EP3229966A1 - Procédé et système de thermorégulation de capillaires exempts d'étanchéité - Google Patents

Procédé et système de thermorégulation de capillaires exempts d'étanchéité

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Publication number
EP3229966A1
EP3229966A1 EP15816700.7A EP15816700A EP3229966A1 EP 3229966 A1 EP3229966 A1 EP 3229966A1 EP 15816700 A EP15816700 A EP 15816700A EP 3229966 A1 EP3229966 A1 EP 3229966A1
Authority
EP
European Patent Office
Prior art keywords
capillaries
tempering
capillary
fluorescence
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP15816700.7A
Other languages
German (de)
English (en)
Inventor
Philipp Baaske
Stefan Duhr
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
NanoTemper Technologies GmbH
Original Assignee
NanoTemper Technologies GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by NanoTemper Technologies GmbH filed Critical NanoTemper Technologies GmbH
Publication of EP3229966A1 publication Critical patent/EP3229966A1/fr
Pending legal-status Critical Current

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/06—Test-tube stands; Test-tube holders
    • B01L9/065—Test-tube stands; Test-tube holders specially adapted for capillary tubes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/14—Process control and prevention of errors
    • B01L2200/142—Preventing evaporation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/18—Transport of container or devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0825—Test strips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1805—Conductive heating, heat from thermostatted solids is conducted to receptacles, e.g. heating plates, blocks
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces

Definitions

  • the invention relates generally to a system and method for controlling the temperature of capillaries filled with samples to be tested.
  • the invention relates to a method for controlling the temperature of capillaries for optical measurements of tempered samples as a function of the temperature.
  • the optical measurement in the UV range is based on a Fluence behavior of the samples to be measured.
  • the invention also relates to a system with which the inventive method is simple and efficient to carry out.
  • a significant advantage of the invention is that it is possible to dispense with a sealing of the capillaries for temperature control and optical measurement of the samples within the capillaries.
  • melting curve analyzes for example, thermal stability measurements, thermal shift assays (TFA) and differential scanning fluorimetry (DSF) are important tools for the qualitative and quantitative assessment of the stability and aggregation behavior of proteins and drug formulations.
  • MicroScale Thermophoresis (Thermo-Optical Particle Characterization), in which, for example, affinities (Kd, EC50) of interactions at different temperatures are measured in order to determine the thermodynamic variables dl i from the measurement results using, for example, a van't Hoff plot and derive dS.
  • Analytics e.g., food analysis, cosmetics, ..
  • the temperature range to be investigated extends, for example, from 0 ° C. to 100 ° C., or to the respective area in which the respective liquid is in its liquid form.
  • Capillaries as Proben aftercr are very interesting for these applications, since they have a very small and very well defined volume.
  • capillaries can be filled independently by capillary action with liquids, and you can therefore, for example, on pumps without.
  • capillaries such as borosilicate 3.3. Quartz, synthetic fused silica. etc., also with regard to their optical properties, in particular their transparency, purity and autofluorescence, advantageous.
  • short capillaries with a small inner and outer diameter for example with an outer diameter not greater than 1 mm and an inner diameter of not greater than 0.8 mm, preferably 0.65 mm outer diameter and 0.5 mm inner diameter, advantageous because they are only a small Have volume and thus save sample material.
  • the capillaries In order to carry out the measurement methods described, such as, for example, a melting curve analysis, it is necessary to temper the capillaries, for example from 10 ° C. to 100 ° C. In this tempering is typically observed a strong evaporation of the liquid at elevated temperatures. This evaporation or evaporation leads to disturbing flows in the liquid and in particular to such a strong loss of fluid that measurements at elevated temperatures over a longer period of time are not possible.
  • seals such as wax
  • a wax can be pushed out of the capillary at elevated temperatures by the vapor pressure in the capillary and thus lose their functionality.
  • capillaries are provided in the form of micro cuvette arrays (MCA). These micro cuvettes are clamped in a frame, whereby this frame seals the cuvettes by means of silicone strips at both ends. To avoid contamination, these silicone strips and / or the frame must be replaced regularly, which incurs additional costs.
  • MCA micro cuvette arrays
  • biomolecules such as proteins, peptides, nucleic acids, DNA, RNA.
  • Antibodies but also cells, bacteria, nanodiscs, vesicles, viruses, etc. is desirable to work with very small volumes on a microliter scale, short, very thin capillaries are beneficial.
  • it is advantageous to use thin-walled capillaries because, for example, autofluorescence and other artifacts can be minimized by this thinness.
  • capillaries i.e. capillaries with a small diameter and a thin wall
  • the disadvantage of being very fragile For this reason, the non-destructive mechanical sealing of the capillaries, for example by a plug or a cap, not or only with considerable, and thus no longer economical effort possible.
  • the present invention relates to a method by which liquids can be tempered in a capillary without sealing the capillary and visually examined.
  • several capillaries are tempered simultaneously without sealing the capillaries and examined simultaneously or successively optically.
  • Preferred advantages of the seal-free method according to the invention can be described in the following manner as follows.
  • the risk that a capillary can break is significantly reduced, since usually the risk of breakage when closing the capillary is greatest or large.
  • work steps are saved for sealing, since not every capillary must be sealed at both ends.
  • the solution according to the invention is thus not only faster, but also less expensive, that is, less expensive, and it is still possible to avoid contamination by viruses.
  • the present invention relates to a method for controlling the temperature of at least one, preferably a plurality of capillaries.
  • the capillary / capillaries are / are placed on a support, for example.
  • the carrier preferably has a length L, width B and height H (see for example Fig. 3).
  • the capillaries are arranged along the width of the carrier on the carrier.
  • the carrier has preferably a recess into which, for example, a tempering element can be inserted.
  • it is preferred that the capillaries are held by the carrier only outside the tempering element, so that the entire width of the tempering element is available for measurements.
  • the capillaries should preferably be tempered in their central area by contact with the tempering, wherein the ends of the capillaries filled with samples are unlocked during the temperature.
  • the tempering element can be heated or heated and / or cooled, wherein the reference point is preferably the ambient temperature.
  • the temperature range of the sample in the capillary extends for example from 0 ° C to 100 ° C, or to the respective area in which the respective liquid is in its liquid form.
  • the sample in a range of 0 ° C to 100 ° C to temper.
  • the sample liquid is a liquid with a lower melting point, for example a liquid containing other solvents, for example organic solvents, for example alcohols, or consisting essentially completely of these substances, then the preferred lower limit of the tempering range may also be lower, for example lower than 0 ° C.
  • the preferred temperature range of an aqueous solution containing, for example, buffers, salts, detergents, lipids, surfactants, polymers, DMSO, sucrose or glycerol can also be a temperature range greater or lesser than 0 ° C to 100 ° C.
  • the preferred lower limit of the tempering range may also be lower, for example below 0 ° C.
  • the preferred upper limit of the tempering range may also be higher, for example above 100 ° C.
  • supercooled liquids can also be used according to the invention.
  • the sealant-free capillaries according to the invention also survive repeated freezing and thawing operations of the aqueous solution which are carried out, for example, to check whether repeated freezing and thawing leads to the unfolding and / or aggregation of biomolecules in the aqueous solution.
  • aqueous solutions with biomolecules are stored at -20 ° C or -80 ° C. Before storage, these aqueous solutions are present with biomolecules in liquid form, when stored at, for example -20 ° C or -80 ° C freeze these aqueous solutions, for use, they are removed from the freezer and thawed to make them back in liquid form to use.
  • the sample to be examined is filled into a capillary, wherein the capillary is usually not filled from end to end with the liquid of the sample.
  • the part of the capillary which is filled with the liquid of the sample is referred to below as the liquid column.
  • the liquid column of the capillary is to be aligned with the temperature-control element such that the two ends of the liquid column protrude beyond the temperature-control element.
  • the tubular capillaries according to the invention have a length between 40-75 mm, preferably between 45-55 mm, more preferably about 50 mm.
  • the width of the tempering is preferably between 5 - 34mm. more preferably between 20 - 0mm. more preferably 20-25mm, more preferably about 25mm.
  • a tempering preferably silicon, preferably pure silicon is used.
  • the capillary or the capillaries may also be advantageous to use the capillary or the capillaries on the To press the tempering, so as to ensure the contact between the capillary and tempering.
  • the cover can be arranged partially above the tempering region and / or exert a force on the capillaries outside the tempering region.
  • the individual capillaries are filled with a liquid, preferably with an aqueous sample solution, in particular buffer solutions for biochemical / biological measurements.
  • aqueous sample solution in particular buffer solutions for biochemical / biological measurements.
  • nonaqueous solvents may also be used or admixed, for example organic solvents.
  • Sample solutions may contain an analyte, preferably a protein, in a suitable aqueous solution, e.g. a buffer solution, but also in an organic solvent (e.g., alcohols such as ethanol, octanol, isopropanol) or in water or a mixture of water with one or more organic solvents (such as ethanol, octanol or isopropanol).
  • a suitable aqueous solution e.g. a buffer solution
  • an organic solvent e.g., alcohols such as ethanol, octanol, isopropanol
  • water or a mixture of water with one or more organic solvents such as ethanol, octanol or isopropanol.
  • sample solution or sample liquid according to the invention which is filled into the capillaries may also be oils, emulsions, dispersions or other substances or mixtures which are present in at least one of the preferred temperature ranges in the liquid phase and can be filled into the capillary.
  • the length of the liquid column in the capillary is preferably at least 1.1 times the width of the tempering element, preferably at least 1.2 times, preferably at least 1.3 times, more preferably at least 1.35 times further preferably at least 1.4 times, more preferably at least 1.45 times, more preferably at least 1.5 times, more preferably at least 1.6 times, more preferably at least 1.7 times the width of the tempering element ,
  • the capillaries preferably have an inner diameter of 0.02 to 0.9 mm.
  • the capillaries Preferably, have an outer diameter of 0.1 to 2 mm.
  • the capillaries may be made of glass, for example, preferably borosilicate 3.3. Quartz or Synthetic Fused Silica may be made without limitation.
  • capillaries are generally tubes with very small inside diameters. Due to the strong surface effects in the foreground compared to larger pipes, capillarity occurs in capillaries. a physical effect. Liquids with high surface tension rise in capillaries.
  • the capillaries of the invention are not limited to a particular cross-sectional shape. Most capillaries are formed around. According to the invention, the cross section of a capillary can also be oval, triangular, quadrangular, pentagonal, hexagonal, octagonal, semicircular, or trapezoidal, or have another irregular shape. It is also preferred according to the invention that the capillaries are made of a solid, preferably non-deformable material such as glass and the cross-sectional shape of the capillaries does not change for or during a measurement. For example, the cross-sectional shape during filling is the same as during the measurement.
  • Compression of the cross section for measurement is preferably avoided, for example because the inner and outer diameter of the capillaries also affect fluorescence measurements, absorbance measurements, absorbance measurements or scattered light measurements. Since the capillaries according to the invention are not closed on at least one side, a Deforaitechnik the capillaries can also lead to a squeezing out of the sample liquid to be examined, which should preferably be avoided.
  • the present invention also relates to a method for optically examining samples filled in capillaries.
  • the capillaries are filled with the sample.
  • the capillaries are positioned on the tempering element for tempering.
  • a plurality of capillaries are preferably initially arranged on a carrier, and the carrier with the plurality of capillaries is then positioned on the temperature control element.
  • the capillaries can be tempered as described above.
  • the samples can be excited with light.
  • the excitation with light is not limited to a certain wavelength of light. According to a preferred embodiment, for example, an excitation by means of UV light take place. Subsequently, the light emitted by the sample is measured. Also in the measurement of the emitted light, the present invention is not limited to a specific wavelength.
  • the present invention also relates to a system for the optical examination of samples in capillaries.
  • the system according to the invention preferably comprises a tempering device for tempering the capillaries.
  • a carrier for holding the capillaries it may be preferable to provide a carrier for holding the capillaries.
  • the inventive system may also include an optical measuring system for emitting light and detecting light.
  • the system may comprise at least one capillary. Preferably a non-deformable and preferably tubular capillary.
  • non-deformable is meant, in particular, that the cross-section of the capillary remains substantially the same under an applied pressure.
  • non-deformable is preferably hard.
  • system according to the invention can also be used to measure thermophoresis effects in samples.
  • biomolecules such as proteins.
  • suitable chemicals such as chaotropes such as urea or guanidinium hydrochloride or organic solvents, or by changing the temperature (ie, for example, "melting” by raising the temperature), changed.
  • tertiary structure of biomolecules such as proteins and nucleic acids is often also dependent on the presence of ligands or cofactors such as ions (eg Mg 2+ or Ca 2+ ), for example by measuring the fluorescence (preferably typtophan fluorescence in the case of proteins) various concentrations of ligands and / or cofactors occur.
  • the biomolecule preferably protein
  • the biomolecule can be denatured chemically or thermally, and structural changes can be measured by intrinsic fluorescence (preferably, typtophan fluorescence in the case of proteins). Thereby, e.g. Changes in fluorescence intensity or shift of fluorescence maxima, etc. can be detected.
  • the melting point of the biomolecule to be examined e.g. Protein
  • the melting point is the state in which the biomolecule to be examined, e.g. Protein, half folded and half unfolded.
  • the tryptophan fluorescence can be measured at a wavelength of 330 nm and / or 350 nm.
  • the change in the intensity of fluorescence may be e.g.
  • the quotient of the fluorescence intensity at 330 nm to the fluorescence intensity at 350 nm is a preferred parameter.
  • the melting point can be determined from the maximum of the first derivative of the F330 / F350 curve.
  • the melting of nucleic acids or their complexes can by means of
  • Fluorescence measurement can be followed.
  • fluorescence measurements e.g. also the measurement of circular dichroism (CD) in question.
  • CD circular dichroism
  • the aggregation behavior of biomolecules can also be measured.
  • the measurement of the aggregation behavior is particularly interesting but not only for the approval of drugs.
  • This aggregation can be measured, for example, by means of the change in the intrinsic fluorescence, for example the change in the fluorescence intensity and / or the shift in the fluorescence emission maximum.
  • this aggregation can also be measured by measuring the fluorescence anisotropy of the biomolecules.
  • the measurement of the fluorescence anisotropy preferably also makes it possible to measure a change in the size of the biomolecules and thus, for example, to measure the size of resulting aggregates or to measure the decay of multimers of biomolecules, for example the thermal decomposition of a tetramer into its four monomers.
  • the thermally, chemically, enzymatically or temporally induced changes in the size of the biomolecules and thus also their aggregations or multimerizations can be measured by means of light scattering.
  • FIG. 4 is a schematic plan view of six different capillaries of different degree of filling, which lie on a tempering element;
  • Fig. 5 is a schematic representation of an optical measurement with several
  • Fig. 6 is a measurement diagram made with an optical measurement of Fig. 5, wherein each peak corresponds to a capillary;
  • FIG. 7 shows the course of a melting curve at an emission window of 330 nm
  • FIG. 9 the quotient of the two optical detection channels from FIGS. 7 and 8; FIG.
  • Fig. 11A shows an example of a typical buffer screening from antibody research
  • Fig. I B shows an example of a change in the thermal stability of a protein by binding of small molecules
  • FIG. 12-17 illustrations of an application example according to the invention
  • FIG. 18 shows, similar to FIG. 5, a schematic representation of an optical measurement with 48 capillaries on a temperature control body.
  • the invention generally relates to a system and a method for controlling the temperature of a capillary, preferably several capillaries simultaneously, which are filled with samples to be examined.
  • the capillaries are made of glass.
  • the capillaries are made of a material having a similar, not much lower and / or not much / significantly higher thermal conductivity than the liquid in the capillaries. Glass is also preferred for this reason because it has a similar thermal conductivity to an aqueous solution.
  • the heat according to the invention is transferred to the solution by means of glass, ie, if the thermal conductivity of the capillary material is too low, the solution in the capillaries is not heated properly and / or fast enough.
  • a material for the capillaries which is in the range of 0.15 W / (m * K) to 60 W / (m * K).
  • materials such as PMMA / Plexiglas, polypropylene, PEEK and Teflon fall into the lower limit range.
  • Another preferred range for glass as material is formed depending on different types of glass and extends, for example, from about 0.5 W / (m * K) to 1.6 W / (m * K).
  • the capillaries can be made of glass and / or a polymer and / or at least one of borosilicate glass, borosilicate 3.3 glass (for example Duranglas), quartz glass such as Suprasil, Infrasil, synthetically produced quartz glass, soda lime glass, Bk-7, ASTM Type 1 Class A glass, ASTM Type 1 Class B glass.
  • the polymers may contain: PTFE, PMMA, Zeonor TM, Zeonex TM, Teflon AF, PC, PE, PET, PPS, PVDF, PFA, FEP and / or acrylic glass.
  • the capillaries be transparent to light of wavelengths from 200 nm to 1000 nm, preferably from 250 nm to 900 nm.
  • this at least one segment is also transparent to light of the following wavelength ranges: from 940nm to 1040nm (preferably 980nm +/- 10nm), from 1150nm to 1210mn, from 1280nm to 1600nm (preferably 1450nm +/- 20nm and / or 1480nm +/- 20nm and / or 1550nm +/- 20nm), from 1900nm to 2000nm (preferably 1930nm +/- 20nm).
  • the transparent region (s) may also extend over the entire tubular structure. In other words, the capillary can be transparent.
  • the light transmission of the segment allows the performance of luminescence / fluorescence / phosphorescence measurements and / or optical
  • the translucency also allowed the performance of fluorescence measurements.
  • it also enables the heating of fluids in the tubular structure by means of electromagnetic radiation, for example light (preferably an infrared (IR) laser), preferably the heating of water and / or organic solvents.
  • electromagnetic radiation for example light (preferably an infrared (IR) laser
  • the capillaries are preferably brought into contact with a tempering element, so that this contact causes a temperature exchange from the tempering to the capillaries and thus to the samples within the capillaries.
  • the capillaries are preferably tempered in the region by means of contact heat, in which the optical measurement also takes place.
  • the thermal contact can be improved by the application of an oil, for example an immersion oil.
  • the optical measurement is preferably not limited to a specific wavelength range, and may for example take place in the IR, visible or UV range. It is also desirable that the temperature element itself emit no or only small amounts of fluorescence, which could falsify the measurement of the sample.
  • the contact material for the tempering element i.e., the element which comes into contact with the capillary (s) and transfers the temperature by direct contact with the capillaries, preferably silicon is used as the contact material for the tempering element.
  • silicon has no or only extremely low autofluorescence, in particular with an excitation light in the range from 260 nm to 700 nm.
  • the excitation is carried out at 260 nm to 300 nm and the emission is measured at> 320 nm.
  • silicon is very well suited for fluorescence measurements, in particular for fluorescence measurements in the UV range (tryptophan, tyrosine, phenylalanine fluorescence).
  • the UV fluorescence region is particularly advantageous since one can thus measure native biomolecules by means of their intrinsic fluorescence, without e.g.
  • silicon can be produced in a highly pure form and can also be acquired. so that any autofluorescence of impurities and thus influencing the measurement results are extremely low. Silicon is also a chemically inert material, so that even a possible contact with a measuring liquid does not cause any reactions that adversely affect the optical measurement.
  • a contact surface of silicon for a Temperierelement kaiin are made very smooth, so that the contact surface with the capillaries can be made as a mirror surface, whereby the excitation light and / or fluorescent light of the sample can be reflected from the mirror surface, which can additionally lead to a gain of the measurement signal.
  • the mirror surface is also broadband, which is also advantageous.
  • silicon has a very good thermal conductivity and is extremely smooth. In the silicon, for example, electronic
  • Circuits / structures can be integrated, for example by doping and / or etching, These structures can be used, for example, to measure a temperature or temperatures.
  • Another exemplary material for the contact material is metal, preferably anodized metal, preferably anodized aluminum.
  • metal preferably anodized metal, preferably anodized aluminum.
  • anodized aluminum for example in black, which shows no autofluorescence in the UV range.
  • silicon has the advantage of high purity over anodized aluminum since the quality of the anodized alloy can often vary.
  • the tempering element itself is preferably tempered by a temperature control device.
  • the tempering element is preferably used only for targeted temperature or heat transfer to the capillary (s).
  • the present invention is not limited to certain tempering devices. Due to the compact design and the appropriate temperature range, for example, Peltier elements offer. As a tempering but can also serve electrical heating elements or liquid-heated heating coils.
  • the capillaries to be tempered are to be arranged so that at least part of the capillaries is in contact with the tempering element.
  • a central region preferably a central region of each capillary, should be in contact with the tempering element, ie, it is preferred that at least one end, more preferably, both ends of the capillary do not come into contact with the tempering element during tempering. come.
  • the central region or center of the capillaries refers to the length of the capillary, i.e., midway between the two ends. In other words, it is preferable that one end, preferably both ends, are not tempered.
  • the capillaries should be held so that each capillary is tempered only within a narrow tempering.
  • the capillaries are arranged so that both ends protrude beyond the tempering element, preferably protrude symmetrically, whereby the ends of the Capillaries are not tempered by the tempering.
  • the individual capillaries are longer than the tempering region by the amount dx.
  • the capillaries are tempered only over a certain part of their length, which in conjunction with the low thermal conductivity of glass capillaries causes the ends of the capillaries practically always remain at room temperature, if only sufficient distance to the temperature range or Temperature control element is present. That Even if the center or the central region of the capillary is tempered by means of the tempering to 90 ° C, observed at the ends of a correspondingly long capillary no greater evaporation than at room temperature. This means that you do not have to seal if the evaporation is acceptable at room temperature.
  • the temperature range should preferably be less than / shorter than 32mm, more preferably less than 25mm, i. not more than 25mm length of the capillary should be tempered in the middle. In other words, on both sides of the tempering should preferably survive 12.5mm non-tempered capillary length.
  • the lower theoretical limit of the tempering range is 1 mm, the length being preferably not less than 5 mm for practical reasons.
  • the examples of the present invention will be discussed with a width of the 25mm tempering range, which width is preferred. However, it has been shown that even a 20mm wide tempering works well or can be handled. Similarly, a tempering of 30mm is still easy to handle.
  • capillary lengths of 50mm this length being preferred. However, it has been shown that even 20mm, 25mm, 30mm, 35mm, 45mm long capillaries work well and are manageable. Likewise, capillary lengths of 55, 60, 65, 70, 75, and 80mm are still easy to handle.
  • the tempering device is set to 20 ° C.
  • the area of the capillary, which rests directly on the tempering device, is approx. 20 ° C.
  • the area of the capillary, measured over the recess, was partially 22 ° C with additional inhomogeneous temperature distributions.
  • the ambient temperature is again assumed to be 25 ° C.
  • the temperature control is this time set to 90 ° C.
  • the area of the capillary, which rests directly on the tempering device, is approx. 90 ° C.
  • the area of the capillary, which is measured over the recess, has about 82 ° C + and an inhomogeneous temperature (depending on the width of the air gap).
  • MST buffer with Tween 20 mixed with blue dye for better measurability
  • MST buffer without Tween 20 green dye added for better measurability
  • MST buffer (kinase buffer) without tween
  • test capillaries were tempered as follows: raising the temperature from 20 ° C to 90 ° C at a heating rate of 1 ° C / min and then remaining at 90 ° C for 30 minutes. This is an exemplary curve for a melt curve measurement to study the thermal stability of a capillary.
  • AD Outside diameter
  • the capillaries In order to ensure a contact between the capillaries and the tempering, it is preferred to press the capillaries against the tempering. This can be done for example with a lid.
  • the lid which holds down the capillaries and thereby ensures good temperature control, preferably the same applies; it should not be wider than 25mm. If you want to make the tempering of the tempering wider, you need longer capillaries to avoid excessive evaporation at the Kapillarenden or prevent. Longer capillaries, in turn, may be inconvenient because they involve greater sample consumption.
  • the tempering surface should not be too narrow, since otherwise the capillaries in their central measuring range are no longer uniformly tempered. or differently tempered molecules diffuse from the outside into the measuring range.
  • Figure 1 shows a diagram in which the evaporation in a 50mm long capillary with an inner diameter of 0.5mm and an outer diameter of 0.65mm examined has been.
  • the diagram shows the percent evaporation (Y-axis) as a function of the width of the tempering element.
  • a preferred width of the tempering surface for 50mm long capillaries is less than 30mm, preferably less than 25mm, thereby providing even higher temperatures, e.g. 100 ° C are possible.
  • the maximum temperature depends on the liquid, in particular on the boiling point of the liquid or of the solvent. In particular, the formation of air bubbles on reaching the boiling point may be disturbing for the optical measurements. Therefore, the upper limit of aqueous solutions is preferably 100 ° C.
  • FIG. 2 shows a diagram in which the vapor evaporation was investigated in a capillary having a diameter of only 0.5 mm and an inside diameter of 0.5 mm and an outside diameter of 0.65 mm.
  • MST typical buffer solution without detergent
  • Tween buffer solution with detergent
  • FIG. 3 shows an exploded view of a device for tempering according to the invention.
  • the heat sink 1 are arranged on a movable unit.
  • a heating block 3 made of metal (for example, aluminum or copper) is arranged.
  • a heat-conducting foil or thermal compound may in turn preferably be located.
  • a special heat-conducting film 4 is preferably arranged.
  • a plastic frame 6 (here macroion) for positioning the capillaries (not shown) is arranged.
  • This cover 7 should preferably not be wider than the silicon wafer 5.
  • the lid 7 preferably presses the capillaries onto the silicon wafer 5. It is also advantageous if lid 7 has a thermally insulating effect.
  • FIG. 4 shows a schematic plan view of six capillaries a) -f) with different degree of filling or different positioning which lie on the plastic frame / carrier 6 in order to be tempered by an underlying silicon wafer 5. All six capillaries in positions a) -f) have the same or substantially the same length here.
  • Position a) shows the capillary centered centered, ie centered with respect to the central axis "M" of the frame 6 and the silicon wafer 5. The capillary is almost completely filled, ie, the capillary is symmetrically filled to the right and left sufficiently filled with liquid beyond the frame 6
  • Position b) also shows a capillary which is arranged symmetrically about the central axis M.
  • the degree of filling of this capillary is less than in the position a), but still sufficient that an evaporation at the ends does not interfere with a longer measurement adversely.
  • Position c) shows similar to the positions a) and b) a symmetrically filled capillary, the degree of filling, however, is even lower than in position b), so that both on the left and on the right-hand side, only a small projection "A" of the liquid column protrudes beyond the frame 6. However, this small projection leads to evaporations at these ends, which can adversely affect an optical measurement in the region of the silicon wafer 5.
  • Positions a) and b) show hooks, ie, these positions and degrees of filling work without problems, whereas positions c) - f) can cause problems, so that the degree of filling in position d) is sufficient and comparable to position a)
  • the positioning with respect to the silicon wafer 5 is such that the protrusion on the right side (B) is not large enough, in the position e), the capillary is indeed arranged correctly, ie symmetrically with respect to the silicon wafer 5 or the central axis M, but the capillary is filled unevenly
  • the distance A on the left side from the end of the liquid column to the frame or to tempered Silicon wafer 5 is sufficiently large, while the distance B on the right side is too small.
  • the position f) shows a symmetrically aligned capillary with symmetrically aligned liquid column, but with too low degree of filling.
  • the samples to be measured are filled in capillaries. This can be done for example by the capillary forces, or the capillaries are filled, for example with a pipette, but without being limited thereto. Then the capillaries are placed on a support. Subsequently, the support with the filled capillaries is placed on the tempering element according to the invention. Preferably, the capillaries are filled at least in a central region of the capillaries over a length which is wider than the width of the tempering element.
  • the measurement of the samples should be done by fluorescence measurement. For this purpose, the sample is first excited by means of an excitation LED in the UV range, for example at 280 nm.
  • an optic is moved into the measuring position.
  • the samples are tempered with the aid of the tempering element.
  • the temperature is driven over a set ramp to the final temperature.
  • the samples are constantly driven under the optics, whereby the fluorescence values are read out (see FIG. 5).
  • the fluorescence emission is measured at 330 and 350nm.
  • the measured data are stored, the temperature control and the LED are switched off and the axes are moved back to their rest positions.
  • a database file is created with the acquired measurement data.
  • the database is converted into a CSV file ("comma- separated-values") and then read into an analysis software, which is able to automatically calculate the melting points by means of an inflection point analysis by forming the quotient of the two fluorescence Channels 330nm and 350nm, a sigmoidal curve is formed (Figure 9).
  • Fig. 6 a total of 15 samples were analyzed. Each color indicates the associated fluorescence intensity at a given temperature. Due to the very large number of colors, the large number of test runs and thus also high temperature resolution, as each test drive represents a temperature. The uppermost peak of a trace represents the fluorescence signal at the start temperature, and the lowermost trace (here light blue) represents the fluorescence signal at the final temperature of a measurement. The low autofluorescence of the silicon (baseline) can also be seen very clearly.
  • the individual curves for the two channels 330 nm (FIG. 7) and 350 nm (FIG. 8) can also be displayed.
  • a so-called “melting curve” / "denaturation curve” is obtained ( Figure 9) .
  • the melting point of the protein under investigation lies at the turning points of the respective measuring curve.
  • FIGS. 10 a) to 10 i) show examples of possible cross-sectional shapes of capillaries.
  • Fig. 10 a) shows a round capillary with the wall 20 and the cavity or the cavity 21.
  • Figures 10 f) and 10 g) also show round embodiments, but with different wall thicknesses and correspondingly different cavities with the same outside diameter.
  • Fig. 10 (b) shows a semicircular embodiment; Fig. 10c) a hexagonal embodiment; Fig. 10d) a quadrangular embodiment; Fig. 10e) an oval embodiment; 10 h) an example of an embodiment in which the outer shape differs from the inner shape, here with a quadrangular outer shape and an oval or round inner shape, and FIG. 10 i) a combination with a plurality of cavities within an outer shape.
  • Figure IIA shows an example of a typical buffer screening from antibody research. Due to the unfolding of a protein / biomolecule, the emission maximum of the fluorescence shifts from the spectral range 330nm +/- 5nm to the spectral range 350nm +/- 5nm. This shift is made clear by measuring and recording the fluorescence ratio at 350nm divided by fluorescence at 330nm. Shown here is the change in tryptophan emission (F350nm +/- 5nm divided by F330nm +/- 5nm) of an antibody by unfolding at elevated temperatures. The thermal unfolding occurs in the case of the antibody shown at pH values ⁇ pH 7 at significantly lower temperatures, which indicates a destabilization of the antibody under acidic conditions.
  • Figure 11B shows an example of a change in the thermal stability of a protein by binding small molecules. Shown is the change in tryptophan emission (F350nm +/- 5nm divided by F330nm +/- 5nm) of a protein by unfolding at elevated temperatures after binding different amounts of a low molecular weight ligand. The more ligand added, the more ligand binds to the protein and the more thermally stable this protein becomes.
  • the device according to the invention and the method according to the invention preferably comprise one or more of the following features, in particular in nanoDSF applications (nano DSF applications).
  • nanoDSF applications nano DSF applications
  • ultra-high resolution protein stability measurements can thus be carried out.
  • the device hereafter called Prometheus NT.48, can accommodate 48 capillaries.
  • the capillaries are filled with the sample by capillary force action, so you simply immerse the capillaries only in the sample and placed them in the instrument.
  • the instrument is preferably maintenance-free and contains no hoses, valves or pumps. Since the capillaries are preferably for single use, no equilibration or purification is required.
  • Buffer and formulation screens are easily accomplished by mixing the protein with the solutions of interest.
  • Capillary fillers are available to fill capillaries from microtiter plates in seconds.
  • Sample annotations can be conveniently entered while the experiment is running.
  • different concentrations of denaturant are mixed with the protein of interest and incubated for equilibration.
  • the samples are filled in capillaries and then analyzed by the Prometheus NT.48.
  • the nanoDSF is an advanced method for differential scanning F 1 uorimet ri e. to measure ultrahigh-resolution protein stability using intrinsic tryptophan 11 fluorescence for applications in antibody engineering, membrane protein research, formulation and quality control.
  • the devices of the invention provide nanoDSi technology, which is the method of choice for easy, rapid and accurate analysis of protein folding and stability in protein engineering, formulation development and quality control applications.
  • a preferred dual UV technology enables on-line / fluorescence detection, providing unsurpassed scanning speed and data point density, and thus ultra-high resolution deconvolution curves, enabling detection of even the smallest deconvolution signals.
  • protein solutions can be analyzed independently of buffer compositions and over a maximum protein concentration range, preferably from 150 mg / ml to only 5 ⁇ g / ml, allowing analysis of detergent-solubilized protein protease as well as highly concentrated antibody formulations.
  • thermal and chemical unfolding experiments Widely used methods for quantifying the structural stability of a protein are thermal and chemical unfolding experiments.
  • thermal unfolding experiments use a constantly rising temperature to To monitor protein conformational changes over time
  • chemical unfolding experiments use concentration gradients of buffering additives. usually chaotropes, / .. B. urea to unfold proteins to varying degrees.
  • melting temperature The midpoint of the transition from unfolded to unfolding, referred to as “melting temperature” or “I m”, serves as a measure of protein stability, and thermal unfolding experiments in protein engineering, in the form of the 1 u and in screening procedures, as they allow a large number of samples to be assessed rapidly in parallel.
  • the device of the invention is preferably equipped with fluorescence detectors which measure the fluorescence intensity at two different wavelengths, 330 nm and 350 nm, thereby being sensitive to both the change in fluorescence intensity and the shift in fluorescence peak upon deployment.
  • Protein denaturation curves are used to derive important stability parameters. Normally, the thermal stability of a given protein is described by the melting temperature Tm at which half of the protein populum is unfolded. Tm can be calculated from the changes in tryptophan fluorescence intensity or from the ratio of tryptophan emission at 330 and 350 nm, which describes the shift in tryptophan emission upon unfolding. Typically, the 350/330 nm quotient yields data with well-defined protein unfolding transitions, whereas Tm can not always be deduced with single wavelength detection. Thus, the dual wavelength system of the device provides for sensitive detection of deployment processes.
  • the device of the invention (e.g., Prometheus NT.48) can be used in formulation and quality control laboratories. Due to the wide concentration range, biopharmaceuticals with very high concentrations can be investigated which are normally used in the formulation. Particularly useful is the nanoDSF technology used by the Prometheus device for antibody engineering applications because ultra-high resolution enables detection and analysis of multiple transitions and unfolding events. Furthermore, with nanoDSF it is possible to measure the stability of membrane proteins in detergents since this method is really label-free and does not require a fluorescent dye.
  • the estimation of protein stability is an integral part of the protein stability
  • the basis of marker-free lluorimetric analysis of protein folding lies in the properties of the fluorescent amino acid tryptophan. Because tryptophan is a hydrophobic amino acid. it is usually present in the hydrophobic core of proteins where it is shielded from the surrounding aqueous solvent. However, after unfolding, tryptophan is released, which alters its p h o s p o sics 1 properties [6]. By detecting changes in the fluorescence intensity of tryptophan and its shift in the emission peak, the transition of a protein from the folded to the unfolded state can be precisely recapitulated. In this way, the melting temperature (Tm) and the rm ody n can be determined by their properties [7].
  • Tm melting temperature
  • rm ody n can be determined by their properties [7].
  • the Prometheus NT.48 instrument can measure up to 48 samples in parallel and uses high-precision capillaries filled with only 10 ⁇ of sample. With the help of a detector specially designed to monitor changes in the emission spectrum of tryptophan with maximum sensitivity and speed, highest data point density and precision is achieved. Proteins of the ⁇ -amylase family have proven useful for the analysis of protein folding [8]. Most amylases have very similar tertiary structures sharing three ( ⁇ / a) Barrels domains and at least one conserved Ca " binding site (Figure 12). Figure 12 shows the structure of porcine pancreatic ⁇ -amylase (PA, green) and ⁇ -amylase from Aspergillus oryzae (TAKA, blue) . The red sphere represents a Ca ion.
  • PA porcine pancreatic ⁇ -amylase
  • TAKA Aspergillus oryzae
  • the Prometheus T.48 instrument monitors the shift in intrinsic tryptophan fluorescence of proteins upon deployment by detecting fluorescence at emission wavelengths of 330 and 350 nm. To determine protein melting point (Tin, where half of the protein is folded and the other half unfolded) the flow can be. It is possible to use one of the two channels or, alternatively, the ratio of fluorescence intensities (F330 / F350 quotient) can be plotted.
  • the fluorescence quotient monitors both the change in tryptophan fluorescence intensity as well as a shift in the emission maximum of the fluorescence to higher Wavelengths ("redshift") or lower wavelengths ("blueshift”).
  • redshift a shift in the emission maximum of the fluorescence to higher Wavelengths
  • blueshift a shift in the emission maximum of the fluorescence to higher Wavelengths
  • the thermal unfolding of PPA and TAKA was performed at a heating rate of 1 ° C / minute, resulting in a data point density of 10 points / ° C, which provided an accurate determination of the onset of protein unfolding as well as an exact adaptation of the transition from folded to mathematical Models possible.
  • Figure 13 shows the changes in tryptophan fluorescence of PPA and TAKA upon thermal unfolding.
  • the raw fluorescence data from both wavelengths show a clear transition from folded to unfolded (Figure 13A, left), which could be used directly for Tm analysis. In contrast, this transition is not evident from the raw data for PPA ( Figure 13B, left).
  • TAKA showed a typical unfolding profile with shift of tryptophan fluorescence to higher wavelengths (redshift), PPA exhibited a less common shift of tryptophan fluorescence to lower wavelengths (blue shift).
  • the instrument Prometheus NT.48 can be used to accurately determine Tm values with a minimum of sample and time expenditure.
  • Prometheus NT.48 has been used to test the effects of various buffering additives that have been shown to increase protein stability; Ii., Glycerol, sucrose, trehalose and sorbitol at concentrations ranging from 10% to 40% (weight per volume) of PPA and TAKA.
  • the formulation screen of 16 different buffer conditions for each amylase isoform was performed in a single run with a temperature range of 20 ° C to 90 ° C and a heating rate of 1 ° C / min. Measurements were taken within about 70 minutes with a total sample consumption of 400 ⁇ (10 ⁇ for each buffer condition plus 4 control experiments for each isoform without additive) and a total protein level of just 80 ⁇ g.
  • Tm values of the amylase proteins could be determined under different conditions. All results show good agreement with published values. However, compared to methods using standard fluorometers, most importantly, both sample consumption and time spent performing the experiments using the Prometheus NT.48 are dramatically reduced.
  • the capillary format of the instrument allows flexible design of experiments, with each number of samples being measured between 1 and 48 simultaneously.
  • the use of Prometheus capillaries provides even greater precision of UV fluorescence detection than high performance quartz cuvettes, with the benefits of low sample consumption, high throughput, and great versatility.
  • the capillary-based approach prevents cross-contamination, and no tedious and time-consuming cleaning steps are required.
  • high scanning speeds and thus a high data density enable a robust analysis of melting curves by mathematical adaptation algorithms and furthermore enable a precise determination of deploying deployments.
  • the direct detection of phosphatofluorescence for monitoring protein unfolding has several benefits compared to other methods routinely used to monitor thermal unfolding, e.g. B. D i ITe ence - Scanning Fluorimetry (DSF) or Thermofluor Assays.
  • DSF D i ITe ence - Scanning Fluorimetry
  • Thermofluor Assays use external fluorophores that bind to hydrophobic sites of the protein that are usually buried in the core of the protein. Upon deployment, these sites are exposed and the fluorophore attaches, resulting in increased fluorescence.
  • these assays are not suitable for a detailed analysis of folding thermodynamics because they interfere with folding-unfolding equilibria by direct interaction with the proteins.
  • external fluorophores are linked to a number of buffers (including, for example, detergents) or protein types, e.g. B. membrane proteins, incompatible.
  • buffers including, for example, detergents
  • protein types e.g. B. membrane proteins, incompatible.
  • DSF is routinely used in primary screening in the drug discovery process
  • external fluorophores can be linked Interact or block binding sites and produce false-negative and false-positive results.
  • the Prometheus NT.48 instrument can also be used to analyze chemical denaturation of proteins in seconds.
  • the results show that the Prometheus NT.48 instrument is extremely well-suited for rapid, accurate and cost-effective characterization of protein stability in both the academic and industrial settings. Its flexibility and speed make it a valuable tool for a plethora of diverse experimental procedures, ranging from thorough characterization of protein folding to high-throughput screening projects.
  • Porcine ⁇ -amylase (porcine pancreas ⁇ -amylase, PPA, Roche) and Aspergillus oryzae ⁇ -amylase (TAKA, Sigma) were dissolved in 30 mM Hepes, 50 mM NaCl, 2 mM CaCl 2, pH 7.4 at concentrations of 10 mg / ml dissolved. Final concentrations in thermal unfolding experiments were 10 ⁇ .
  • buffer exchange was performed by means of buffer exchange centrifugal columns (NanoTemper Technologies).
  • a second buffer exchange to buffer without CaCF, but with 5 mM EDTA.
  • the proteins were in 20 mM Na citrate buffer. pH 5.9 with the respective concentrations of sucrose. Sorbitol. Trehalose or glycerol transferred.
  • the proteins were diluted to a final concentration of 10 ⁇ . For each condition, 10 ⁇ sample per capillary was prepared. The samples were placed in UV capillaries (NanoTemper Technologies) and experiments were performed using the Prometheus NT.48. The temperature gradient was set at a rise of 1 ° C / min in a range of 20 ° C to 90 ° C. Protein unfolding was measured by detecting the temperature-dependent change in tryptophan fluorescence at emission wavelengths of 330 and 350 nm.
  • Tryptophan fl uores / enz on thermal unfolding of TAKA (left).
  • the transition from the folded to the unfolded state is already visible in the fluorescence raw data at emission wavelengths of 330 and 350 nm.
  • the picture shows the high data density of Prometheus NT.48.
  • Tm two methods can be used. Median analysis (center) defines a median line between an upper and lower baseline. Their cross section with the experimental data represents Tm. Alternatively, the experimental data can be adjusted with a polynomial function. Its first derivative shows a peak at the point of maximum steepness corresponding to Tm (right).
  • B Equivalent analysis of Tm for PPA. It should be noted that, unlike TAKA, the transition from folded to unfolded protein is not visible in the fluorescence data (left), while Tm can be easily determined from fluorescence quotient (right) plots.
  • FIG. 14 Accuracy and reproducibility of the unfolding data of the Prometheus NT.48.
  • A The plots represent a superposition of 10 independently registered melting curves of PPA and TAKA, respectively.
  • B The Tm determination for both proteins shows a small value for this difference between the experiments ( ⁇ 0.2 ° C) and a good correlation with published results [9].
  • Fig. 15 Ca 2+ effects on the amylase stability. By removal of Ca 2 ions, there is a marked destabilization of both A my 1 as e- 1 so form. whereupon the shift from Tm refers to lower values.
  • Fig. 16 Formulation screening of PPA.
  • Tm thermal precipitation
  • Fig. 1 7 Formal tion screening of TAKA.
  • TAKA has been optimized thermal unfolding monitored under 16 different additive conditions. A significant shift from Tm to higher values can be observed in the plots of the fluorescence quotients for each additive. By quantifying Tm under different conditions, it can be seen that the addition of 40% sucrose is most effective, while glycerol and trehalose have the weakest effect.
  • NanoTemper Technologies offers the nanoDSF technology. h., the method of choice for easy, rapid, and accurate analysis of protein folding and stability with applications in protein engineering, formulation development, and quality control.
  • the nanoDSF is an advanced differential scanning technology based on the detection of minute changes in the intrinsic fluorescence of the amino acid tryptophan.
  • the fluorescence of tryptophans in a protein depends strongly on their close environment. By tracking changes in the fluorescence of the amino acid tryptophan, chemical and thermal stability can be truly assessed without marker.
  • protein solutions can be analyzed independently of buffer compositions and over a maximum protein concentration range of 150 mg / ml to just 5 ⁇ g / ml, allowing the analysis of detergent-dense membrane proteins as well as highly concentrated antibody formulations.
  • NanoTemper's dual UV technology enables oxy-fluorescence detection for unsurpassed scanning speed and data point density and, therefore, for a high-performance fluorescence detection Ultra-high resolution of unfolding curves ensures that even the smallest development signals can be detected.
  • Protein derivation curves are used to derive important stability parameters. Normally, the thermal stability of a given protein is described by the melting temperature Tm at which one-half of the protein population is unfolded.
  • Tm can be calculated from the changes in tryptophan fluorescence intensity or from the ratio of tryptophan emission at 30 and 350 nm, which is the
  • the 350/330 nm quotient yields data with well-defined transitions in protein unfolding, whereas single-wavelength Tm is not always can derive.
  • the dual-wavelength system of the Prometheus NT.48 provides for sensitive detection of unfolding processes.
  • the invention also includes the exact or exact terms, features, numerical values or ranges, etc. when, above or below, these terms, features, numerical values or ranges are used in conjunction with terms such as, for example.

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Abstract

L'invention concerne un procédé de thermorégulation de plusieurs capillaires (10) qui sont disposés sur un support (6), le support (6) de longueur (L), de largeur (B) et de hauteur (H) recevant des capillaires (1) dans le sens de la largeur dudit support (6). Le support (6) présente un évidement (61) destiné à loger un élément thermorégulateur (5) de manière telle que les capillaires (10) puissent être thermorégulés dans leur zone centrale par contact avec l'élément de thermorégulation (5). Selon l'invention, les extrémités (11, 12) des capillaires (10) remplis d'échantillons sont non fermés pendant la thermorégulation.
EP15816700.7A 2014-12-12 2015-12-11 Procédé et système de thermorégulation de capillaires exempts d'étanchéité Pending EP3229966A1 (fr)

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