynamics. This release kinetics can be influenced essentially by the stability of the archaeosomes.
- Both lipids have a sufficient number of functional groups in the head groups for efficient modification with linkers for use in targeted drug delivery (e.g. attachment of antibody frag ments, aptamers, etc.) .
- the highest possible number of rings is necessary for the lowest possible membrane permeability and thus gastric resistance, which, as the present invention has shown, therefore requires a cultivation at the highest possible temperature.
- the associated loss of temperature as being a suitable means for establishing the Cal: Arc ratio, however, can be controlled ac cording to the present invention via the growth rate.
- the archaeosomes may not be too stable, because the drug otherwise cannot be delivered.
- the modifiability of the release kinetics of archaeosomal formulations according to the present invention is maintained and drugs produced on archaeosomes from the tailored lipid mix tures provided by the present invention, for example, after oral administration, have a different duration of action in the in testine .
- the present invention is based on at least two significant results which have been obtained in the course of the present invention.
- the present invention provides a controlled production method which allows reproducible and defined compo sitions of archaeal lipids.
- Bioreactor cultivation methods have been developed in the recent past together with new analyzation tools, computer-di rected process-monitoring and software-guided process-control- ling to achieve break-through technology in cultivation of mi croorganisms on completely different levels. This has specifi cally impacted the cultivation technology for microorganisms which require complex cultivation conditions, such as archae- bacteria which e.g. require cultivation temperatures signifi cantly above common cultivations temperatures, such as 30 or 37 °C or even room temperature.
- the preparation of a lipid mix ture with the highest possible number of rings in the phytanyl chains of the caldarchaeol species is possible while maintaining the adjustability of the ratio Cal: Arc (within the physiological limits) .
- 2Hex-Cal and Pi-Arc are specifically preferred target species according to the present invention.
- the present invention it is there fore possible to provide compositions with archaeosomes with a high number of rings due to increased growth temperature with simultaneous adjustment of the Cal: Arc ratio over the growth rate.
- the present inven tion makes it possible to obtain a lipid extract which can be either used directly or with minimum purification effort for the production of archaeosomes with high gastric juice resistance and tailor-made release kinetics for different pharmaceutical active ingredients.
- oral retard therapy and “oral acute therapy” are used according to the present invention in their usual meaning: Whereas a “retard” (or “time-release”, “sustained release”, “extended release” oral therapy shows a “retard ef fect” which enables a continuous release even after some time after delivery, e.g.
- an absolutely acute release oral therapy is immediately released after administration and has a release maximum immediately after de livery, e.g. within 3 h after 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 . consciousControlling drug delivery” in Pharma ceutics: drug Delivery and Targeting” (2012), Pharmaceutical Press ) .
- the lipids produced for the production of oral archaeosomes with sustained-release effect are specifically suited for the needs of oral administration and therefore exhibit a high gastric juice resistance.
- This resistance is safeguarded by the presence of a high number of cyclopentane rings.
- this can be achieved by applying high process temperatures, especially in the range of 75 to 85°C.
- For the retard effect necessary for sustained-release a slow release of the active substance over a longer period is needed, which therefore requires temporal stability.
- this can be achieved by the presence of less destabilizing Pi-Arc.
- the present invention provides the teaching that this requires a high growth rate, i.e.
- the present method provides a composition for use for oral retard therapy which comprises a ratio of dihexose-caldarchaeol (2Hex-Cal) to phosphatidyl inositol-archaeol (Pi-Arc) of 1:5 to 1:7, espe cially of 1:5.5 to 1:6.5.
- a composition for use for oral retard therapy which comprises a ratio of dihexose-caldarchaeol (2Hex-Cal) to phosphatidyl inositol-archaeol (Pi-Arc) of 1:5 to 1:7, espe cially of 1:5.5 to 1:6.5.
- the lipids produced for the production of oral archaeosomes for acute therapy are, on the one hand, also specifically suited for the needs of oral administration and therefore exhibit a high gastric juice resistance.
- This resistance is - as in the retard case - safeguarded by the presence of a high number of cyclopentane rings.
- a composition for acute therapy requires a rapid release of the drug in the intestine. Therefore, a lower stability of the composition is necessary. According to the present inven tion, this can be achieved by the presence of more destabilizing Pi-Arc.
- the present invention provides the teaching that this requires a low growth rate, i.e. preferably a growth rate of 0.005 to 0.015 per hour, especially of 0.007 to 0.013 per hour.
- the present method provides a composition for use for oral acute therapy which comprises a ratio of dihexose-caldarchaeol (2Hex-Cal) to phosphatidyl ino sitol-archaeol (Pi-Arc) of 1:2 to 1:4, especially of 1:2.5 to 1:3.5.
- compositions obtainable by the present method tailor-made archaeosomes can be provided wherein drugs to be delivered can be wrapped for administration, preferably for oral administration to human patients in need of an effective amount of this drug.
- a preferred Sulfolobus strain is Sulfolobus acidocaldarius cells; this is one of the established strains in the present field of technology. Deposi taries for this Sulfolobus organism used may be, for example German strain collection for microorganisms (Deutsche Sammlung von Mikroorganismen und Zellkulturen, DSMZ), catalogue number DSM 639. An alternative source of supply is e.g. American Type Culture Collection (ATCC) , catalogue number ATCC 33909. Other archaebacteria are also available e.g. at the DMSZ or the ATCC
- the Sulfolobus cell lipids are extracted. This can be done in the way as disclosed in the art.
- the cells are homogenised after culturing, prefera bly via homogenization, especially high-pressure homogenization, or sonication, so that the lipids are extractable from the aque ous culture.
- a preferred method for purifying the lipids accord ing to the present invention is the supercritical fluid extrac tion, especially the extraction with supercritical CO2 (see e.g. Hanif et al . , Int. J. Mol. Sci. 13 (2012), 3022-3037) .
- a buffer e.g. a buffer known and described in the art to be suitable for Sulfolobus cell lipids extraction, for example with ammonium acetate buffer, especially with an ammo nium acetate buffer having 100 to 200 mM ammonium acetate.
- the archaeal lipids are obtained by extraction of the aqueous phase comprising the cells and/or the homogenised cells into an organic phase, preferably by extraction into a chloroform/meth anol organic phase, and phase separation of the organic phase from the aqueous phase.
- the organic phase then contains the lipids in the desired, "tailor-made" ratio.
- the archaeal lipids can then be obtained by evaporation of the organic phase, pref erably by evaporation of the organic phase to dryness.
- the archaeal lipids obtained are combined with a pharmaceutically active drug, preferably in combination with a pharmaceutically acceptable carrier, so as to obtain a compo sition comprising archaeal lipids and the pharmaceutically ac tive drug.
- a pharmaceutically active drug preferably in combination with a pharmaceutically acceptable carrier
- This is preferably done after the purified composition comprising archaeal lipids ( archaeosomes ) are provided; it is, however, also possible to add the drug and/or further components already in the process of purification and co-purify it/them with the lipids.
- the archaeal lipids are obtained by supercritical fluid extraction, especially extraction with supercritical CO2.
- the present invention also relates to a composition comprising archaeal lipids obtainable by a method according to the present invention.
- These "tailor- made" archaeosomal compositions are novel and of high utility for packaging drugs to be delivered to patients, especially for delivering drugs which are already known to be suitable for human therapeutic and prophylactic use.
- compositions according to the present invention comprise either a ratio of dihexose-caldar- chaeol (2Hex-Cal) to phosphatidyl inositol-archaeol (Pi-Arc) of 1:5 to 1:7 (retard oral use) or a ratio of dihexose-caldarchaeol (2Hex-Cal) to phosphatidyl inositol-archaeol (Pi-Arc) of 1:2 to 1:4 (acute oral use) .
- Both compositions as defined by this specific 2Hex-Cal/PI-Arc ratio) have a high resistance against gastric juice which allows them an efficient passage of the stomach after oral administration.
- the retard oral composition further comprises less destabilising Pi-Arc and has therefore a retarded release property in the intestine allowing a timely delayed release.
- the acute oral compositions are less stable in the intestine and the packaged drug can therefore immediately released from the liposomes, enabling a quick delivery for in testinal uptake of the drug.
- compositions according to the present invention are suitable to package any drug which is suitable for liposomal packaging. Accordingly, the compositions according to the pre sent invention are specifically suited for further comprising a drug, preferably a drug for human medical use, especially a drug which is subject to an authorization to place the product on the market as a medicinal product issued in the EU or in the US.
- a drug preferably a drug for human medical use
- any drug can be packaged in the lipid compositions according to the present invention.
- compositions according to the present invention comprise at least 5 % w/w, preferably at least 10 % w/w, Pi-Arc and at least 50 % w/w 2Hex-Cal (% w/w as % w/w of total lipid) .
- the present invention provides a method for producing a composition comprising ar- chaeal lipids from an archaeal cell culture, comprising growing an archaeal cell culture at controlled conditions and extracting the archaeal lipids from the archaeal culture so as to obtain a composition comprising archaeal lipids.
- the present invention provides a controlled production method which allows reproduci ble and defined compositions of archaeal lipids.
- the controlled conditions are selected from pH, growth rate, tem perature, and process mode.
- the method according to the present invention is in principle applicable to all archaeal cells.
- the archaeal cell culture according to the present invention is a culture of S. acidocaldarius,.
- Preferred temperatures for growing the archaeal culture can be optimised using the teachings according to the present in vention and are preferably in the range at a temperature of 55 to 90°C, preferably of 70 to 85°C, especially of 75 to 85°C.
- Specifically preferred embodiments of the present invention comprise growing the archaeal culture at a growth rate of 0.025 to 0.035 per hour, especially of 0.027 to 0.033 per hour, or at a growth rate of 0.005 to 0.015 per hour, especially of 0.007 to 0.013 per hour .
- Specifically preferred embodiments of the present invention comprise growing the archaeal culture at a pH of 2 to 4, pref erably of 2.5 to 3.5.
- the present invention can be applied by growing the archaeal culture at continuous mode and by growing the archaeal culture in batch mode, especially in fed-batch mode.
- the method according to the present invention preferably comprises extracting the archaeal lipids by two phase extrac tion, organic extraction, especially Soxhlet, high pressure or high temperature extraction; by ionic liquids extraction, or by supercritical fluid extraction.
- the present method comprises extracting the archaeal lipids by a solvent selected from the group consisting of water, hexane, chloroform, acetone, diethyl ether, methanol, ethanol, ethyl acetate, di- chloromethane, MTBE, CO2, and mixtures thereof.
- a solvent selected from the group consisting of water, hexane, chloroform, acetone, diethyl ether, methanol, ethanol, ethyl acetate, di- chloromethane, MTBE, CO2, and mixtures thereof.
- Fig. 1 shows the composition of the lipid pattern depending on growth temperature
- Fig. 2 shows the composition of the lipid pattern depending on growth rate
- Fig. 3 shows the composition of the lipid pattern depending on process mode
- Fig. 4 shows the structures main lipids of Sulfolobus aci- docaldarl us
- Fig. 5 shows the influence of growth temperature on the number of cyclopentane rings
- Fig. 6 shows the spectral data of biomass sample grown at
- Fig. 7 shows the spectral data of biomass sample grown at
- Fig. 8 shows the spectral data of biomass sample grown at
- Fig. 9 shows the spectral data of biomass sample grown with a growth rate of 0.010 hr 1 ;
- Fig. 10 shows the spectral data of biomass sample grown with a growth rate of 0.020 hr 1 ;
- Fig. 11 shows the spectral data of biomass sample grown with a growth rate of 0.034 hr 1 ;
- Fig. 12 shows the spectral data of biomass sample grown in fed-batch mode
- Fig. 13 shows the spectral data of biomass sample grown in continuous mode.
- the present invention describes a technology for the pro duction of distinct lipid patterns in Sulfolobus acidocaldarius with a defined and tunable composition via variation of process parameters during the upstream process.
- the present examples show that via variation of critical process parameters during the upstream, e.g. temperature, growth rate, process mode, the organism Sulfolobus acidocaldarius pro prised different lipid patterns (ratio of distinct lipid frac tions) .
- critical process parameters e.g. temperature, growth rate, process mode
- the organism Sulfolobus acidocaldarius pro prised different lipid patterns (ratio of distinct lipid frac tions) .
- lipid patterns ratio of distinct lipid frac tions
- Cultures were grown in a controlled bioreactor environment on a defined medium.
- S. aci- docaldarius was cultivated in a continuous process mode to ensure constant process conditions over at least 3 generation times.
- the continuous phase was preceded by a batch and fed-batch phase for buildup of biomass.
- the volumes in the following paragraphs are exemplarily and can be scaled up or down if required.
- the preculture was transferred aseptically to a culture vessel containing a defined medium according to Quehenberger et al . (2019), yielding a total starting volume for the batch phase of 1.5 L.
- the fed-batch phase was followed by a con tinuous cultivation at the desired growth temperature and growth rate. To ensure enough time for the organism to adapt to the specific growth conditions during the continuous phase biomass was harvested after at least 3 generation times.
- Formula 4 Calculation of the Feedrate during the fed-batch phase .
- Feeding was controlled via the weight of the Feed vessel and a PID-controller was used to compensate for deviations of the feed pump .
- V R working volume
- Dissolved oxygen levels were monitored online and main tained above 15% by aerating with 0.25 to 0.5 vvm (volumes gas per culture volume per minute) pressurized air. Air was substi tuted with pure oxygen if necessary. pH was monitored online and kept at 3.0 +/- 0.1 by addition of H2SO4 (9.6% v/v) . Feed medium was supplied via a peristaltic pump module following a feed forward controlled exponential feeding strategy. Mixing was per formed at 300 to 500 rpm. CO2 content in the offgas was monitored.
- Cells were harvested via centrifugation at 14,000g for 15 min. After discarding the supernatant, the cell pellet was stored at -20 °C for further processing.
- a cell pellet from 100 mL culture broth per sample (dry cell weight at the time of harvest is shown in Table 1) was thawed and resuspended in 10 mL ice cold ammonium acetate buffer (155 mM) .
- the suspension was ho mogenized in a glass beaker on an ice/water bath via sonication (6x1 min with 30 sec pause between pulses) . After homogenization the sample was diluted to an OD600 of 1.5 with ice cold ammonium acetate buffer (155 mM) .
- the dried lipid fractions were reconstituted in a 1:3 CHCls/MeOH mixture. These fractions were immediately used for MALDI sample preparation using 2-4-, 6-trihydroxy-acetophenon (THAP) as MALDI matrix (roughly 15 mg THAP/mL without or with the addition of sodium chloride for promotion of sodium adduct ions) .
- THAP 6-trihydroxy-acetophenon
- E LAB 20 keV, collision gas: helium
- the present invention provides for the follow ing preferred embodiments: 1. Method for producing a composition comprising archaeal li pids from an archaeal cell culture, comprising growing an ar chaeal cell culture at controlled conditions and extracting the archaeal lipids from the archaeal culture so as to obtain a composition comprising archaeal lipids.
- Method according to any one of the embodiments 1 to 3, comprising growing the archaeal culture at a temperature of 55 to 90°C, preferably of 70 to 85°C, especially of 75 to 85°C.
- Method according to any one of the embodiments 1 to 4 comprising growing the archaeal culture at a growth rate of 0.025 to 0.035 per hour, especially of 0.027 to 0.033 per hour, or at a growth rate of 0.005 to 0.015 per hour, especially of 0.007 to 0.013 per hour .
- Method according to any one of the embodiments 1 to 5, comprising growing the archaeal culture at a pH of 2 to 4, preferably of 2.5 to 3.5.
- Method according to any one of the embodiments 1 to 6, comprising growing the archaeal culture at continuous mode.
- Method according to any one of the embodiments 1 to 6, comprising growing the archaeal culture in batch mode, espe cially in fed-batch mode.
- Method for producing a composition comprising archaeal li pids from a Sulfolobus cell culture, comprising
- composition for use for oral retard therapy comprises a ratio of dihexose-caldarchaeol (2Hex-Cal) to phosphatidyl inositol- archaeol (Pi-Arc) of 1:5 to 1:7, especially of 1:5.5 to 1:6.5.
- composition for use for oral acute therapy comprises a ratio of dihexose-caldarchaeol (2Hex-Cal) to phosphatidyl inositol- archaeol (Pi-Arc) of 1:2 to 1:4, especially of 1:2.5 to 1:3.5.
- composition comprising archaeal lipids obtainable by a method according to any one of embodiments 1 to 20.
- composition comprising archaeal lipids, preferably accord ing to embodiment 21, wherein the composition comprises a ratio of dihexose-caldarchaeol (2Hex-Cal) to phosphatidyl inositol- archaeol (Pi-Arc) of 1:5 to 1:7 or of 1:2 to 1:4.
- composition according to any one of embodiment 21 to 24, wherein the archaeosomes comprise as a drug to be administered to a patient, an antigen, an oral drug, a vaccine, an antioxi dant, a virus-like particle, a nucleic acid, especially a DNA, an RNA, a pDNA, an antibiotic, a polypeptide or a protein.
- composition according to any one of embodiment 21 to 26 wherein in a liposomal composition which is subject to an au thorization to place the product on the market as a medicinal product issued in the EU or in the US, the liposomes are replaced by the archaeosomes provided by a method according to any one of embodiments 1 to 20.
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Priority Applications (10)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
KR1020217024480A KR20210141452A (en) | 2019-03-15 | 2020-02-21 | Method for producing a composition comprising archaea lipids in Sulfolobus cell culture |
CN202080012143.8A CN113365605A (en) | 2019-03-15 | 2020-02-21 | A method for producing composition containing archaebacteria lipid from sulfolobus cell culture |
AU2020242577A AU2020242577A1 (en) | 2019-03-15 | 2020-02-21 | Method for producing a composition comprising archaeal lipids from a Sulfolobus cell culture |
ES20705088T ES2946033T3 (en) | 2019-03-15 | 2020-02-21 | Method for producing a composition comprising archaeal lipids from a Sulfolobus cell culture |
SG11202107250SA SG11202107250SA (en) | 2019-03-15 | 2020-02-21 | Method for producing a composition comprising archaeal lipids from a sulfolobus cell culture |
US17/423,863 US20220098629A1 (en) | 2019-03-15 | 2020-02-21 | Method for producing a composition comprising archaeal lipids from a sulfolobus cell culture |
CA3130657A CA3130657A1 (en) | 2019-03-15 | 2020-02-21 | Method for producing a composition comprising archaeal lipids from a sulfolobus cell culture |
JP2021556210A JP2022526485A (en) | 2019-03-15 | 2020-02-21 | Methods for Producing Compositions Containing Paleobacterial Lipids from Sulforobus Cell Cultures |
EP20705088.1A EP3937901B1 (en) | 2019-03-15 | 2020-02-21 | Method for producing a composition comprising archaeal lipids from a sulfolobus cell culture |
IL286344A IL286344A (en) | 2019-03-15 | 2021-09-13 | Method for producing a composition comprising archaeal lipids from a sulfolobus cell culture |
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EP4342460A1 (en) | 2022-09-21 | 2024-03-27 | NovoArc GmbH | Lipid nanoparticle with nucleic acid cargo |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP4342460A1 (en) | 2022-09-21 | 2024-03-27 | NovoArc GmbH | Lipid nanoparticle with nucleic acid cargo |
WO2024062001A1 (en) | 2022-09-21 | 2024-03-28 | Novoarc Gmbh | Lipid nanoparticle with nucleic acid cargo |
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CA3130657A1 (en) | 2020-09-24 |
AU2020242577A1 (en) | 2021-07-22 |
JP2022526485A (en) | 2022-05-25 |
US20220098629A1 (en) | 2022-03-31 |
EP3937901B1 (en) | 2023-03-15 |
CN113365605A (en) | 2021-09-07 |
KR20210141452A (en) | 2021-11-23 |
SG11202107250SA (en) | 2021-07-29 |
EP3937901A1 (en) | 2022-01-19 |
IL286344A (en) | 2021-10-31 |
EP3708151A1 (en) | 2020-09-16 |
ES2946033T3 (en) | 2023-07-12 |
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