METHOD FOR SEAL-FREE TEMPERING OF CAPILLARIES

Description

[0001] The invention generally relates to a method for controlling the temperature of capillaries filled with samples to be analyzed. In particular, the invention relates to a method for controlling the temperature of capillaries for optical measurements of temperature-controlled samples as a function of temperature. The optical measurement is preferably carried out in the UV range based on the fluorescence behavior of the samples to be measured. A system is also disclosed with which the method according to the invention can be carried out simply and efficiently. A significant advantage of the invention is that sealing the capillaries is not necessary for the temperature control and optical measurement of the samples within the capillaries. BACKGROUND OF THE INVENTION

[0002] In biophysics, biochemistry, biology, pharmacy, molecular diagnostics and analytics in general, samples are often exposed to different temperatures in order to characterise them based on their behaviour at different temperatures.

[0003] For example, melting curve analyses, thermal stability measurements, thermal shift assays (TFA) and differential scanning fluorimetry (DSF) are important tools for qualitatively and quantitatively assessing the stability and aggregation behavior of proteins and drug formulations.

[0004] Another example is MicroScale Thermophoresis (thermo-optical particle characterization), in which, for example, affinities (Kd, EC50) of interactions are measured at different temperatures in order to derive the thermodynamic quantities dH and dS from the measurement results, for example using a van't Hoff plot.

[0005] In biophysics, biochemistry, biology, pharmacy, molecular diagnostics, and analytics (e.g., food analysis, cosmetics, etc.), aqueous solutions such as buffers, lysates, urine, sera, whole blood, etc., or liquids in general, are primarily used. In this area, the temperature range to be examined extends, for example, from 0°C to 100°C, or to the respective range in which the respective liquid exists in its liquid form.

[0006] Capillaries are very interesting as sample containers for these applications because they have a very small and well-defined volume. Furthermore, capillaries can be filled with liquids independently through capillary action, eliminating the need for pumps. Furthermore, capillaries made of materials such as borosilicate 3.3, quartz, synthetic fused silica, etc., are also advantageous in terms of their optical properties, particularly their transparency, purity, and autofluorescence. Short capillaries with a small inner and outer diameter, for example, with an outer diameter of no more than 1 mm and an inner diameter of no more than 0.8 mm, preferably with an outer diameter of 0.65 mm and an inner diameter of 0.5 mm, are particularly advantageous because they have a small volume and thus save sample material.Temperature control of capillaries is also known from US 5 720 923 A, WO 97 / 48818 A1, DE 10 2004 022263 A1, JP 2004 187678 A, WO 2007 / 082466 A1.

[0007] To perform the described measurement methods, such as melting curve analysis, the capillaries must be heated to a temperature ranging from 10°C to 100°C, for example. At this temperature, significant evaporation of the liquid is typically observed at elevated temperatures. This evaporation or evaporation leads to disruptive flow in the liquid and, in particular, to such a significant loss of liquid that measurements at elevated temperatures over extended periods are not possible.

[0008] This evaporation can be avoided or reduced by sealing the ends of the capillaries with wax, for example, or by welding them with a flame. However, these sealing methods have significant disadvantages. Welding the capillaries, especially with quartz (which, due to its good optical properties, particularly low autofluorescence, is advantageous for measurements with electromagnetic radiation in the UV range), results in temperatures so high that the molecules under investigation are altered or destroyed during the welding process, making them impossible to study. Furthermore, almost no user has the necessary equipment to generate flames hot enough and defined enough to weld the ends of quartz capillaries together in a defined and localized manner.

[0009] Sealing the capillaries with an additional material, such as wax, always carries the risk of contaminating the liquid / sample with the sealing material and thus distorting the measurements. Furthermore, it has been observed that sealants such as wax can be forced out of the capillary at elevated temperatures due to the vapor pressure within the capillary, thus losing their functionality.

[0010] Systems are also known in which capillaries in the form of Micro Cuvette Arrays (MCA). These Micro Cuvettes are clamped in a frame, which seals the cuvettes with silicone strips at both ends. To prevent contamination, these silicone strips and / or the frame must be replaced regularly, which incurs additional costs.

[0011] Since it is particularly desirable to work with very small volumes on the microliter scale, especially for biomolecules such as proteins, peptides, nucleic acids, DNA, RNA, antibodies, but also cells, bacteria, nanodiscs, vesicles, viruses, etc., short, very thin capillaries are advantageous. Furthermore, it is advantageous to use thin-walled capillaries, as this thin wall design minimizes autofluorescence and other artifacts.

[0012] However, thin-walled capillaries, i.e., capillaries with a small diameter and a thin wall, have the disadvantage of being very fragile. For this reason, non-destructive mechanical sealing of the capillaries, for example, with a plug or cap, is not possible or only possible with considerable and therefore no longer economical effort.

[0013] There is therefore a need for a simple or improved method with which optical measurements can be carried out at higher temperatures and over a longer period of time. SUMMARY OF THE INVENTION

[0014] The method according to the invention is defined by the features of the independent claims. Advantageous embodiments emerge from the subclaims.

[0015] In particular, the present invention relates to a method with which liquids in a capillary can be temperature-controlled and optically examined without sealing the capillary. Preferably, several capillaries are temperature-controlled simultaneously without sealing the capillaries and optically examined simultaneously or one after the other. Preferred advantages of the seal-free method according to the invention can be briefly described as follows. The risk of a capillary breaking is significantly reduced, since the risk of breakage is usually greatest when the capillary is closed. In addition, sealing steps are saved because not every capillary has to be sealed at both ends. The solution according to the invention is therefore not only faster, but also less cost-intensive, i.e. cheaper, and contamination by sealing material can also be avoided.

[0016] The present invention relates to a method for controlling the temperature of at least one, preferably several, capillaries. For easier handling, the capillary(s) are arranged, for example, on a carrier. The carrier preferably has a length L, width B, and height H (see, for example, Fig. 3). The capillaries are preferably arranged along the width of the carrier. The carrier preferably has a recess into which, for example, a temperature control element can be inserted. In addition, it is preferred that the capillaries are held by the carrier only outside the temperature control element, so that the entire width of the temperature control element is available for measurements. The capillaries should preferably be temperature-controlled in their central region by contact with the temperature control element, wherein the ends of the capillaries filled with samples are unsealed during temperature control. It is also preferably advantageous to consider the arrangement of the capillaries in relation to the temperature control element with regard to the fill quantity. According to the invention, the temperature control element can be warmed or heated and / or cooled, wherein the reference point is preferably the ambient temperature.

[0017] According to a preferred embodiment, the temperature range of the sample in the capillary extends, for example, from 0°C to 100°C, or to the respective range in which the respective liquid is present in its liquid form. In other words, if the sample is an aqueous solution and the measurement of the sample is to be carried out in the liquid phase, then it is preferable to temperature-control the sample in a range from 0°C to 100°C. If the sample liquid is a liquid with a lower melting point, for example a liquid that contains other solvents, for example organic solvents, for example alcohols, or consists essentially entirely of these substances, then the preferred lower limit of the temperature-control range can also be lower, for example below 0°C.For example, 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 larger or smaller temperature range than 0°C to 100°C. For example, for aqueous solutions with a high content of salts and / or detergents, the preferred lower limit of the temperature range can also be lower, for example below 0°C. For example, for aqueous solutions with a high content of salts and / or detergents, the preferred upper limit of the temperature range can also be higher, for example above 100°C.

[0018] For example, supercooled liquids can also be used according to the invention.

[0019] Furthermore, according to a further preferred embodiment, the lower limit of the temperature range for aqueous liquids can be below 0°C, or below the freezing point if freezing of the liquid is desired. Since the capillaries according to the invention are not sealed or closed, temperatures below 0°C can also be used without the capillary bursting due to the expansion of the aqueous solution (anomaly of the water). In contrast, a frozen aqueous solution in a sealed capillary could burst the capillary due to the increase in volume. However, with the seal-free capillaries according to the invention, the increase in volume does not pose a problem, since expansion of the frozen liquid is possible due to the lack of a seal.The seal-free capillaries according to the invention also survive repeated freezing and thawing processes of the aqueous solution, which are carried out, for example, to test whether repeated freezing and thawing lead to the unfolding and / or aggregation of biomolecules in the aqueous solution. For example, aqueous solutions containing biomolecules are stored at -20°C or -80°C. Before storage, these aqueous solutions containing biomolecules are in liquid form. When stored at, for example, -20°C or -80°C, these aqueous solutions freeze. They are removed from the freezer and thawed for use in liquid form.For the denaturation / unfolding and / or aggregation of the biomolecules in the aqueous solution, for example, not only the absolute temperature of freezing plays a role, but also, for example, the cooling and warming rate at which the freezing and thawing takes place and / or how often this process is carried out / repeated.

[0020] According to the invention, the sample to be examined is filled into a capillary, whereby the capillary is usually not filled from end to end with the sample liquid. The part of the capillary filled with the sample liquid is referred to below as the liquid column. Preferably, the liquid column of the capillary is aligned with the temperature control element such that both ends of the liquid column protrude beyond the temperature control element.

[0021] Preferably, the tubular capillaries according to the invention have a length between 40 - 75 mm, preferably between 45 - 55 mm, more preferably about 50 mm.

[0022] The width of the temperature control element is preferably between 5 and 34 mm, more preferably between 20 and 30 mm, more preferably 20 and 25 mm, more preferably approximately 25 mm. Silicon, preferably pure silicon, is preferably used as the temperature control element.

[0023] According to particular embodiments, it may be advantageous to form the tempering element in one piece along the width, or to form a plurality of separate tempering regions along the width, wherein these plurality of tempering regions can contact one another or a gap can be formed therebetween.

[0024] To ensure reliable temperature control of the capillaries, it may also be advantageous to press the capillary(s) onto the temperature control element using a cover to ensure contact between the capillary and the temperature control element. The cover can be positioned partially over the temperature control area and / or exert a force on the capillaries outside the temperature control area.

[0025] According to the invention, the individual capillaries are filled with a liquid, preferably with an aqueous sample solution, in particular buffer solutions for biochemical / biological measurements. Additionally or alternatively, non-aqueous solvents, such as organic solvents, can also be used or added.

[0026] 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).

[0027] The sample solution or sample liquid according to the invention which is filled into the capillaries can also be oils, emulsions, dispersions or other substances or mixtures which are in the liquid phase in at least one of the preferred temperature ranges and can be filled into the capillary.

[0028] 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, more 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.

[0029] 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.

[0030] The capillaries can be made of, for example, glass, preferably borosilicate 3.3, quartz, or synthetic fused silica, but are not limited to these.

[0031] As is well known, capillaries are generally small tubes with very small inner diameters. Due to the surface effects, which are more prominent in capillaries than in larger tubes, capillarity, a physical effect, occurs in capillaries. Liquids with high surface tension rise in capillaries.

[0032] Furthermore, the capillaries according to the invention are not limited to a specific cross-sectional shape. Most capillaries are round. According to the invention, the cross-section of a capillary can also be oval, triangular, square, pentagonal, hexagonal, octagonal, semicircular, or trapezoidal, or have another irregular shape.

[0033] It is also preferred according to the invention that the capillaries are made of a solid, preferably non-deformable material such as glass, and that the cross-sectional shape of the capillaries does not change for or during a measurement. For example, the cross-sectional shape is the same during filling as it is during the measurement. Compressing the cross-section for measuring is preferably avoided, for example because the inner and outer diameter of the capillaries also influence fluorescence measurements, absorption measurements, extinction measurements, or scattered light measurements. Since the capillaries are not closed on at least one side according to the invention, deformation of the capillaries can also lead to the sample liquid to be examined being squeezed out, which should preferably be avoided.

[0034] The present invention also relates to a method for the optical examination of samples filled in capillaries. First, the capillaries are filled with the sample. Then, the capillaries are positioned on the temperature control element for temperature control. For this purpose, several capillaries are preferably first arranged on a carrier, and the carrier with the several capillaries is then positioned on the temperature control element. The capillaries can then be temperature-controlled as described above. To finally carry out the optical measurement, the samples can be excited with light, for example. Excitation with light is not limited to a specific wavelength of light. According to a preferred embodiment, excitation can be carried out using UV light, for example. The light emitted by the sample is then measured. Even when measuring the emitted light, the present invention is not limited to a specific wavelength.

[0035] Also disclosed is a system for the optical examination of samples in capillaries. The system preferably comprises a temperature control device for controlling the temperature of the capillaries. Furthermore, it may be preferable to provide a support for holding the capillaries. Additionally or alternatively, the system according to the invention may also comprise an optical measuring system for emitting and detecting light. According to a further preferred embodiment, the system may comprise at least one capillary. Preferably, this capillary is non-deformable and preferably tubular.

[0036] The term "non-deformable" is understood in particular to mean that the cross-section of the capillary remains essentially constant under an applied pressure. Preferably, the term "non-deformable" is understood to mean "macroscopically non-deformable." In particular, the capillary is preferably hard. Furthermore, it is preferred if no pressure is applied to the capillary during a measurement, or if the pressure is so low that the cross-section of the capillary remains essentially unchanged.

[0037] For example, the system presented can also be used to measure thermophoresis effects in samples.

[0038] The methods according to the invention are particularly suitable for protein folding and unfolding experiments and for investigating the stability of biomolecules such as proteins. Here, the structure of the biomolecule under investigation, in particular a protein or protein complex, is altered by adding suitable chemicals, e.g., chaotropes such as urea or guanidinium hydrochloride or organic solvents, or by changing the temperature (e.g., "melting" by increasing the temperature). The secondary and tertiary structure of biomolecules such as proteins and nucleic acids often also depends on the presence of ligands or cofactors such as ions (e.g., Mg 2+ or Ca 2+ ). This can be achieved, for example, by measuring fluorescence (preferably typophan fluorescence in the case of proteins) at different concentrations of the ligands and / or cofactors.

[0039] The biomolecule, preferably a protein, can be chemically or thermally denatured, and structural changes can be measured by intrinsic fluorescence (preferably tryptophan fluorescence in the case of proteins). This can detect, for example, changes in fluorescence intensity or shifts in fluorescence maxima, etc. The melting point of the biomolecule under investigation, e.g., a protein, can also be determined. The melting point is the state in which the biomolecule under investigation, e.g., a protein, is half folded and half unfolded. In the case of proteins, for example, tryptophan fluorescence can be measured at a wavelength of 330 nm and / or 350 nm. The change in fluorescence intensity can be determined, e.g., as a function of temperature or the addition of a denaturant or cofactor / ligand, and / or a time course can be recorded.The quotient of fluorescence intensity at 330 nm to fluorescence intensity at 350 nm (F330 / F350) is a preferred measurement parameter. For example, the melting point can be determined from the maximum of the first derivative of the F330 / F350 curve.

[0040] The melting of nucleic acids or their complexes can also be monitored using fluorescence measurements. In addition to fluorescence measurements, the measurement of circular dichroism (CD) is also possible.

[0041] In addition to the thermal, chemical, enzymatic, or temporal denaturation of biomolecules, especially proteins such as membrane proteins or antibodies, the aggregation behavior of biomolecules can also be measured. Measuring aggregation behavior is particularly, but not only, of interest for drug approval. This aggregation can be measured, for example, by changing the intrinsic fluorescence, such as changing the fluorescence intensity and / or shifting the fluorescence emission maximum. This aggregation can also be measured by measuring the fluorescence anisotropy of the biomolecules.Preferably, the measurement of fluorescence anisotropy also allows to measure a change in the size of the biomolecules and thus, for example, to measure the size of emerging aggregates or to measure the disintegration of multimers of biomolecules, for example the thermally induced disintegration of a tetramer into its four monomers.

[0042] For example, thermally, chemically, enzymatically or temporally induced changes in the size of biomolecules and thus also their aggregations or multimerizations can be measured by light scattering.

[0043] Possible applications of the methods according to the invention can be found in the field of protein engineering (especially antibody engineering) or in the investigation of membrane proteins, in quality control or in the development of biologics in the pharmaceutical industry. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Preferred embodiments of the present invention are described below with reference to the figures. They show: Fig. 1 a diagram of a percentage evaporation for a 50mm capillary as a function of the width of the support surface of a temperature control body / temperature control element; Fig. 2 similar to Fig. 1 a diagram of a percentage evaporation as a function of the width of the support surface of a temperature control body / temperature control element, but with a capillary with a length of 32 mm; Fig. 3 an exploded view of a temperature control device with a carrier for holding the capillaries; Fig. 4 a schematic plan view of six different capillaries with different filling levels lying on a temperature control element; Fig. 5 a schematic representation of an optical measurement with several capillaries on a temperature control element and an optical excitation at 280 nm using an LED; Fig. 6 a measurement diagram using an optical measurement according to Fig. 5was created, where each peak corresponds to a capillary; Fig. 7 the course of a melting curve with an emission window of 330 nm; Fig. 8 the corresponding course of the melting curve after Fig. 7 , but with an emission window of 350mn; Fig. 9the quotient of the two optical detection channels from Figs. 7 and 8 ; Fig. 10a-10i Capillaries of different geometries or cross-sections; Fig. 11A An example of a typical buffer screening from antibody research; Fig. 11B Example of a change in the thermal stability of a protein by binding small molecules; Fig. 12-17 Illustrations from an application example according to the invention; and Fig. 18 Similar to Fig. 5 a schematic representation of an optical measurement with 48 capillaries on a temperature control body. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

[0045] The invention generally relates to a method for controlling the temperature of a capillary, preferably several capillaries simultaneously, which are filled with samples to be examined. According to the invention, the capillaries are made of glass. Preferably, the capillaries are made of a material with a similar, not much lower and / or not very much / significantly higher thermal conductivity than the liquid in the capillaries. Glass is also preferred for this reason, as it has a similar thermal conductivity to an aqueous solution. In particular, it is preferred because, according to the invention, the heat is transferred to the solution by means of glass, i.e., if the thermal conductivity of the capillary material is too low, the solution in the capillaries is not heated correctly and / or not heated quickly enough. If the thermal conductivity is too high, the heat is transported to the ends of the capillary and then leads to increased evaporation.The heat capacity of some materials is listed below purely as an example: Polypropylene (PP) 0.23 W / (m*K); Water: 0.5562 W / (m*K); Glass: 0.76 W / (m*K); Quartz: 1.2 W / (m*K) to 1.4 W / (m*K); Steel: 48 W / (m*K) to 58 W / (m*K).

[0046] Depending on the measurement and the measurement duration, materials with thermal conductivities that differ significantly from water can also be used according to the invention. Thus, it is generally preferred to use a material for the capillaries that lies in the range of 0.15 W / (m*K) to 60 W / (m*K). For example, materials such as PMMA / Plexiglas, polypropylene, PEEK, and Teflon fall into the lower limit. Another preferred range for glass as a material depends on the various types of glass and extends, for example, from approximately 0.5 W / (m*K) to 1.6 W / (m*K).

[0047] 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, Duran glass), 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 can include: PTFE, PMMA, Zeonor™, Zeonex™, Teflon AF, PC, PE, PET, PPS, PVDF, PFA, FEP, and / or acrylic glass.

[0048] In particular, it is preferred that at least one region of the capillaries is transparent to light with a wavelength of 200 nm to 1000 nm, preferably from 250 nm to 900 nm. Particularly preferred, but not limited to, this at least one segment is also transparent to light in the following wavelength ranges: from 940 nm to 1040 nm (preferably 980 nm + / -10 nm), from 1150 nm to 1210 nm, from 1280 nm to 1600 nm (preferably 1450 nm + / -20 nm and / or 1480 nm + / -20 nm and / or 1550 nm + / -20 nm), from 1900 nm to 2000 nm (preferably 1930 nm + / -20 nm). One skilled in the art will understand that the transparent region(s) may also extend over the entire tubular structure. In other words, the capillary may be transparent.

[0049] The light transmittance of the segment allows for luminescence / fluorescence / phosphorescence measurements and / or optical investigations / measurements (e.g., interference, polarization, absorption, dichroism, ellipsometry, anisotropy, Raman, microscopy, dark field, light scattering, FRET, microscale thermophoresis, thermo-optical particle characterization) and / or manipulation of the solution / liquid in the capillary cavity. The light transmittance can also allow for fluorescence measurements. According to a preferred embodiment, it also enables the heating of fluids in the tubular structure using electromagnetic radiation, for example, light (preferably an infrared (IR) laser), preferably the heating of water and / or organic solvents.

[0050] According to the present invention, the capillaries are preferably brought into contact with a temperature control element, so that this contact results in a temperature exchange from the temperature control element to the capillaries and thus to the samples within the capillaries. The capillaries are preferably temperature-controlled by means of contact heat in the area in which the optical measurement also takes place. For example, the thermal contact can be improved in this area by applying an oil, for example an immersion oil. The optical measurement is preferably not restricted to a specific wavelength range and can, for example, take place in the IR, visible or UV range. It is also desirable that the temperature element itself emits no or only a small amount of fluorescence that could falsify the measurement of the sample. According to the invention, the contact material for the temperature control element, i.e., the element that comes into contact with the capillary(s) and transfers the temperature to the capillaries by direct contact, preferably silicon.

[0051] The use of silicon has a number of advantages, although only a few are listed here as examples. Firstly, silicon has no or only extremely low autofluorescence, especially with excitation light in the range of 260 nm to 700 nm. In a typical measurement of tryptophan fluorescence, for example, excitation is carried out at 260 nm to 300 nm and emission is measured at >320 nm. This makes silicon very well suited for fluorescence measurements, especially for fluorescence measurements in the UV range (tryptophan, tyrosine, phenylalanine fluorescence). The UV fluorescence range is particularly advantageous because it allows native biomolecules to be measured using their intrinsic fluorescence without having to modify them, for example, with a dye. Without the inventive use of silicon, an air gap was required in the prior art to avoid autofluorescence effects.This leads to the precise area that one wants to measure optically using fluorescence not being properly tempered. The non-fluorescent silicon allows the measurement area in the capillary to be heated / cooled (tempered) directly.

[0052] Furthermore, silicon can be produced and purchased in a highly pure form, so any autofluorescence from impurities and the resulting influence on the measurement results are extremely minimal. Silicon is also a chemically inert material, so even potential contact with a measuring liquid does not trigger any reactions that could negatively impact the optical measurement. A silicon contact surface for a temperature control element can be manufactured very smoothly, allowing the contact surface with the capillaries to be designed as a mirror surface, allowing the excitation light and / or fluorescent light from the sample to be reflected by the mirror surface, which can further amplify the measurement signal. The mirror surface is also broadband, which is an additional advantage. Furthermore, silicon has very good thermal conductivity and is extremely smooth.Electronic circuits / structures can also be integrated into the silicon, for example, through doping and / or etching. These structures can be used, for example, to measure a temperature or temperatures.

[0053] Another example of a suitable contact material is metal, preferably anodized metal, preferably anodized aluminum. For example, anodized aluminum, for example, in black, does not exhibit autofluorescence in the UV range. Silicon, for example, has the advantage of high purity over anodized aluminum, since the quality of the anodized coating can often fluctuate.

[0054] The temperature control element itself is preferably controlled by a temperature control device. In other words, the temperature control element preferably serves only for the targeted transfer of temperature or heat to the capillary(s). The present invention is not limited to specific temperature control devices. Due to their compact design and suitable temperature range, Peltier elements, for example, are suitable. However, electrical heating elements or liquid-temperature-controlled heating coils can also be used as temperature control devices.

[0055] The capillaries to be temperature-controlled are to be arranged such that at least some of the capillaries are in contact with the temperature-control element. Preferably, only a central region, preferably a middle region of each capillary should be in contact with the temperature-control element, i.e., it is preferred that at least one end, more preferably both ends, of the capillary do not come into contact with the temperature-control element during temperature control. In the present application, the central region or the middle of the capillaries refers to the length of the capillary, i.e., midway between the two ends. In other words, it is preferred that one end, preferably both ends, is / are not temperature-controlled.

[0056] According to the invention, the capillaries are to be held in such a way that each capillary is only tempered within a narrow tempering range. According to a preferred embodiment, the capillaries are arranged in such a way that both ends protrude beyond the tempering element, preferably symmetrically, whereby the ends of the capillaries are not tempered by the tempering element. According to a further preferred embodiment, the individual capillaries are arranged by the amount dx longer than the tempering range.

[0057] Thus, the invention ensures that the capillaries are only tempered over a specific portion of their length. This, combined with the low thermal conductivity of glass capillaries, means that the ends of the capillaries practically always remain at room temperature, provided there is sufficient distance from the tempering area or the tempering element. This means that even if the center or central region of the capillary is tempered to 90°C by the tempering element, no greater evaporation is observed at the ends of a correspondingly long capillary than at room temperature. This means that sealing is not necessary if evaporation at room temperature is acceptable.

[0058] For 50mm long capillaries, for example, the temperature control area should preferably be less than / shorter than 32mm, and more preferably less than 25mm, meaning that no more than 25mm of the capillary length should be temperature controlled in the center. In other words, 12.5mm of non-temperature-controlled capillary length should preferably protrude on both sides of the temperature control area.

[0059] The theoretical lower limit of the temperature control range is 1 mm, although for practical reasons, the length is preferably no less than 5 mm. The examples of the present invention are discussed with a temperature control range width of 25 mm, although this width is preferred. However, it has been shown that a 20 mm wide temperature control range also works well and is manageable. A temperature control range of 30 mm is also manageable.

[0060] The examples of the present invention are discussed with a capillary length of 50 mm, although this length is preferred. However, it has been shown that capillaries of 20 mm, 25 mm, 30 mm, 35 mm, and 45 mm also work well and are manageable. Capillary lengths of 55, 60, 65, 70, 75, and 80 mm are also manageable.

[0061] Since one works with low sample / substance concentrations, the interfering autofluorescence of the material of the state-of-the-art temperature control elements is often much greater than the fluorescence of the sample itself, i.e. measurement is impossible. Measuring directly on pure, untreated aluminum or with aluminum as a base is almost impossible. In the state of the art, one therefore always had to leave a recess / measuring gap / air gap under the area being measured. However, this recess meant that the capillaries in this recessed measuring area assumed a different temperature than in the area in which they rest and via which they are temperature-controlled. This effect from the state of the art is illustrated by the following two examples. A) The room temperature / device temperature / ambient temperature is assumed to be 25°C. The temperature control device is set to 20°C. The area of ​​the capillary that rests directly on the temperature control device is approximately 20°C. The area of ​​the capillary measured above the recess was partially 22°C with additional inhomogeneous temperature distributions. B) The ambient temperature is again assumed to be 25°C. This time the temperature control device is set to 90°C. The area of ​​the capillary that rests directly on the temperature control device is approximately 90°C. The area of ​​the capillary measured above the recess is approximately 82°C+ and has an inhomogeneous temperature (depending on the width of the air gap).

[0062] Within the scope of the present invention, several evaporation tests were conducted using capillaries with various inner and outer diameters. A 25 mm wide temperature control element was used as the test bench. 50 mm long capillaries were used.

[0063] Two solutions were used for the measurements: MST buffer with Tween 20 (added with blue dye for better measurability) and MST buffer without Tween 20 (added with green dye for better measurability).

[0064] MST buffer (kinase buffer) without Tween 50mM Tris-HCl 150mM NaCl 10mM MgCl2 pH 7.8

[0065] With Tween: +0.25% Tween20

[0066] The test capillaries were heated as follows: the temperature was increased from 20 °C to 90 °C at a heating rate of 1 °C / min and then held at 90 °C for 30 minutes. This is an example of a melting curve measurement / measurement to investigate the thermal stability of a capillary. Result Evaporation in [%] Evaporation in [%] Round capillaries, length 50mm MST buffer Tempering 25mm wide Control group at room temperature ID 0.5mm, OD 0.65mm Borosilicate 3.3 m. Tween 9,78 10,67 o.Tween 7,57 8,00 ID 0.5mm, OD 1.00mm Borosilicate 3.3 m. Tween 9,90 11,33 o.Tween 8,12 7,67 ID 0.2mm, OD 1.00mm Borosilicate 3.3 m. Tween 9,04 8,33 o.Tween 8,55 8,00 ID 0.8mm, OD 1.00mm Borosilicate 3.3 m. Tween 10,00 10,00 o.Tween 6,91 8,00 ID 0.100mm, OD 0.360mm Synthetic Quartz Glass m. Tween 7,34 6,86 Rectangular capillaries, Length 50mm

[0067] ID 0.05mm x 0.5mm m. Tween 12,79 14,00 ID 0.02mm x 0.2mm m. Tween 13,12 11,70

[0068] The tests were conducted with capillaries of different inner diameters (ID) and outer diameters (OD). For round capillaries in the range of 0.1 mm to 0.8 mm ID, virtually no significant dependence on the inner diameter was observed.

[0069] With rectangular capillaries (very thin-walled, therefore no information on the outer diameter or OD is known), a slightly higher evaporation can be measured, but this is still within the limits, or rather, with the rectangular capillaries, practically no difference can be measured between the tempered capillary and the non-tempered capillary at room temperature (= control group).

[0070] To ensure contact between the capillaries and the temperature control element, it is preferable to press the capillaries against the temperature control element. This can be achieved, for example, with a lid. The same preferably applies to the lid, which holds the capillaries down and thus ensures good temperature control; it should also be no wider than 25 mm. If you want to make the temperature control surface of the temperature control element wider, longer capillaries are required to avoid or prevent excessive evaporation at the capillary ends. Longer capillaries, in turn, can be disadvantageous, as they result in greater sample consumption.

[0071] However, the temperature control surface should not be too narrow, as otherwise the capillaries in their central measuring area are no longer evenly tempered, or molecules with a different temperature can diffuse into the measuring area from the outside.

[0072] Therefore, upper and / or lower limits for advantageous widths of the temperature control element and / or lengths of advantageous capillaries, and in particular their mutual dependence, were determined experimentally.

[0073] Figure 1 shows a diagram in which the evaporation was investigated in a 50 mm long capillary with an inner diameter of 0.5 mm and an outer diameter of 0.65 mm. The diagram shows the percentage evaporation (Y-axis) as a function of the width of the temperature control element.

[0074] For these studies, a typical buffer solution without detergent (="MST") and the same buffer solution with detergent (="Tween") were tested. The tests were conducted with and without detergent, as detergent can influence evaporation behavior. The control group was not subjected to temperature control. The temperature control of the other group corresponded to a typical melting curve: i.e., an increase in temperature from 20°C to 90°C within 70 minutes, followed by a 30-minute residence time at 90°C.

[0075] For example, it can be seen that with a 40 mm wide temperature control element (temperature control body), evaporation of between 25 and 30% occurs. At a width of approximately 36 mm, evaporation is already in the range of 10 to 15%. If the width is 34 mm or less, evaporation is below 10% and comparable to evaporation without a temperature control element. This means that from a temperature control area width of approximately 30 mm, the samples in the control group without temperature control and the temperature-controlled samples are identical (evaporation is minimal). In particular, evaporation of <10% is typically acceptable; i.e., if evaporation is <10%, sealing can preferably be omitted.

[0076] Based on this study, a preferred width of the temperature control surface for 50 mm long capillaries is less than 30 mm, preferably less than 25 mm, which allows for even higher temperatures such as 100°C. A person skilled in the art will recognize that the maximum temperature depends on the liquid, particularly the boiling point of the liquid or solvent. In particular, the formation of air bubbles upon reaching the boiling point may interfere with optical measurements. Therefore, the upper limit for aqueous solutions is preferably 100°C.

[0077] Figure 2shows a diagram in which evaporation was investigated in a capillary only 32 mm long with an inner diameter of 0.5 mm and an outer diameter of 0.65 mm. A typical buffer solution without detergent (="MST") and the same buffer solution with detergent (="Tween") were investigated. The control group was not temperature-controlled. The temperature control of the other group corresponded to a typical melting curve: i.e., an increase in temperature from 20°C to 90°C within 70 minutes, followed by a residence time of 30 minutes at 90°C. It is very clear to see that for a short capillary with a length of 32 mm, it is practically impossible to control evaporation without sealing. Even with a temperature-controlled area width of 8 mm, evaporation of more than 10% occurs.

[0078] Figure 3shows an exploded view of a device for temperature control according to the invention. Heat sinks 1 for dissipating waste heat are arranged at the very bottom. For example, the heat sinks 1 are arranged on a movable unit. For good thermal conductivity, at least one thermally conductive film or thermally conductive paste is preferably present between the heat sink 1 and the Peltier element 2. A heating block 3 made of metal (for example aluminum or copper) is arranged above the Peltier element 2. In between, there can again preferably be a thermally conductive film or thermally conductive paste. A preferably thin and narrow silicon wafer 5 for temperature control of the capillaries by contact is arranged above the heating block 3. A special thermally conductive film 4 is preferably arranged between the heating block 3 and the silicon wafer 5. A plastic frame 6 (here Makrolon) for positioning the capillaries (not shown) is arranged around the silicon wafer 5.

[0079] Finally, a narrow, thin cover 7 with a measuring gap for optical measurements is placed on top. This cover 7 should preferably be no wider than the silicon wafer 5. The cover 7 preferably presses the capillaries onto the silicon wafer 5. It is also advantageous if the cover 7 has a thermally insulating effect.

[0080] The capillaries are to be positioned centrally on the plastic frame 6 (ie the capillaries protrude sufficiently on both sides so that evaporation is minimal). For example, Fig. 4A schematic top view of six capillaries a) - f) with different filling levels or different positions, which lie on the plastic frame / carrier 6 to be tempered by an underlying silicon wafer 5. All six capillaries in positions a) - f) have the same or essentially the same length. Position a) shows the capillary centered, i.e., centered with respect to the central axis "M" of the frame 6 or the silicon wafer 5. The capillary is almost completely filled, i.e., the capillary extends symmetrically to the right and left beyond the frame 6, sufficiently filled with liquid.

[0081] Position b) also shows a capillary arranged symmetrically around the central axis M. The filling level of this capillary is lower than in position a), but still sufficient that evaporation at the ends does not adversely affect even a longer measurement.

[0082] Position c) shows a symmetrically filled capillary, similar to positions a) and b), but its filling level is even lower than in position b), so that on both the left and right sides, only a small overhang "A" of the liquid column protrudes beyond the frame 6. This small overhang, however, leads to evaporation at these ends, which can negatively influence optical measurements in the area of ​​the silicon wafer 5. Accordingly, hooks are only shown at positions a) and b), i.e., this position and filling level function without problems, whereas positions c) - f) can lead to problems. Thus, the filling level in position d) is sufficient and comparable to position a), but the positioning with respect to the silicon wafer 5 is such that the overhang on the right side (B) is not large enough. In position e), the capillary is positioned correctly, i.e., symmetrical with respect to the silicon wafer 5.the central axis M, but the capillary is unevenly filled. Distance A on the left side from the end of the liquid column to the frame or to the tempered silicon wafer 5 is sufficiently large, whereas distance B on the right side is too small. Finally, position f) shows a symmetrically aligned capillary with a symmetrically aligned liquid column, but with an insufficient filling level.

[0083] An optical measurement according to the present invention is described below by way of example.

[0084] The samples to be measured are filled into capillaries. This can be done, for example, by capillary force, or the capillaries can be filled, for example, with a pipette, but is not limited to this. The capillaries are then placed on a carrier. The carrier with the filled capillaries is then placed on the temperature control element according to the invention. Preferably, the capillaries are filled, at least in a central region of the capillaries, over a length that is wider than the width of the temperature control element. The samples are to be measured using fluorescence measurement. For this purpose, the sample is first excited using an excitation LED in the UV range, for example, at 280 nm.

[0085] At the start of the measurement, an optic is moved into the measuring position. The samples are tempered using the temperature control element. The temperature is preferably increased to the final temperature via a preset ramp. During this time, the samples are continuously moved under the optic, and the fluorescence values ​​are read out (see Fig. 5 In this example, the fluorescence emission is measured at 330 and 350 nm. This yields fluorescence values ​​in two wavelength ranges versus temperature. Once the final temperature is reached, the measurement data are saved, the temperature control and LED are turned off, and the axes are returned to their rest positions.

[0086] After a measurement is completed, a database file containing the acquired measurement data is created. Using conversion software, the database is converted into a CSV file ("comma-separated values") and then imported into analysis software. This software is capable of automatically calculating the melting points using inflection point analysis. By forming the quotient of the two fluorescence channels, 330 nm and 350 nm, a sigmoidal curve is created ( Fig. 9 ).

[0087] In Fig. 6A total of 15 samples were analyzed. Each color indicates the corresponding fluorescence intensity at a specific temperature. The very large number of colors reflects the large number of measurement runs and thus the high temperature resolution, since each measurement run represents a temperature. The top peak of a measurement curve represents the fluorescence signal at the starting temperature, and the bottom curve (here light blue) represents the fluorescence signal at the end temperature of a measurement. The low autofluorescence of the silicon (baseline) is also clearly visible.

[0088] It is also possible to display the individual curves for the two channels 330 nm ( Fig. 7 ) and 350 nm ( Fig. 8 ) are displayed. By forming the quotient of the two channels 350nm / 330nm, one obtains a so-called "melting curve" / "denaturation curve" ( Fig. 9 ). The melting point of the protein under investigation lies at the inflection points of the respective measurement curve.

[0089] Finally, the Figures 10 a) to 10 i) Examples of possible cross-sectional shapes of capillaries. Fig. 10 a ) a round capillary with the wall 20 and the hollow space or cavity 21. The Figures 10 f) and 10 g ) also show round designs, but with different wall thicknesses and correspondingly different cavities with the same outer diameter. Fig. 10 b ) shows a semicircular design; Fig. 10 c ) a hexagonal embodiment; Fig. 10 d ) a square design; Fig. 10e ) an oval design; Fig. 10 h ) an example of an embodiment in which the outer shape differs from the inner shape, here with a square outer shape and an oval or round inner shape and Fig. 10 i ) a combination with several cavities within an outer mold.

[0090] Figure 11Ashows an example of a typical buffer screening from antibody research. The unfolding of a protein / biomolecule shifts the emission maximum of the fluorescence from the spectral range 330 nm + / - 5 nm to the spectral range 350 nm + / - 5 nm. This shift is made clear by measuring and recording the ratio of fluorescence at 350 nm divided by fluorescence at 330 nm. Shown here is the change in the tryptophan emission (F350 nm + / - 5 nm divided by F330 nm + / - 5 nm) of an antibody due to unfolding at elevated temperatures. Thermal unfolding occurs for the antibody shown at pH values <pH 7 bei deutlich niedrigeren Temperaturen auf, was auf eine Destabilisierung des Antikörpers unter sauren Bedingungen hindeutet.

[0091] Figure 11 Bshows an example of a change in the thermal stability of a protein due to binding of small molecules. Shown is the change in the tryptophan emission (F350 nm + / - 5 nm divided by F330 nm + / - 5 nm) of a protein due to unfolding at elevated temperatures after binding different amounts of a small molecule ligand. The more ligand added, the more ligand binds to the protein and the more thermally stable this protein becomes.

[0092] The device according to the invention and the method according to the invention preferably comprise one or more of the following features, particularly in nanoDSF applications. In particular, ultra-high-resolution protein stability measurements can be performed. Preferred features

[0093] Native DSF: no dye required Dual UV system: 330 nm and 350 nm fluorescence is detected 48 samples each Ultra-high resolution: measure 48 capillaries in 7 seconds and observe more unfolding transitions Wide concentration range: from 5 µg / ml to 150 mg / ml Temperature range: from 15 °C to 100 °C Thermal and chemical denaturation Maintenance-free instrument and / or Easy handling: simple sample preparation and software with intuitive user interface

[0094] The device, hereinafter referred to as Prometheus NT.48, can accommodate 48 capillaries. The capillaries are preferably filled with the sample by capillary action, so the capillaries are simply immersed in the sample and placed in the instrument. The instrument is preferably maintenance-free and contains no tubing, valves, or pumps. Since the capillaries are preferably single-use, no equilibration or cleaning is required.

[0095] Thermal unfolding experiments require no assay development or laborious sample preparation. Simply immerse the capillaries in the protein solutions to fill them and load them onto the capillary carrier. A high-speed discovery scan is performed to determine optimal excitation and detection settings. Then, simply set the temperature ramp and start the experiment.

[0096] Buffer and formulation screens can be easily performed by mixing the protein with the solutions of interest. Capillary filling devices are available to fill capillaries from microtiter plates in seconds.

[0097] Sample annotations can be conveniently entered while the experiment is running.

[0098] For chemical unfolding experiments, different concentrations of denaturant are mixed with the protein of interest and incubated for equilibration. The samples are filled into capillaries and then analyzed by the Prometheus NT.48.

[0099] For example, scanning a chemical denaturation series with 48 samples takes only 7 seconds.

[0100] nanoDSF is an advanced differential scanning fluorimetry technique for measuring protein stability with ultra-high resolution using intrinsic tryptophan fluorescence for applications in antibody engineering, membrane protein research, formulation and quality control.

[0101] The devices of the invention provide nanoDSF technology, which is the method of choice for easy, rapid and accurate analysis of protein folding and stability in applications in protein engineering, formulation development and quality control.

[0102] By monitoring changes in the fluorescence of the amino acid tryptophan, chemical and thermal stability can be assessed in a truly label-free manner. Furthermore, a preferred dual-UV technology allows on-the-fly-Fluorescence detection, which provides unsurpassed scanning speed and data point density and thus ultra-high resolution of deconvolution curves, allowing even the smallest deconvolution signals to be detected.

[0103] Furthermore, since no secondary reporter fluorophores are required, protein solutions can be analyzed independent of buffer compositions and over a maximum protein concentration range, preferably from 150 mg / ml to only 5 µg / ml, allowing the analysis of detergent-solubilized membrane proteins as well as highly concentrated antibody formulations.

[0104] Widely used methods for quantifying protein structural stability are thermal and chemical unfolding experiments. While thermal unfolding experiments use a constantly increasing temperature to monitor protein conformational changes over time, chemical unfolding experiments use concentration gradients of buffer additives, usually chaotropes such as urea, to unfold proteins to varying degrees.

[0105] Many proteins undergo thermal unfolding over a narrow temperature range. The midpoint of the folded-to-unfolded transition, referred to as the "melting temperature" or "Tm," serves as a measure of protein stability. Thermal unfolding experiments are particularly popular in protein engineering, formulation development, and screening procedures because they allow large numbers of samples to be rapidly evaluated in parallel.

[0106] Similar unfolding curves can be obtained from chemical denaturation experiments, which, in addition to thermal unfolding experiments, can provide information on thermodynamic parameters and equilibria in protein folding and unfolding.

[0107] The fluorescence of tryptophans in a protein depends strongly on their immediate environment. Typically, changes in protein structure affect both the intensity and emission wavelength of tryptophan fluorescence. The device of the invention is preferably equipped with fluorescence detectors that measure fluorescence intensity at two different wavelengths, 330 nm and 350 nm, making it sensitive to both the change in fluorescence intensity and the shift in the fluorescence maximum upon unfolding.

[0108] Protein denaturation curves are used to derive important stability parameters. The thermal stability of a given protein is typically described by the melting temperature (Tm) at which 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 330 and 350 nm, which describes the shift in tryptophan emission upon unfolding. Typically, the 350 / 330 nm ratio yields data with well-defined transitions during protein unfolding, whereas Tm cannot always be derived using single-wavelength detection. Thus, the device's dual-wavelength system ensures sensitive detection of unfolding processes.

[0109] The device of the invention (e.g., Prometheus NT.48) can be used in laboratories for formulation and quality control. The wide concentration range allows biopharmaceuticals to be tested at very high concentrations, which are typically used in formulation. The nanoDSF technology used by the Prometheus device is particularly suitable for applications in antibody engineering, as the ultrahigh resolution allows for the detection and analysis of multiple transitions and unfolding events. Furthermore, nanoDSF makes it possible to measure the stability of membrane proteins in detergents, as this method is truly label-free and does not require a fluorescent dye.

[0110] In addition, a preferred application example is discussed below.

[0111] A detailed analysis of protein stability is a prerequisite for both the fundamental understanding of protein folding mechanisms and the successful development of biologics in the pharmaceutical industry. The following demonstrates the performance of the new Prometheus NT.48 instrument, which detects intrinsic protein fluorescence changes during thermal or chemical unfolding of up to 48 samples in parallel. Introduction

[0112] Estimation of protein stability is an integral part of basic research, drug discovery, and drug development [1]. For example, shifts in the melting temperature (Tm) of a target protein upon binding to a small molecule ligand are routinely used in primary screens in the drug discovery process [2]. In addition, the thermal and chemical stability of biologics, e.g., antibodies, is often monitored to ensure optimal conditions for large-scale production and long-term storage [3, 4]. Furthermore, careful analysis of protein unfolding and refolding mechanisms can provide important insights into the thermodynamic origins of protein folding, helping to elucidate the molecular basis of degenerative diseases such as Alzheimer's, Parkinson's, or diabetes.

[0113] The basis of label-free fluorimetric analysis of protein folding lies in the properties of the fluorescent amino acid tryptophan. Because tryptophan is a hydrophobic amino acid, it is usually located in the hydrophobic core of proteins, where it is shielded from the surrounding aqueous solvent. However, after unfolding, tryptophan is exposed, which changes its photophysical 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 thermodynamic properties can be determined [7].

[0114] The following demonstrates the performance of the Prometheus NT.48 in monitoring the thermal unfolding of proteins in a formulation screening project. The Prometheus NT.48 instrument can measure up to 48 samples in parallel and uses high-precision capillaries filled with only 10 µl of sample. Using a detector specifically designed to monitor changes in the emission spectrum of tryptophan with maximum sensitivity and speed, the highest data point density and precision are achieved. Proteins of the α-amylase family have proven useful for analyzing protein folding [8]. Most amylases share very similar tertiary structures with three (β / α)-barrel domains and at least one conserved Ca 2+ binding site ( Fig. 12). Fig. 12 shows the structure of α-amylase from porcine pancreas (PPA, green) and α-amylase from Aspergillus oryzae (TAKA, blue). The red sphere represents a Ca 2+ ion.

[0115] At the same time, they exhibit an extremely wide melting temperature range (from 40 °C to 110 °C), making them perfect candidates for basic research on the determinants of protein thermal stability [9]. In addition to their value for basic medical research, amylases are used commercially in the large-scale production of ethanol from sugars.

[0116] In this example, the thermal unfolding of mammalian α-amylase (porcine pancreatic α-amylase, PPA) and fungal α-amylase (Aspergillus oryzae α-amylase, TAKA) was investigated. The stabilizing effects of calcium ions on protein conformation were reviewed, and finally, formulation screens were conducted with different additives that improve thermal stability to varying degrees.

[0117] The Prometheus NT.48 instrument monitors the shift in the intrinsic tryptophan fluorescence of proteins upon unfolding by detecting fluorescence at an emission wavelength of 330 and 350 nm. To determine the protein melting point (Tm at which half of the protein is folded and the other half is unfolded), the fluorescence change in either channel can be used, or alternatively, the ratio of the fluorescence intensities (F330 / F350 ratio) can be plotted.

[0118] For most proteins, the latter approach is preferred, as the fluorescence quotient monitors both the change in tryptophan fluorescence intensity and a shift of the fluorescence emission maximum to higher wavelengths ("redshift") or lower wavelengths ("blueshift"). 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, allowing for precise determination of the onset of protein unfolding as well as accurate fitting of the folded-to-unfolded transition by mathematical models.

[0119] Fig. 13 shows the changes in tryptophan fluorescence of PPA and TAKA upon thermal unfolding. Especially for TAKA, the raw fluorescence data from both wavelengths show a clear transition from folded to unfolded ( Fig. 13A, left), which could be used directly for Tm analysis. In contrast, this transition is not evident from the raw data for PPA ( Fig. 13B , left). Furthermore, while TAKA showed a typical unfolding profile with a shift of tryptophan fluorescence to higher wavelengths (redshift), PPA showed a less widespread shift of tryptophan fluorescence to lower wavelengths (blueshift).

[0120] Plotting the fluorescence quotient F330 / F350 of both proteins as a function of temperature yielded clear melting curves that could be used to analyze the respective melting temperatures of the amylase isoforms. The melting temperature can be determined using different methods: For median analysis, a lower and an upper baseline are first defined, and a median line is inserted. The intersection point between the experimental curve and the median line is defined as Tm ( Fig. 13A and B, middle). An alternative approach is to determine the maximum of the first derivative of the absorbance signal. This method avoids the somewhat subjective determination of the baseline values ​​( Fig. 13A and B , right), and it also allows the determination of multiple melting points, e.g., for antibody unfolding or for more complex multi-domain proteins.

[0121] Most importantly, the standard deviation of the results in the table is Fig. 14B from the first derivative analysis within the range of the fitting error, demonstrating maximum reproducibility of the results. Thus, the Prometheus NT.48 instrument can be used to accurately determine Tm values ​​with minimal sample and time expenditure.

[0122] The results show that both the reproducibility and accuracy of the thermal unfolding experiments with PPA and TAKA were very high ( Fig. 14A and BThe Tm values ​​obtained were very similar to those cited in the literature [9]. Ca 2+< -dependence of thermal amylase stability

[0123] A second set of experiments aimed to recapitulate the stabilizing effects of Ca 2+< ions on both α-amylase isoforms. It has previously been demonstrated that Ca 2+< ions are required for increased Tm values ​​of different amylase isoforms, ranging from virtually no effect for amylase from Alteromonas to a Tm increase of 50 °C for amylase from Bacillus licheniformis [9].

[0124] To investigate the effects of Ca 2+< ions on PPA and TAKA stability, both proteins were incubated in buffer containing 5 mM EDTA to remove bound Ca 2+< for 30 minutes prior to thermal unfolding experiments. As expected, the removal of Ca 2+< ions by EDTA resulted in a significant increase in Tm for both amylase isoforms ( Fig. 15). For PPA, ΔTm was more pronounced (-16.6 °C) than for TAKA (-12 °C), which correlates well with previously published results (PPA -17 °C, TAKA -14 °C) [10, 11]. Effects of buffer additives on thermal amylase stability

[0125] Screening for additives and buffer conditions that improve protein stability, also called formulation screening, is crucial for maximizing the shelf life of antibodies and other biologics. Using the Prometheus NT.48, the effects of various buffer additives previously shown to increase protein stability were tested, namely, glycerol, sucrose, trehalose, and sorbitol, with concentrations ranging from 10% to 40% (weight per volume) of PPA and TAKA.

[0126] 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 performed within approximately 70 minutes with a total sample consumption of 400 µl (10 µl for each buffer condition plus four control experiments for each isoform without additive) and a total protein amount of just 80 µg.

[0127] The plots of the tryptophan fluorescence quotients clearly show that each additive increased the Tm of PPA and TAKA in a concentration-dependent manner. For PPA, trehalose was already most effective at concentrations of 30% (+12 °C), while glycerol was the least effective, increasing the Tm by only 7.5 °C at a concentration of 40% ( Fig. 16A and B). For TAKA, the addition of 40% sucrose was most effective in increasing Tm (+12 °C), while glycerol and trehalose showed the smallest effect (+7.5 °C and +8 °C, respectively) ( Fig. 17A and B These results are in good agreement with a previous study investigating the effect of additives on the thermal unfolding of Bacillus α-amylase

[12] . Conclusions

[0128] In this case study, the performance of the Prometheus NT.48 instrument was demonstrated in determining thermal unfolding properties of two α-amylase isoforms in screening applications.

[0129] By detecting changes in tryptophan fluorescence at two defined wavelengths, Tm values ​​of the amylase proteins could be determined under different conditions. All results show good agreement with published values. Most importantly, compared to methods using standard fluorimeters, both sample consumption and the time required to conduct the experiments are dramatically reduced using the Prometheus NT.48.

[0130] The instrument's capillary format allows for flexible experimental design, measuring any number of samples between 1 and 48 simultaneously. Importantly, the use of Prometheus capillaries offers even higher precision in UV fluorescence detection than high-performance quartz cuvettes, with the advantages of low sample consumption, high throughput, and great versatility. Furthermore, the capillary-based approach prevents cross-contamination and eliminates the need for laborious and time-consuming cleaning steps. Furthermore, high scanning speeds and thus high data density enable robust analysis of melting curves using mathematical fitting algorithms and also enable precise determination of incipient unfolding.

[0131] In addition, the direct detection of tryptophan fluorescence for monitoring protein unfolding has several advantages compared to other methods routinely used to monitor thermal unfolding, such as differential scanning fluorimetry (DSF) or Thermofluor assays. These assays utilize external fluorophores that bind to hydrophobic sites on the protein, usually buried within the protein core. Upon unfolding, these sites are exposed, and the fluorophore attaches, resulting in increased fluorescence. However, these assays are not suitable for detailed analysis of folding thermodynamics because they disrupt folding-unfolding equilibria by directly interacting with the proteins. Furthermore, the external fluorophores are incompatible with a number of buffers (including, for example, detergents) or protein types, such as membrane proteins.Finally, although DSF is routinely used in primary screening in the drug discovery process, external fluorophores can interact with compounds or block binding sites, producing false negative and false positive results.

[0132] In addition to its capabilities for parallel monitoring of the thermal unfolding of a large number of samples, the Prometheus NT.48 instrument can also be used to analyze chemical denaturation of proteins in a matter of seconds. In summary, the results demonstrate that the Prometheus NT.48 instrument is exceptionally well-suited for rapid, precise, and cost-effective characterization of protein stability in both academic and industrial settings. Its flexibility and speed make it a valuable tool for a wide range of different experimental procedures, ranging from in-depth characterization of protein folding to high-throughput screening projects. Materials and processes Sample preparation

[0133] Porcine α-amylase (porcine pancreatic α-amylase, PPA, Roche) and Aspergillus oryzae α-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. Final concentrations in thermal unfolding experiments were 10 µM. To remove residual traces of ammonium sulfate or other contaminants, buffer exchange was performed using buffer exchange spin columns (NanoTemper Technologies). To determine the Ca 2+< dependence of α-amylase stability, a second buffer exchange was performed to buffer without CaCl 2 but containing 5 mM EDTA.

[0134] For the formulation screen, the proteins were transferred into 20 mM Na citrate buffer, pH 5.9, with the respective concentrations of sucrose, sorbitol, trehalose or glycerol. Thermal unfolding experiments

[0135] For thermal unfolding experiments, proteins were diluted to a final concentration of 10 µM. For each condition, 10 µl of sample was prepared per capillary. Samples were loaded into UV capillaries (NanoTemper Technologies), and experiments were performed using a Prometheus NT.48. The temperature gradient was set to increase by 1 °C / min over 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. Data analysis

[0136] Melting temperatures were determined by detecting the maximum of the first derivative of the fluorescence ratios (F330 / F350). For this purpose, an 8th-order polynomial fit was calculated for the transition region. Next, the first derivative of the fit was calculated, and the peak position (at Tm) was determined.

[0137] Fig. 13: Analysis of TAKA and PPA melting curves. (A) Plot of the decay of tryptophan fluorescence upon thermal unfolding of TAKA (left). The transition from the folded to the unfolded state is already visible in the raw fluorescence data at emission wavelengths of 330 and 350 nm. The inset demonstrates the high data point density of the Prometheus NT.48. Two methods can be used to determine Tm. In median analysis (center), a median line is defined between an upper and lower baseline. Its cross-section with the experimental data represents Tm. Alternatively, the experimental data can be fitted 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, in contrast to TAKA, the transition from folded to unfolded protein is not visible in the raw fluorescence data (left), whereas Tm can be easily determined from the fluorescence quotient plots (right).

[0138] Fig. 14 : Accuracy and reproducibility of the Prometheus NT.48 unfolding data. (A) The plots represent an overlay of 10 independently recorded melting curves of PPA and TAKA, respectively. (B) The Tm determination for both proteins shows a small standard deviation between experiments (≤0.2 °C) and a good correlation with published results [9].

[0139] Fig. 15 : Ca 2+< effects on amylase stability. Removal of Ca 2+< ions results in a marked destabilization of both amylase isoforms, as indicated by the shift of Tm to lower values.

[0140] Fig. 16: Formulation screening of PPA. To determine optimal conditions for increased thermal stability of PPA, its thermal unfolding was monitored under 16 different additive conditions. A clear shift in Tm to higher values ​​is observed in the fluorescence quotient plots for each additive. Quantification of Tm under different conditions shows that the addition of 30% trehalose is most effective, while glycerol has the weakest effect.

[0141] Fig. 17: Formulation screening of TAKA. To determine optimal conditions for increased thermal stability of TAKA, its thermal unfolding was monitored under 16 different additive conditions. A clear shift in Tm to higher values ​​is observed in the fluorescence quotient plots for each additive. Quantification of Tm under different conditions shows that the addition of 40% sucrose is most effective, while glycerol and trehalose have the weakest effects. literature literature

[0142] A preferred embodiment of a device or system according to the invention, hereinafter referred to as Prometheus NT.48 with nanoDSF technology, is described. With the Prometheus series, NanoTemper Technologies offers nanoDSF technology, the method of choice for the easy, rapid, and accurate analysis of protein folding and stability with applications in protein engineering, formulation development, and quality control.

[0143] Preferred benefits of nanoDSF: Benefit from native DSF - no dye, buffer and detergent independence Observe more transitions - due to high resolution Get results faster - work with smaller sample volumes Measure in a wide concentration range from 5 µg / ml to 150 mg / ml

[0144] nanoDSF is an advanced differential scanning fluorimetry technology based on the detection of minute changes in the intrinsic fluorescence of the amino acid tryptophan.

[0145] The fluorescence of tryptophans in a protein depends strongly on their immediate environment. By tracking changes in the fluorescence of the amino acid tryptophan, chemical and thermal stability can be assessed in a truly label-free manner.

[0146] Furthermore, since no secondary reporter fluorophores are required, protein solutions can be analyzed independently of buffer compositions and over a maximum protein concentration range from 150 mg / ml down to as low as 5 µg / ml, allowing the analysis of detergent-solubilized membrane proteins as well as highly concentrated antibody formulations.

[0147] NanoTemper’s dual UV technology enables on-the-fly-Fluorescence detection, which provides unsurpassed scanning speed and data point density and thus ultra-high resolution of deconvolution curves, allowing even the smallest deconvolution signals to be detected.

[0148] The following table summarizes preferred technical features: Prometheus NT.48 Samples per measurement run 48 samples Fluorescence detection 330 / 350 nm Marking required No marking, no dye Concentration of the fluorescent molecule 5 µg / ml to > 150 mg / ml Molecular mass range (Da) 101 - 107 Volume per measurement 10 µl Temperature control 4 °C - 98 °C Heating speed 0.1 - 10 °C / min Biophysical parameters Denaturing centers Tm and Cm Tryptophan fluorescence required Yes Measurement in detergents Yes Time for experiment and analysis Minutes - Hours Immobilization required No Maintenance required No

[0149] Protein denaturation curves are used to derive important stability parameters. The thermal stability of a given protein is typically described by the melting temperature Tm at which half of the protein population is unfolded.

[0150] 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.

[0151] Typically, the 350 / 330 nm ratio yields data with well-defined transitions during protein unfolding, whereas single-wavelength detection does not always allow for the determination of Tm. Thus, the dual-wavelength system of the Prometheus NT.48 provides sensitive detection of unfolding processes.

[0152] The invention also encompasses the precise or exact terms, features, numerical values, or ranges, etc., when mentioned above or below in connection with terms such as "about, approximately, about, substantially, generally, at least, at least," etc. (thus, "about 3" is also intended to include "3," or "substantially radial" is also intended to include "radial"). The term "or" also means "and / or."

Claims

1. A method for tempering at least one capillary (10), which is at least partially filled with a liquid column and positioned on a carrier (6), wherein the carrier (6) having a length (L), width (W) and height (H) receives the capillary (10) along the width of the carrier (6), and the liquid column of the capillary (10) comprises two ends and is aligned to a tempering element (5) in such a manner that at least one end of the liquid column projects beyond the tempering element (5) and the capillary (10) is in contact with the tempering element (5) so that at least part of the capillary and the liquid column contained therein is tempered, characterized in that the ends (11, 12) of the capillary (10) are unsealed during tempering and project beyond the tempering element (5) to such an extent that no higher evaporation than at room temperature can be observed at the ends of the capillary.

2. The method according to claim 1, wherein the at least one capillary (10) comprises a length between 40-75 mm, preferably between 45-55 mm, further preferred approximately 50 mm.

3. The method according to any one of claims 1 or 2, wherein silicon is used as tempering element (5).

4. The method according to any one of the preceding claims, wherein the width of the tempering element (5) is between 5-34 mm, preferably between 20-30 mm, further preferred 20-25 mm, further preferred approximately 25 mm.

5. The method according to claim 4, wherein the tempering element (5) is formed integrally along its width or comprises several tempering regions separated from each other, which may adjacently contact each other or cover the width of the tempering element (5) with at least one spacing.

6. The method according to any one of the preceding claims, wherein the at least one capillary (10) is pressed onto the tempering element (5) by a lid, in order to guarantee a contact between capillary (10) and tempering element (5).

7. The method according to any one of the preceding claims, wherein the capillary (10) is filled with an aqueous sample solution or a solvent, in particular with buffer solvents for biochemical / biological measurements.

8. The method according to any one of the preceding claims, wherein the length of the liquid column in the capillary (10) is at least 1.1-fold the width of the tempering element (5), preferably at least 1.2-fold, preferably at least 1.3-fold.

9. The method according to any one of the preceding claims, wherein the capillaries (10) i) have an inner diameter of 0.02 to 0.9 mm and / or ii) an outer diameter of 0.1 to 2 mm.

10. The method according to any one of the preceding claims, wherein the capillaries (10) are made of glass, preferably borosilicate 3.3, quartz, synthethic fused silica.

11. The method according to any one of the preceding claims, wherein the cross-section of a capillary may be round, oval, triangular, quadrangular, pentagonal, hexagonal, octagonal, semi-circle or trapezoidal, or comprise any other irregular shape.

12. The method for optically examining samples filled in capillaries comprising the steps of: filling capillaries (10) with samples; arranging the capillaries (10) on a carrier (6); tempering the capillaries (10) according to a method of the preceding claims; exciting the samples with light, preferably with UV light; and measuring the light which is emitted by the samples in the capillaries.

13. The method according to claims 1-12, for differential scanning fluorimetry (DSF) to measure protein stability using intrinsic tryptophan fluorescence.