Characterization of particles in solution

Abstract

The present invention relates to a method for measuring the properties of multiple particles in a solution and an apparatus for performing the method, wherein the method comprises the following steps: providing a container containing a sample of the multiple particles in the solution, wherein the sample has a volume preferably between 0.1 μL and 15 μL; providing a monochromatic light source and a photodetector; transmitting light from the monochromatic light source into the container containing the sample; detecting the light emitted from the container using the photodetector; and determining the properties of the multiple particles in the solution contained in the sample based on dynamic light scattering (DLS) measurements.

Description

Technical Field

[0001] Generally, the present invention relates to a method for characterizing multiple particles in solution based on dynamic light scattering. Specifically, the present invention relates to a method that allows characterization of the three-dimensional structures of multiple proteins and variations in the three-dimensional structures of multiple proteins in solution (preferably including their stability), such as temperature dependence, especially in the case of multiple small sample volumes using dynamic light scattering (DLS), preferably (e.g., in conjunction with differential scanning fluorimetry (DSF)) with measurement times of less than 1 second; and an apparatus for performing the same operation. The present invention preferably provides a method and system that provide enhanced and accurate measurements of aggregated and intrinsic properties (e.g., folding of protein particles) within a short time and a single system. Background Technology

[0002] Determining product characteristics quickly and accurately is crucial for many applications. For example, in the pharmaceutical or biotechnology fields, as well as the food industry and materials science, it is important to ensure that products maintain high purity and / or stable quality during production and potential storage. Quality control is typically performed based on product samples and / or samples containing the product (e.g., multiple particles in a solution, such as proteins). These controls are often time-consuming and can cause considerable delays until results are obtained. Therefore, to reduce costs associated with impurities and / or undesirable product characteristics, it is desirable to have a solution readily available that provides high-precision, preferably real-time, evaluation of product characteristics (e.g., the characteristics of particles in solution).

[0003] One example of such particles is a protein. Proteins are involved in almost all cellular processes and are therefore essential for the function of cellular organisms, including humans. Depending on their function, proteins can be classified as structural proteins that determine the structure of cells and tissues; proteins with catalytic functions, i.e., enzymes; ion channels that regulate cellular ion concentrations, thereby modulating osmotic homeostasis and signal transduction; transport proteins; regulatory proteins, including hormones; and proteins involved in immune responses, such as antibodies. The number and / or activity of proteins can be affected, for example, under conditions of physiological stress, including high temperatures and / or in the presence of genetic diseases.

[0004] Due to their ubiquity and their impact on cellular processes, changes in protein quantity and / or activity can have a significant impact on the survival and health of organisms.

[0005] Proteins have been extensively studied due to their relevance, given their structure, function, distribution, levels, and potential uses in a variety of applications, including medicine. Proteins are large molecular compounds composed of amino acids linked by peptide bonds. The specific amino acid sequence, also known as the primary structure of a protein, is determined by genes. Due to hydrogen bonds between amino acid residues in a protein, the amino acid sequence can fold to form a conformation, also known as secondary structure, where the spatial arrangement of the amino acid sequence is called tertiary structure. The tertiary structure and three-dimensional folding of proteins is a particularly interesting problem because its study not only provides information about the molecular structure of proteins, but it can also provide detailed information about the spatial arrangement of active amino acid residues, such as in the catalytic active site of an enzyme or the antigen-binding site of an antibody, thus providing detailed information about their activity. Furthermore, some proteins possess quaternary structure, which refers to the aggregation and / or binding of proteins to form stable (oligomeric) proteins; individual proteins are called subunits of (oligomeric) proteins. Since deviations from the native conformations of (multiple) proteins are often associated with reduced efficacy, the three-dimensional structure of proteins, in particular, can be considered key to their biological effects.

[0006] For example, optimizing protein availability (especially in bioactive conformations) represents a promising approach in therapeutic settings. If a subject's protein level is lower than that observed in other subjects (such as controls), obtaining a predetermined amount of the protein is beneficial to that subject. Particularly in therapeutic applications, it is necessary to obtain high purity of the protein of interest in a given conformation to avoid undesirable immune responses upon administration. For the same reason, active substances (such as the active ingredients of protein-based biopharmaceuticals) must remain stable during storage; that is, the protein of interest contained in the biopharmaceutical should remain intact and in the intended conformation, and therefore must not degrade, alter its three-dimensional structure, or form aggregates during storage.

[0007] Because active substances (such as antibodies) are developed in a way that makes them active only in their natural form, denatured active substances are generally ineffective and must be avoided. Denaturation refers to structural changes in biomolecules (such as proteins), and in most cases involves the loss of these molecules' biological functions. Denaturation can be caused by physical or chemical factors.

[0008] Denaturation of particles (such as antibodies) should be avoided as it reduces efficacy. Therefore, active ingredient formulations must be developed to prevent drug denaturation, i.e., to stabilize the drug through heat, chemistry, and / or time. Furthermore, aggregation of active substances can also lead to ineffectiveness. In addition, aggregated and / or denatured particles (such as administered aggregated antibodies) can trigger a response from the body's immune system and must therefore be avoided in drugs, or their proportion in the drug should be minimized. Therefore, particle aggregation, such as that in antibody therapy, should be avoided because it triggers an immune system response and also leads to reduced efficacy.

[0009] However, it is often unclear why particles aggregate and / or denature: do particles aggregate because of their denature, i.e., not existing in their native form, or do they aggregate in their native form and then denature?

[0010] Various methods have been developed to characterize the three-dimensional structure of proteins (oligomers) under different environmental conditions, including natural and chemical and / or thermal denaturation conditions. Studying the stability of protein three-dimensional folding, such as its dependence on temperature, pH, and / or the presence of other chemical components, is highly advantageous for optimizing storage conditions (e.g., in protein-based biopharmaceuticals). To determine protein stability, differential scanning fluorescence (DSF) methods, including nanoscale differential scanning fluorescence, can be applied. For example, qualitative methods, including backscattering, can be used to analyze the presence of larger particles (such as unwanted protein aggregates), while dynamic light scattering (DLS)-based analyses can be used to investigate the size distribution of proteins in solution.

[0011] For comprehensive characterization of particles, analyzing aggregation or denaturation alone is often insufficient. However, existing systems cannot simultaneously measure protein aggregation and (un)folding with high sensitivity, although the interaction of these two parameters is of interest in most cases. For example, Prometheus… TM The device (Microcalorimetry) can measure protein aggregation and (un)folding in solution, but its sensitivity is limited in the case of aggregation, making it impossible to distinguish the presence of some small aggregates from the presence of a few large aggregates. For example, the latter can be distinguished by comparing the device with another device (such as...). This is achieved by combining a plate reader (Wyatt Technologies). However, when using the latter, additional technical limitations, including a lower heating rate, can lead to the loss of information about whether unfolding or aggregation occurs first when particles are exposed to heat.

[0012] Therefore, alternative solutions are still needed to comprehensively and efficiently characterize particles such as proteins in solution at high throughputs, ensuring reproducible quantitative results even at low particle concentrations. Summary of the Invention

[0013] Among other things, this invention is based on the discovery that by performing dynamic light scattering (DLS) measurements, the three-dimensional structure of particles in solution can be analyzed with high sensitivity and reproducibility at high throughput, preferably combined with nano-differential scanning fluorescence (nano-DSF) measurements based on the same sample containing multiple particles in solution, even with small sample volumes over short periods of time. Based on these measurements, the aggregation and free energy of protein folding can be determined rapidly and accurately, preferably within a single device. Furthermore, using the methods and systems of this invention, information about the presence of particle aggregates and / or expected particle size distribution deviations in solution can be obtained from approximately 48 samples, for example, in two seconds or even less, thereby significantly accelerating quality control. For comparison, existing methods using dynamic light scattering require at least 12 seconds for a single sample. Therefore, even real-time measurements, such as measurements of liquid flow, can be performed using the methods and devices of this invention to characterize particles in solution.

[0014] Specifically, using the system and method according to the invention, "intraparticle" and "interparticle" effects can be measured, for example, the denaturation and aggregation of particles in solution can be measured, and even approximately simultaneously (i.e. substantially simultaneously) or simultaneously.

[0015] For example, "intraparticle" effects are preferably related to folding / unfolding and are generally related to the 3D structure of the particles (primary, secondary, tertiary, and quaternary structures). Particle denaturation is also an "intraparticle" effect and can be measured in the procedure and system according to the invention by measuring the inherent particle fluorescence (e.g., tryptophan fluorescence, tyrosine fluorescence). Meanwhile, particle aggregation, a type of "interparticle" effect that alters particle size, can be measured by scattering unabsorbed light.

[0016] Simultaneous measurement of thermally induced denaturation and aggregation of particles / molecules is beneficial, for example, to determine whether particle denaturation is caused by aggregation or vice versa. In other words, it is beneficial to understand whether particles aggregate in their native configuration or in an unfolded or partially unfolded configuration. This knowledge helps to better understand aggregation mechanisms, thereby improving the development of stable formulations of the particles under study (e.g., buffer solutions containing stabilizing components). Since the above processes may occur in different sequences at different timescales (e.g., from about 1 second to several hours), it is particularly beneficial to simultaneously measure nanoscale differential scanning fluorescence and dynamic light scattering over time to monitor the kinetics of these interdependent processes.

[0017] This invention is defined by the features of the independent claims. Preferred embodiments of the invention are defined by the dependent claims.

[0018] This invention relates to a method for measuring the properties of multiple particles in a solution, the method comprising the step of providing a container comprising a sample of multiple particles in the solution. The sample has a small volume, for example, between 0.1 μL and 15 μL. The method preferably comprises the steps of: providing a light source that emits substantially monochromatic light, providing a photodetector, and emitting light from the monochromatic light source onto the container containing the sample. In other words, the multiple particles in the container are illuminated by light from the light source, and the scattered light from the multiple particles is detected by the photodetector. That is, the light from the container, i.e., the light emitted by the container, is detected by the photodetector, wherein the properties of the multiple particles in the solution contained in the sample can be determined based on measurements of dynamic light scattering (DLS). Hereinafter, the light emitted / emitted from the container is also referred to as “emitted light” and, after its interaction with the sample, is used interchangeably with the terms “scattered light” or “backscattered light” from the sample.

[0019] Preferably, the sample has a volume between 0.1 μL and 15 μL, more preferably between 1 μL and 15 μL, and even more preferably between 8 μL and 12 μL. The sample is preferably provided in a small-volume container, such as in the pores of a multi-well plate or in a capillary, which provides the further advantage of easy filling of the capillary by capillary forces. For example, a single capillary or even multiple capillary tubes mounted on an array (support) can be immersed in the solution along with the sample, so that each capillary tube can be completely immersed within seconds due to capillary forces.

[0020] Preferably, the light from the monochromatic light source is coherent, and preferably has a wavelength between 350 nm and 500 nm, more preferably 405 nm, 445 nm, or 488 nm. Those skilled in the art will understand that a real light source can never be completely monochromatic, i.e., have zero optical bandwidth. Therefore, the term monochromatic light source refers to a light source with a very limited wavelength range or bandwidth as described above. In other words, in this application, the terms monochromatic light and quasi-monochromatic light are used interchangeably.

[0021] For example, laser sources are typically monochromatic or quasi-monochromatic, meaning that the optical bandwidth is small enough that some behaviors of the light are difficult to distinguish from those of truly monochromatic light.

[0022] Preferably, the (quasi)monochromatic light source is a coherent light source, such as a laser, preferably a diode laser, and more preferably a diode laser selected from the group consisting of: frequency-stabilized diode lasers, DPSS lasers, PPLN frequency-doubled diode lasers, frequency-doubled DPSS lasers, diode-pumped fiber lasers, frequency-doubled diode-pumped fiber lasers, and diode-pumped up-conversion fiber lasers. Preferably, the laser has a coherence length of at least 0.1 mm. The laser preferably has a coherence length of at least 0.1 mm, more preferably at least 1 mm.

[0023] Preferably, the laser has a power between 1 milliwatt and 200 milliwatts, more preferably between 10 milliwatts and 100 milliwatts, more preferably between 45 milliwatts and 80 milliwatts, and even more preferably between 50 milliwatts and 70 milliwatts. The laser may also have a power between 1 milliwatt and 200 milliwatts, preferably between 10 milliwatts and 180 milliwatts, more preferably between 50 milliwatts and 150 milliwatts, and even more preferably between 70 milliwatts and 120 milliwatts, for example, at 100 milliwatts. Preferably, a dynamic light scattering laser with electronically / digitally variable / controllable output can be used.

[0024] Preferably, monochromatic light is transmitted from a monochromatic light source to the container / sample via a laser wavelength single-mode fiber. Preferably, a polarization-maintaining (PM) fiber, such as a laser wavelength polarization-maintaining single-mode fiber, is used. According to another embodiment, it may be advantageous to use a non-polarization-maintaining fiber. Preferably, the light is further focused (e.g., via an objective lens) onto a focal point within the container.

[0025] Preferably, the light emitted by the monochromatic light source is perpendicular to a longitudinal axis of the container. The material is delivered to the container at one angle, wherein... Preferably, the angle is between 0 and 45 degrees, and preferably, a focal point is located within the container. Preferably, the light detected by the photodetector is at an angle to a longitudinal axis of the container. An angle (or scattering, backscattering) is emitted from the container, wherein The range is between 0 and 45 degrees, where The value is preferably the same as The values ​​are the same. Preferably, the angle between the light transmitted from the monochromatic light source to the container and the light emitted from the container and detected by the photodetector is... The angle is between 0 and 150 degrees, preferably between 10 and 150 degrees, and more preferably between 10 and 60 degrees. Since the position of the container can be determined within the device, the angle of the emitted or scattered light is preferably measured against the container. However, those skilled in the art will understand that the light scattered or emitted from particles within the container is light containing particle information that can be used for its characterization. For example, the method of the present invention can be used to perform dynamic light scattering measurements based on light scattered by particles in a solution. Furthermore, the method of the present invention can also be used to perform differential scanning fluorescence (DSF) measurements based on fluorescence emitted by particles. Macroscopically, the scattered light and fluorescence are emitted / exposed from the container.

[0026] Preferably, the transmitted monochromatic light is focused into the container containing the sample using an objective lens, wherein light emitted from the container is preferably also focused by the objective lens. Preferably, the objective lens has a focal length between 10 mm and 200 mm. Preferably, the transmitted monochromatic light is focused in the container with a full width at half maximum (FWHM) of a focal spot between 3 μm and 30 μm, preferably resulting in a measurement volume between 0.01 nanoliters and 0.1 nanoliters, preferably between 0.01 nanoliters and 0.02 nanoliters, and more preferably about 0.016 nanoliters.

[0027] Preferably, the photodetector is a photomultiplier tube (PMT), silicon photomultiplier tube (SiPM), or avalanche photodiode (APD) photon counting detector, and more preferably a photomultiplier tube or silicon photomultiplier tube.

[0028] Preferably, the dynamic light scattering measurement is obtained in less than 5 seconds, more preferably less than 1 second, more preferably between 200 milliseconds and 800 milliseconds, and more preferably within about 500 milliseconds.

[0029] Preferably, dynamic light scattering measurement is performed only once per sample at a specific temperature, for example, to improve measurement speed. However, dynamic light scattering measurements may be performed multiple times per sample / container at the same and / or different temperatures, for example, to calculate the average of the measurements (e.g., at the same temperature) and / or to account for different conditions of the particles (e.g., at different temperatures).

[0030] Preferably, the dynamic light scattering measurement includes the step of performing at least one correlation operation, preferably at least one autocorrelation operation.

[0031] Preferably, the dynamic light scattering measurement includes the following steps: acquiring an analog output signal from the photodetector; and processing the acquired analog output signal. Preferably, the step of processing the acquired analog output signal includes the following step: digitizing the acquired analog output signal to convert it into a digital output signal.

[0032] Digitizing the detector's (entire) analog output signal offers several advantages over existing techniques, which will be discussed in detail below. Specifically, the resulting digitized output data can be viewed as the digitized raw signal; however, it is easier to process subsequently, depending on the intensity of the light detected by the detector. In short, the digitized output data can then be processed using either analog or digital processing devices, for example, to distinguish individual peaks of individual photons.

[0033] Therefore, the step of processing the output signal further includes the following steps: i) processing the digitized output signal into a digitized single-photon pulse signal, preferably when the intensity of the detection light emitted from the container is less than 2 million photons per second; and / or ii) processing the digitized output signal into multiple discrete values ​​of an analog signal, preferably when the intensity of the detected light is greater than 2 million photons per second. The step of processing the obtained output signal includes step i) or step ii), and wherein the time for deciding whether to process the digitized output signal into a digitized signal according to step i) or step ii) is less than 1 second, preferably a maximum of 0.05 seconds, preferably using an FPGA (Field Programmable Gate Array). Furthermore, both signals (i.e., the photon count signal and the discrete value signal) can be processed simultaneously. Thus, the decision on whether to process according to step i) or ii) after measurement can be satisfied.

[0034] Preferably, the step of processing the obtained output signal further includes the following steps: storing the processed digital output signal obtained from step i) or step ii); or storing the processed digital output signal obtained from steps i) and ii); and further processing one of the stored output signals.

[0035] As an example, if the signal is processed as a photon-counting signal and / or an analog signal, the way the signal is processed is not predetermined by the photon-detecting detector, such as when using a dedicated photon-counting detector, but rather predetermined in a later stage by an algorithm in a device such as an FPGA (Field-Programmable Gate Array), ASIC (Application-Specific Integrated Circuit), or other programmable means. Preferably, these algorithms can be modified (e.g., improved) without changing the hardware. For example, the 2 million photon limit can be updated and / or changed via firmware and / or software, under which the algorithm changes from labeling the signal as a photon-counting signal to labeling it as an analog signal. For example, the limit can be switched from 2 million photons per second to 1 million photons per second or 4 million photons per second. This is preferred for existing solutions where the hardware (e.g., the photon counter) must be swapped to change the aforementioned limit.

[0036] Preferably, the method further includes a step of measuring fluorescence. Preferably, the measured fluorescence is the fluorescence of the plurality of particles in the solution contained in the sample and / or the fluorescence of the material of the container itself, wherein the fluorescence is preferably the autofluorescence of the plurality of particles / container material. Preferably, the method further includes the steps of: determining the location of the container based on the measured fluorescence, and optionally, repositioning the container relative to a light source (i.e., repositioning the container and / or the light source) based on the measured fluorescence and the determined container location. Preferably, the step of determining the container location is also applicable to samples without (autofluorescent) particles, by using containers that themselves exhibit sufficient autofluorescence. In other words, the method of the present invention preferably determines the location of a container (e.g., a capillary) by fluorescence measurement, wherein the detected fluorescence may be based on the material of the particles and / or the container. For example, a container made of glass or quartz glass already produces sufficient autofluorescence radiation to determine the location of the container based on the fluorescence measurement, even if the container contains no sample or a sample with particles that do not (autofluorescently) fluoresce.

[0037] Alternatively or additionally, the method according to the invention preferably further includes the step of measuring the back reflection of the container containing the sample, the step of which can be used for characterizing sample particles and / or for determining the position of the container.

[0038] Preferably, the method further includes the step of tempering the container over time at at least a first time point at a first temperature and at a second time point at different second temperatures. Therefore, if the characteristics of the particles are determined at two different temperatures, the change in characteristics can be measured. Thus, the method of the present invention can be performed at a first temperature T1, and then at different second temperatures T2 or multiple different temperatures Txy, to measure the change in particle characteristics in the solution, and to determine the change in particle characteristics by comparing T1 with T2 or temperature Txy.

[0039] Preferably, the present invention relates to a method for measuring the properties of multiple particles in a solution, the method comprising the following steps:

[0040] (a) Provide a sample containing a plurality of particles in a solution;

[0041] (b) Provide a temperature control system for generating a defined temperature for sample probing by contact heating and / or cooling;

[0042] (c) Measure the plurality of particles at a first temperature;

[0043] (d) A second temperature is generated within the sample by means of the temperature control system described above;

[0044] (e) Measuring the plurality of particles in the sample at the second temperature, and

[0045] (f) The plurality of particles are characterized based on the two measurements above.

[0046] According to the present invention, measurement steps c) and e) are the dynamic light scattering measurement steps disclosed in this application and / or the nanoscale differential scanning fluorescence measurement steps disclosed in this application. According to a preferred embodiment, both measurement steps c) and e) include a dynamic light scattering measurement step, an additional first fluorescence reading, and a second fluorescence reading.

[0047] Preferably, the step of tempering the container over time at at least a first time point at a first temperature and a second time point at a second temperature includes tempering the container at a tempering rate between 0.01°C / min and 30°C / min, preferably between 0.1°C / min and 10°C / min, and / or wherein the first temperature and the second temperature are between -20°C and 160°C.

[0048] Preferably, a specific temperature, preferably within the above-mentioned range, and the temperature is maintained for a specific time, such as more than 10 seconds, more than 1 minute, or longer, for example in an isothermal mode, with a specific accuracy preferably between + / -0.01°C and + / -1.5°C, preferably between + / -0.01°C and + / -0.5°C, provides certain advantages in terms of uniformity, reproducibility, and precision of the measurement.

[0049] Preferably, the method further includes the following steps(s): performing nano-differential scanning fluorescence (nano-DSF) measurements; and / or measuring the back reflection of the container containing the sample.

[0050] Preferably, the method further includes the steps of: providing another photodetector, and using the other photodetector to measure the static scattered light of the container containing the sample, preferably at an angle to a longitudinal axis of the container. in Preferably between 10 degrees and 150 degrees, more preferably between 10 degrees and 60 degrees.

[0051] Preferably, the container for containing small sample volumes is a small container, such as a capillary tube and / or a multi-well plate. Preferably, the capillary is a capillary tube sample container or simply a capillary tube. Preferably, the capillary of the present invention includes a substantially constant inner diameter and / or a substantially constant outer diameter along its entire length.

[0052] Preferably, the capillary is made of glass, preferably glass that has no or only slight autofluorescence properties, such as borosilicate glass and / or quartz glass and / or synthetic fused silica.

[0053] The capillary preferably has a circular cross-section, with an inner diameter preferably between 0.1 mm and 1 mm, more preferably between 0.15 mm and 0.5 mm, an outer diameter preferably between 0.2 mm and 1.2 mm, more preferably between 0.65 mm and 1 mm, and a length between 5 mm and 70 mm, more preferably between 32 mm and 50 mm, and more preferably around 50 mm. Based on the diameter, a compromise is further preferably selected between capillary wall thicknesses that are large enough to allow for stable manual operation and thin enough to minimize optical refraction within the desired range. Therefore, the preferred capillary wall thickness can be selected according to individual needs, such as manual handling, automatic handling, etc.

[0054] Furthermore, depending on the application, shorter or longer capillaries may be advantageous. The effect based on capillary length preferably depends on the desired application. For example, very short capillaries have the advantage of very small volume, which is advantageous for applications requiring very small amounts of material (efficiency). Very short capillaries can be completely filled by capillary forces (when they have a correspondingly fitted diameter). If they are short enough, the capillaries do not even need to be tilted relative to gravity (g), as the capillary forces themselves completely fill the capillaries that are antiparallel to g. Short capillaries also have advantages in terms of space; therefore, more capillaries can be placed on a limited surface.

[0055] The advantage of longer capillaries is that they do not require sealing at one or both ends; for example, according to the invention, the effect of solution evaporation is minimal relative to the measurement time. For instance, capillaries longer than 32 mm provide a low evaporation rate during the preferred measurement duration of the invention.

[0056] Preferably, multiple containers are provided, each container containing a sample of multiple particles in a solution, and multiple properties of the multiple particles in the solution are measured in each container according to the method of the invention. Preferably, a fluorescence measurement for each container is followed by a dynamic light scattering measurement for each container; or a dynamic light scattering measurement for each container is followed by a fluorescence measurement for each container; or a fluorescence measurement and a dynamic light scattering measurement are performed on one of the multiple containers, and then a fluorescence measurement and a dynamic light scattering measurement are performed on another of the multiple containers. More preferably, a fluorescence measurement and a dynamic light scattering measurement are performed simultaneously on each of the multiple containers.

[0057] Preferably, the plurality of characteristics are selected from the group consisting of: particle size distribution, aggregation temperature, melting temperature, transition temperature, unfolding temperature, liquid-liquid phase separation temperature (TLLPS), change in unfolding free energy f, second virial coefficient (B22 / A2), particle self-interaction, colloidal stability, hydrodynamic radius, repulsive or attractive interactions between particles (kD), solubility, long-term protein stability, and critical denaturant concentration.

[0058] For example, by determining the onset time of size increase (from measurements of radius as a function of temperature) compared to the start of aggregation, characteristics of unfolding / oligomery / aggregation can be derived.

[0059] The activation energy of the unfolded colloid can be derived, for example, from subsequent experiments at different heating rates, and can be used to evaluate the stability of the colloid.

[0060] The present invention also relates to an apparatus for measuring the properties of multiple particles in a solution, preferably according to the method of the invention, wherein the apparatus comprises a means for accommodating at least one container, the at least one container containing a sample of the multiple particles in the solution, preferably the at least one container being used to accommodate 0.1 to 15 microliters of the multiple particles; a monochromatic light source and a photodetector; means for performing a dynamic light scattering measurement; and a control means adapted to control the means for accommodating at least one container, control the monochromatic light source for transmitting light from the monochromatic light source to the at least one container, control the photodetector for detecting multiple signals from the at least one container, and control the means for performing the dynamic light scattering measurement.

[0061] Preferably, the apparatus further includes a means for performing a correlation operation, the correlation operation being preferably an autocorrelation operation, wherein the autocorrelation operation is preferably an autocorrelation logic implemented in hardware and / or software.

[0062] Preferably, the apparatus further includes means for digitizing signals obtained from the photodetector, wherein the means preferably includes a field-programmable gate array, and the control means is preferably further adapted to control the apparatus for digitizing multiple signals obtained from the photodetector.

[0063] Preferably, the apparatus further includes a means for measuring the fluorescence of the plurality of particles in a solution of the sample, wherein the control means is further adapted to control the apparatus for measuring the fluorescence of the plurality of particles in a solution contained in the sample.

[0064] Preferably, the apparatus further includes a positioning device for positioning the sample containing the plurality of particles in the solution, wherein the control device is further adapted to control the positioning of the device for containing the sample.

[0065] Preferably, the apparatus further includes a temperature control system for tempering the container at a first temperature at at least a first time point and at a second temperature over time at a second time point, wherein the control device is also adapted to: control the temperature control system for tempering the container at a first temperature at at least a first time point and at a second temperature over time at a second time point.

[0066] Preferably, the apparatus further includes a device for performing a nanometer differential scanning fluorescence measurement and / or a device for measuring back reflection, wherein the control device is also adapted to: control the device for performing the nanometer differential scanning fluorescence measurement and / or the device for measuring back reflection.

[0067] Preferably, the apparatus further includes another photodetector; and means for performing a static scattered light measurement, wherein the control means is further adapted to control the means for performing the static scattered light measurement.

[0068] Preferably, the device further includes a single-mode optical fiber; and means for transmitting monochromatic light from the monochromatic light source through the single-mode optical fiber, wherein the control means is further adapted to: control the device to transmit monochromatic light from the monochromatic light source through the single-mode optical fiber. Attached Figure Description

[0069] In the following description, preferred embodiments of the invention will be described in detail with reference to the accompanying drawings. The accompanying drawings show:

[0070] Figure 1: A) Angle between the main beam and the capillary axis. B) The dynamic light scattering optics are tilted on the axis of the capillary to avoid detecting reflections.

[0071] Figure 2 The function of a pulse phase detector.

[0072] Figure 3 A comparison graph of the signal strength of a photon counting signal and the signal strength of an analog signal (dashed lines represent extrapolation fitting of the linear range).

[0073] Figure 4: A) Signal layout with one correlator whose input switches between photon counting and analog data. B) Signal layout with two correlators operating in parallel, one in analog mode and the other in photon counting mode.

[0074] Figure 5The measurement system according to the present invention.

[0075] Figure 6: A) Scheme of dynamic light scattering optical element. B) Part of the CAD model (cross-section) of dynamic light scattering optical element with two collimating lenses.

[0076] Figure 7: A) Schemes for dynamic light scattering optics with one excitation and two detectors, such as a photodiode with multimode fiber and a photomultiplier tube with single-mode fiber. B) A confocal version of a dynamic light scattering optics. C) A combined fluorescent optics and dynamic light scattering optics in confocal (x-distance = 0). D) Design of a dynamic light scattering optics with a free-space coupled source. E) Design of a dynamic light scattering optics with a lens cover, astigmatism correction, fluorescence blocking filter, and polarization filter. F) Design of a dynamic light scattering optics with two dynamic light scattering arms and a confocal fluorescent optics for simultaneous measurement at a single point. G) Design of a lensless, two-arm dynamic light scattering optics.

[0077] Figure 8: A) Binding measurement of fluorescence shift and particle size distribution of IgG in sodium acetate buffer as a function of temperature. B) Binding measurement of fluorescence shift and average particle size of IgG in sodium acetate buffer as a function of temperature. C) Binding measurement of fluorescence shift and particle size distribution of IgG in HEPES buffer as a function of temperature. D) Binding measurement of fluorescence shift and average particle size of IgG in HEPES buffer as a function of temperature.

[0078] Figure 9 A summary of the dynamic light scattering quality parameters of different samples.

[0079] Figure 10 The attenuation rate (inversely proportional to the particle radius) is expressed as a function of the measurement location. White circles indicate the measurement locations analyzed.

[0080] Figure 11 The preferred embodiment of the present invention.

[0081] Figure 12 Exemplary protein molecular weight distributions (UniProt database, human; https: / / smith.chem.wisc.edu / content / proteomics-technologies).

[0082] Figure 13 : Schemes for characterizing exemplary proteins using the methods and apparatus according to the present invention

[0083] (http: / / book.bionumbers.orq / how-biq-is-the-averaqe-protein / ).

[0084] Figure 14 This describes the signal quality of dynamic light scattering measurements at different locations within the container, in order to determine the optimal measurement point within the container.

[0085] Figure 15 Measurement of antibody buffer screening and antibody candidate selection.

[0086] Figure 16 Further measurements of antibody buffer screening and antibody candidate selection.

[0087] Figure 17 Further measurements of antibody buffer screening and antibody candidate selection.

[0088] Figure 18 : Based on an example with random clustering, understand the measurement of clustering paths.

[0089] Figure 19 Based on the example of structured aggregation, we can further measure the aggregation path. Detailed Implementation

[0090] The general principles of the invention will be discussed in further detail below, and illustrative examples or preferred embodiments of the invention will be discussed.

[0091] Specifically, in a first aspect, the present invention relates to a method for measuring the properties of multiple particles in a solution, the method comprising the steps of providing a container comprising a sample of multiple particles in the solution, wherein the sample has a volume preferably between 0.1 μL and 15 μL; providing a monochromatic light source and a photodetector; transmitting light from the monochromatic light source to the container containing the sample; and using the photodetector to detect light emitted from the container; and determining multiple properties of the multiple particles in the solution contained in the sample based on a dynamic light scattering measurement. An advantage of the present invention is that known dynamic light scattering measurements can now be performed in very small sample volumes, for example, dynamic light scattering measurements of samples within capillaries, which has never been disclosed or suggested in the prior art. Conversely, the prior art tells us that dynamic light scattering measurements preferably require a volume larger than the aforementioned sample probe. Furthermore, the method of the present invention has the advantage of obtaining accurate measurement results with high throughput.

[0092] Preferably, the method further includes the step of performing a nanometer differential scanning fluorescence measurement. The advantage of this step is that, based on the dynamic light scattering and nanometer differential scanning fluorescence measurement results obtained by the method, particle size distribution and potential particle aggregation can be rapidly and accurately assessed, preferably within a single device. To improve the accuracy of measurements performed according to the method of the invention, such as dynamic light scattering measurements, preferably and nanometer differential scanning fluorescence measurements, the method preferably further includes the step of measuring fluorescence to determine the container location. For example, the (auto)fluorescence of particles in the solution and / or the autofluorescence of the container itself can be used, wherein the location of the container can be determined by using the fluorescence signal thus obtained. Therefore, the location of the container containing a sample with multiple particles in the solution under study can be accurately determined, and optionally, the container can be (re)positioned to optimize the accuracy of the respective (multiple) measurements.

[0093] Therefore, the method of the present invention is highly advantageous for measuring the properties of multiple particles in a solution, and exhibits high sensitivity even with small sample volumes, using emitted monochromatic light and scattered light for dynamic light scattering measurements. Preferably, the method according to the present invention is executed automatically.

[0094] Dynamic light scattering (DLS) is a method for analyzing the diffusion coefficient of particles, a measure of particle migration in solution. In DLS measurements, a sample in a container containing a solution of particles is illuminated with monochromatic light, and the intensity of the scattered light is detected at a predetermined angle. The path length of the monochromatic light to the particles and the path length of the scattered light to the detector differ, depending on the position of each particle within the container. By superimposing the light waves scattered from different particles contained in the sample, a specific interference pattern of the sample can be obtained based on the detected light. Therefore, the intensity of the detected signal depends on the position and motion of the particles within the container, i.e., Brownian molecular movement / motion, which alters the length of the light path and thus the obtained interference pattern. For example, the faster the particles move, the faster the signal intensity changes, resulting in a higher diffusion coefficient. Determining the diffusion coefficient of particles in a solution contained in a sample based on DLS measurements is advantageous because it can be used to determine the hydrodynamic radius of the particles, including, for example, their hydration film. Therefore, by applying the method according to the invention, even small particles, such as particles with a hydrodynamic radius in the range of 0.1 nm to 3000 nm, preferably in the range of 0.5 nm to 1000 nm or 2 nm to 3000 nm, can be detected and characterized with high sensitivity and accuracy.

[0095] The particles, whose properties can be measured using the method according to the invention, can be naturally occurring, biochemically and / or synthetically modified, or synthetic particles or combinations thereof. Preferably, the particles have an average diameter of 1000 nanometers or less (preferably 0.1 nanometers to 700 nanometers, more preferably about 1 nanometer to 500 nanometers). The particles can be at least partially or entirely biological particles (also called bioparticles), therefore, the term "particle" as used herein preferably does not refer to, for example, dust, gold particles, etc. The latter example, including gold particles, is preferably not included in the sample under study, nor added to the sample under study, for example, not included in the sample processing and / or preparation steps prior to the method according to the invention. Their presence in the sample is not advantageous because they will scatter light more strongly than biological particles, and thus may mask the scattered light signal of the (biological) particles under study. Preferably, so-called "probe particles" are not added to the sample, as the sample is preferably studied with its original composition.

[0096] In the context of this invention, and particularly in the claims, it should be noted that the term "particle" or "multiple particles" also refers to a variety of beads, particularly a variety of microbeads, a variety of vesicles, a variety of molecular clusters, a variety of nanoparticles, or a variety of molecules, particularly a variety of biomolecules, such as a variety of nucleic acids (e.g., DNA, RNA, LNA, PNA), a variety of proteins, and other biopolymers and combinations thereof, as well as a variety of biological cells (e.g., bacteria, prokaryotic or eukaryotic cells) or a variety of subcellular fragments, a variety of viral particles, a variety of virus-like particles, or a variety of viruses and a variety of organelles, etc. Examples of molecules include, but are not limited to, fluorescent dyes, peptides, particularly polypeptides, sugars, particularly polysaccharides, compounds, small molecules, fragments, or surfactants.

[0097] The terms "modified particles" or "modified beads" specifically refer to beads or particles that include or are linked to molecules, preferably biomolecules or fluorescent dyes. This also includes coating these beads or particles with these (bio)molecules.

[0098] Further examples of particles according to the invention include various viral particles, various virus-like particles, various cells (especially cells with a diameter less than 20,000 nanometers), various DNA molecules, various RNA molecules, DNA origami, various small molecules (e.g., molecules with a molecular weight less than 900 Daltons), various liposomes, various proteins, various protein complexes, such as in the case of various oligomeric proteins, and / or various protein aggregates, wherein the type of particles, such as proteins contained in the solution to be studied, can be the same or different. Preferably, the particles in the solution are of the same type, such as proteins or complexes and / or aggregates thereof.

[0099] Preferably, the sample under study is obtained directly, for example, during or after product manufacturing. Therefore, the sample is preferably unmodified and / or amended before applying the method according to the invention. If, for example, gold particles are added to the sample, the resulting effects must be considered when processing and / or interpreting the results of the performed dynamic light scattering (preferably additional fluorescence measurements). Therefore, it is advantageous to investigate the particles under study in a directly obtained and unmodified sample, as information about the characteristics of the particles in the solution can be obtained without potentially time-consuming and / or costly pretreatment and / or post-treatment.

[0100] In this document, the terms "protein" and "oligomeric protein" are used interchangeably unless otherwise specified. Specifically, the term "protein" as used herein includes any kind of amino acid sequence, i.e., a chain of two or more amino acids linked by peptide bonds. More specifically, the term "protein" as used herein refers to any amino acid sequence of interest. Preferably, the amino acid sequence is at least 5 amino acids long, more preferably at least 10 amino acids, and even more preferably at least 50, 100, 200, or 500 amino acids. Therefore, the term "protein" includes short peptides, such as oligopeptides, polypeptides, proteins, protein fragments, i.e., portions of known proteins, such as biologically active portions or antigenic portions, including epitopes. There are no limitations regarding the function of the protein.

[0101] The concentration range of the particles is preferably between a single particle (e.g., nanoparticles, for gold nanoparticles) and 500 mM (e.g., in the case of small molecules).

[0102] The particles in the solution are preferably proteins. See Table 1 below (minimum R for different protein masses; https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC3055910 / ) and Figure 12As shown, most proteins have a molecular weight of, for example, less than 500 kDa, and therefore have a radius of less than 5 nanometers and / or a diameter of less than 10 nanometers.

[0103] Therefore, the preferred proteins have a radius of less than 5 nanometers and / or a diameter of less than 10 nanometers. Furthermore, Figure 13 Some preferred examples are shown in the figure.

[0104]

[0105] Table 1

[0106] Examples of low protein concentrations are lysozyme at 0.5 mg / mL, insulin at 0.4 mg / mL, and IgG at 0.06 mg / mL. Preferred protein concentrations range from 0.001 mg / mL to 250 mg / mL, more preferably from 0.01 mg / mL to 200 mg / mL.

[0107] Particles such as proteins can be isolated from biological materials, such as cell cultures used for in vitro protein production, and / or produced artificially. The latter has several advantages, as it is time- and / or cost-effective and ensures high purity of the protein of interest. Preferably, the particles in solution are aggregates of the same protein or complex and / or its constructs, wherein the protein is artificially produced. Therefore, highly accurate and sensitive measurements can be obtained to characterize multiple particles in solution.

[0108] Several properties of particles in solution have commercial and academic significance. For example, particle size distribution in solution is useful for assessing the particle composition in solution, including the presence and / or quantity of undesirable particles, such as proteins that have degraded and / or aggregated due to instability under specific conditions. Since different particles may aggregate under different conditions, the aggregation conditions of a given particle can be assessed to characterize the particle, for example, the temperature at which the particle is exposed when a given proportion (e.g., half) of the particles are aggregated. Thus, the aggregation temperature can be used to define the onset (Tagg) of aggregation. The same logic applies to the application of temperature dependence on particle folding and unfolding, starting with a particle-specific unfolding temperature. The melting temperature / point (Tm) is of greater interest, defining the temperature at which a particle (e.g., a protein) has a potential folding state that is thermodynamically no more favorable than another temperature. Thus, the particle exists in comparable amounts in either configuration, i.e., the naturally folded configuration and the unfolded and thus denatured configuration. Therefore, the melting temperature can serve as an indicator of particle thermal stability. Furthermore, the free folding energy, which is directly related to protein stability and depends on the protein's amino acid composition, can be studied. The second virial coefficient (B22, also known as A2) is an indicator of the colloidal stability of particles and can be further used to predict protein aggregation. A positive value indicates that the colloidal interaction between proteins is mainly negative, while a negative value indicates the net attraction interaction (kD; diffusion interaction parameter) between proteins.

[0109] Therefore, the properties of particles in solution can be measured using the method according to the invention, preferably selected from the group consisting of: particle size distribution, aggregation temperature, melting temperature, unfolding temperature onset, free folding energy, second virial coefficient (B22 / A2), particle self-interaction, colloidal stability, hydrodynamic radius, repulsive or attractive interactions between particles (kD), and critical denaturant concentration.

[0110] Furthermore, by determining the onset time of size increase (e.g., derived from measurements of radius as a function of temperature) compared to the start of aggregation, characteristics of unfolding / oligomery / aggregation can be derived. Additionally, the activation energy of unfolding can be derived, for example, from subsequent experiments at different heating rates; this is a useful application, for example, in evaluating colloidal stability.

[0111] The method according to the invention can be used to measure the properties of the plurality of particles in a solution (preferably an aqueous solution) by providing a container containing a sample of the plurality of particles in the solution, wherein the sample has a volume preferably between 0.1 μL and 15 μL.

[0112] Therefore, a sample containing some or all of the particles in the solution under study is analyzed, wherein the sample is contained in a container. In the case of the container, for example, a capillary tube, the sample preferably has a volume between 0.1 μL and 15 μL, more preferably between 1 μL and 15 μL, and even more preferably between 8 μL and 12 μL. In the case of the container, for example, a multi-well plate, specifically a 384-well plate, the sample preferably has a volume between 5 μL and 40 μL; in the case of a 96-well plate, it is preferably between 50 μL and 200 μL; and in the case of a 1536-well plate, it is preferably between 1 μL and 15 μL. Such a small sample volume is advantageous when the number of available particles is limited and / or the cost of the particles under study is high. Furthermore, analyzing small sample volumes using the method according to the invention has at least the following additional advantages: i) better masking of scattering on the container walls, i.e., minimizing the effect of light scattering on the container walls (e.g., capillary walls); ii) stronger scattering signals from samples containing particles in the solution under study, thereby reducing measurement time; iii) reduced probability of multiple scattering at high concentrations, which depends on the number of particles in the solution in the sample; and iv) the availability of cheaper monochromatic light sources (e.g., lasers with shorter coherence lengths) to obtain high-quality measurement results, thereby accurately characterizing the particles under study.

[0113] The sample containing particles in the solution under study is contained in a container, preferably in the pores of a capillary or porous plate. In the case of a capillary, the capillary may be made of resin, plastic, ceramic, polymer, or glass. Preferably, the capillary has open ends at both longitudinal ends. Preferably, the capillary is a glass capillary. This method, according to the invention, is advantageous for improving accuracy and reproducibility by reducing artifacts and signal noise that can affect dynamic light scattering measurements, which are based on light signals emitted from a container containing a sample of multiple particles in the solution. Therefore, a glass container is advantageous because glass has less influence on light transmission and scattering than other materials such as resin, and is stable and inert even under high temperatures and extreme solution conditions (such as acidic conditions). Therefore, the capillary is preferably a glass capillary with a circular cross-section, having an inner diameter between 0.1 mm and 1 mm, preferably between 0.15 mm and 0.5 mm, and preferably an outer diameter between 0.2 mm and 1.2 mm, preferably between 0.65 mm and 1 mm, and preferably a length between 5 mm and 70 mm, preferably between 32 mm and 50 mm, more preferably about 50 mm. Preferably, the inner diameter and / or outer diameter are constant along the entire length of the capillary, and preferably both are constant. Such a capillary is often referred to as a tube capillary to further emphasize its form. The capillary wall acts similarly to a lens, thus exhibiting optical effects. Therefore, the thickness of the capillary wall affects the accuracy and sensitivity of the measurement. The use of capillaries with an inner diameter between 0.1 mm and 1 mm, preferably between 0.15 mm and 0.5 mm, and an outer diameter between 0.2 mm and 2 mm, preferably between 0.3 mm and 0.7 mm, has been found to be the preferred method for obtaining accurate and sensitive measurements. Specifically, the inner diameter described above is advantageous in ensuring a short path length of the beam within the sample under study; furthermore, with a smaller diameter, thermal conductivity between the container material (and the sample) and the heating element (preferably made of silicon) is better. However, in the case of dynamic light scattering measurements, a larger diameter is considered preferable.

[0114] According to the method of the invention, a monochromatic light source is provided, preferably a coherent light source, such as a laser. Monochromatic light describes light radiation containing a single optical frequency. As is known in the art, no true laser is truly monochromatic, and all lasers emit light within a range of frequencies, which are referred to as the linewidth of the laser transition. In most lasers, the linewidth is narrow. Therefore, for simplicity, the invention refers to it as monochromatic light. Monochromatic light is particularly advantageous for characterizing particles in solution because its use ensures defined, constant measurement conditions, thereby ensuring precise and highly accurate analysis.

[0115] Preferably, the light from the monochromatic light source has a wavelength of less than 500 nm, more preferably between 350 nm and 500 nm, and even more preferably 405 nm, 445 nm, or 488 nm. Measurements at wavelengths less than 500 nm (e.g., 445 nm, 405 nm, or 488 nm) are particularly advantageous for reducing costs while maintaining high-quality results, as cheaper and less complex lasers can be used. Furthermore, using light from a monochromatic light source with a wavelength less than 500 nm (e.g., 445 nm, 405 nm, or 488 nm) has the advantage of resulting in a stronger scattered light signal and low absorption by most particles (including most proteins) compared to using light from a monochromatic light source with a wavelength of 500 nm or higher.

[0116] Monochromatic light can be provided by a laser, preferably a diode laser, and even more preferably a diode laser selected from the group consisting of: frequency-stabilized diode lasers, DPSS lasers, PPLN frequency-doubled diode lasers, frequency-doubled DPSS lasers, diode-pumped fiber lasers, frequency-doubled diode-pumped fiber lasers, and diode-pumped up-conversion fiber lasers. Preferably, the laser has a power between 1 milliwatt and 200 milliwatts, more preferably between 10 milliwatts and 100 milliwatts, more preferably between 45 milliwatts and 80 milliwatts, and even more preferably between 50 milliwatts and 70 milliwatts.

[0117] Monochromatic light sources can be characterized by their coherence length. The coherence length is the maximum difference in path length or flight time between two beams from the same monochromatic light source, so that a stable interference pattern is produced when they are superimposed. The interference pattern can be caused by interference effects, which can be caused by the phase difference in the light waves arriving at the detector due to different path lengths and / or flight times. The monochromatic light source (preferably a laser) preferably has a coherence length of at least 0.1 mm.

[0118] According to the method of the invention, light from a monochromatic light source (preferably a laser) is transmitted into a container containing a sample (including particles in the solution under study). Therefore, the light is preferably transmitted from the laser into the container containing the sample under study in the form of a beam to characterize the particles in the solution contained in the sample. In this document, the terms "light," "light beam," and "beam" are used interchangeably.

[0119] Light can be transmitted in a variety of ways. Preferably, monochromatic light is transmitted from a monochromatic light source via a single-mode fiber. More preferably, monochromatic light is transmitted from a laser via a single-mode fiber at the laser wavelength (e.g., a polarization-maintaining single-mode fiber at the laser wavelength).

[0120] Monochromatic light transmitted from a monochromatic light source to the container, i.e., the transmitted monochromatic light, is preferably focused in the container containing the sample using an objective lens. Furthermore, light emitted from the container is also preferably collected by the objective lens. Preferably, the objective lens has a focal length between 10 mm and 200 mm. Alternatively or additionally, the transmitted monochromatic light is preferably focused in the container, having a full width at half maximum (FWHM) of a focal spot between 3 μm and 30 μm, preferably resulting in a measurement volume smaller than the sample volume. In particular, the measurement volume that can be investigated using the method according to the invention is preferably between 0.01 nanoliters and 0.1 nanoliters, more preferably between 0.01 nanoliters and 0.02 nanoliters, and even more preferably about 0.016 nanoliters.

[0121] Therefore, it is particularly preferred that the monochromatic light is provided by a powerful laser through a wide aperture, for example, an aperture having a width between 0.5 mm and 10 mm, preferably between 1 mm and 5 mm, the light having a wavelength of 405 nm, 445 nm, or 488 nm, and focused into a container containing the sample using an objective lens. Thus, for example, approximately one million photons per second are transmitted to the container. For this case, methods for measuring the particle characteristics in the solution are known in the art. To ensure accurate results, the method uses, for example, an optical filter located between the light source and the container to reduce the amount of light transmitted to the container, and / or includes the steps of diluting the sample to reduce the particle concentration in the solution, thereby reducing the amount of emitted light. Conversely, the method according to the invention can be applied without diluting the sample or using such a filter by providing at least two different analytical modes, as described below (detailed description of obtaining and processing the output signal).

[0122] For high-quality characterization of particles in solution, it is also important to accurately transmit light into the container containing the sample. More specifically, it is advantageous to place the optical element at an angle along the capillary axis to avoid detecting reflections from the container. Therefore, light from a monochromatic light source is preferably transmitted to the container (e.g., the capillary) at a predetermined angle. Specifically, the light from the monochromatic light source is preferably at an angle perpendicular to a longitudinal axis of the container (e.g., the longitudinal axis of the capillary). The angle at which the signal is transmitted to the container. Preferably, the temperature is between 0 and 45 degrees.

[0123] According to the method of the invention, a photodetector is also provided for detecting light emitted from a container, wherein the light emitted from the container refers to scattered light from the container (e.g., a capillary). Furthermore, the emitted light is preferably detected at a predetermined angle. Therefore, the light detected by the photodetector is preferably at an angle to a longitudinal axis of the container (e.g., the capillary). An angle emanates from the container (e.g., a capillary), wherein Preferably, the temperature is between 0 and 45 degrees Celsius. Preferably, The value and The values ​​are the same. Furthermore, the angle between the light transmitted from the monochromatic light source to the container (e.g., capillary) and the light emitted or scattered from the container (e.g., capillary) and detected by the photodetector. Preferably, the temperature is between 0 and 150 degrees, more preferably between 10 and 150 degrees, and even more preferably between 10 and 60 degrees.

[0124] like Figure 1A As shown in the example, light from a monochromatic light source (such as a laser) can be used as an excitation beam 50 at an angle to the longitudinal axis 10 of the container 11. An angled transmission of light from excitation beam 50 to container 11 (e.g., capillary 11) encompasses sample 12 containing particles in the solution under study. The light from excitation beam 50 is scattered by the multiple particles contained in the solution under study within sample 12, and thus emitted from container 11. The emitted or scattered light can be detected as detection beam 51, forming an angle with the longitudinal axis 10 of container 11. 20. The angle between the excitation (light) beam 50 and the detection (light) beam 51 is called... 54 can also be described as the angle between the light transmitted from the monochromatic light source to the container 11 and the light emitted from the container and detected by the photodetector.

[0125] like Figure 1B As shown in the example, it is advantageous to position the dynamic light scattering optical element 15 so that the excitation beam 50 is at an angle. 53 is transmitted to the longitudinal axis 10 of the container 11 (e.g., tilted to the capillary axis) to avoid detecting the reflected beam 19 (reflected by the wall of the capillary 11). Figure 1B The angle 53' is shown to be 90°. The advantage of this invention is that the excitation beam 50 is focused onto a focal point 17 located within the container by using a suitable dynamic light scattering optics 15. Therefore, by using suitable focusing optics and emitting light at an appropriate angle relative to the longitudinal axis 10 of the capillary, interference reflections from the capillary wall can be avoided. Preferably, an optical fiber between the optics 15 and the capillary 11 is not necessary, allowing for a small air distance between them, which facilitates relative movement between the optics 15 and the capillary 11, advantageous when measuring multiple capillaries in a short time. Positioning the focal point 17 within the capillary can be achieved through powerful focusing with a small focal length.

[0126] Specifically, to ensure that focus 17 is located within the small capillary, the present invention provides a method for enhancing the measurement of capillary position, and optional feedback control to reposition the capillary and / or the position for measurement and / or subsequent measurements. Furthermore, according to the present invention, not only can the measurement be ensured to be performed within the container (at some point), but it is also preferably performed at an optimal location within the container.

[0127] For example, if the container is a capillary, such capillaries typically have an inner diameter of 500 micrometers, but the preferred location has a size (e.g., diameter) of only about 50 micrometers. Using the focusing method described above, an accuracy of 10 micrometers can be achieved. Based on this high accuracy, not only can the location of the container / capillary be precisely determined, but also the measurement volume within the container can be determined, such as the volume located at a certain location "in the middle" of the container, some distance from the container walls(s).

[0128] This location or site can be found, for example, by performing multiple dynamic light scattering measurements at several different locations within the container / capillary using a weakly scattering sample. The preferred location is where the autocorrelation function has the highest signal-to-noise ratio (SNR) (SNR = (acf amplitude) / (sum of squared fitted residuals)). For example... Figure 14 The scans of capillaries with different SNR values ​​in different colors are shown, which allows, for example, the identification of the enclosed area as the location of the measurement. For example, when using a lysozyme sample with a concentration of 2 mg / mL and a measurement duration of 200 ms, the typical SNR value is 50.

[0129] The provided photodetector is preferably a photomultiplier tube (PMT), silicon photomultiplier tube (SiPM), or avalanche photodiode (APD) photon counting detector. The raw output signal of such a detector is typically an analog output signal, such as a variable current or voltage, which can be converted into a variable voltage or current, respectively. According to the invention, the variable output signal is preferably further processed as discussed in more detail below. Conversely, traditionally, dynamic light scattering measurements have been limited to the use of avalanche photodiode photon counting detectors. However, a disadvantage of avalanche photodiode photon counting detectors is that the intensity of the detected light must be very weak in order to be detected. Some state-of-the-art devices attempt to overcome this disadvantage, for example, by using additional filters to reduce the light intensity, which takes time and thus reduces processing power. Therefore, to overcome these disadvantages, the photodetector of the present invention is preferably a photomultiplier tube or silicon photomultiplier tube, which offers further advantages, as discussed in more detail below.

[0130] According to the method of the present invention, the light detected by the photodetector can be used for dynamic light scattering measurement, therefore,

[0131] The characteristics of multiple particles in a solution contained in a sample can be determined based on dynamic light scattering measurements. For a given sample, the dynamic light scattering measurement is preferably obtained in less than 5 seconds, more preferably less than 1 second, more preferably between 200 milliseconds and 800 milliseconds, and more preferably within about 500 milliseconds. Therefore, even with a small sample volume, high throughput can be ensured while maintaining high accuracy and sensitivity in the analysis.

[0132] The dynamic light scattering measurement preferably includes the step of performing at least one correlation operation, preferably at least one autocorrelation operation. The autocorrelation operation (or function) evaluates the similarity between a signal at a given time and a signal after a specific delay time, where the intensity fluctuations of large particles are slower than those of small particles. Therefore, the autocorrelator calculates a normalized autocorrelation function describing the similarity of the signal after a specific delay time, and thus correlates the intensity fluctuations of the scattered light with time to determine the rate of intensity fluctuation, which is related to the diffusion behavior of the particles. Therefore, performing at least one correlation operation, preferably at least one autocorrelation operation, is advantageous for determining the diffusion coefficient of particles in solution based on the fluctuations in the light intensity reaching the detector. Preferably, the respective correlation (preferably autocorrelation) functions are transferred to a personal computer for further analysis and / or stored on a storage medium such as a hard disk.

[0133] Dynamic light scattering measurement preferably includes the following steps: acquiring an analog output signal from the photodetector; and processing the acquired analog output signal. Preferably, the step of processing the acquired analog output signal includes the step of digitizing the acquired analog output signal to convert it into a digital output signal. Specifically, the acquired analog output signal can be digitized into a digital output signal with a different data rate, preferably a high data rate of at least 40 MS / s. This is particularly advantageous when the provided photodetector is a photomultiplier tube or a silicon photomultiplier tube, and therefore, according to the method of the invention, it can even be used for continuous operation.

[0134] Preferably, the step of processing the obtained analog output signal into a digitized output signal further includes the following steps: i) processing the digitized output signal into a digitized single-photon pulse signal, preferably when the intensity of the detection light emitted from the container is less than 2 million photons per second; and / or ii) processing the digitized output signal into multiple discrete values ​​of an analog signal, preferably when the detected light intensity is greater than 2 million photons per second. The advantage of this is that the obtained analog output signal can be processed according to the signal intensity and / or signal intensity fluctuations of the sample, thereby ensuring optimal processing of the obtained analog output signal and optimized characterization of particles in the solution contained in the sample under study.

[0135] Specifically, photons emitted from the container containing the sample under study can be detected by a photodetector (e.g., a photomultiplier tube), and preferably the obtained analog output signal is digitized.

[0136] Therefore, if the intensity of the detection light emitted from the container is less than 2 million detection photons per second, the digitized signal of the low-scattering sample is preferably processed into a discretized signal, thus becoming a digitized signal. This allows for the detection of individual photons emitted from the container with high precision. The advantage of counting individual photons in the low-brightness range is based on the knowledge that all photons have the same energy content, so all other signal components can be inferred as noise or caused by other random factors such as offset drift and / or gain fluctuations.

[0137] For example, a pulse phase detector can be used to count individual photons. (See also: Regarding...) Figure 4A and 4B In a further detailed discussion, it is preferred that the analog output signal (directly) from detector 30 is digitized, preferably by an analog-to-digital converter (ADC). Figure 2 The left side shows an example of the generated digital output signal 39.

[0138] Specifically, signal 39 shown is the digital output signal of the photomultiplier tube. For example... Figure 2 As exemplarily shown, the pulse detector 32 can be used to discretize the digitized output signal 39 (e.g., obtained from a photodetector after digitization) by applying a threshold such as a photon counting threshold 36. Thus, if the digitized output signal exceeds the threshold 36, a discretized signal is created indicating, for example, the presence of a photon. In other words, the digitized output signal 39 is processed as a digital (single) photon pulse signal. If the signal 39 does not exceed the threshold, the corresponding discretized signal indicates, for example, the absence of a photon.

[0139] However, when the acquired signal becomes too bright, resulting in too much photon signal overlap, single-photon counting becomes nearly impossible or even impossible. In such cases, the digitized output signal is preferably processed as an "analog signal," for example, as multiple discrete values ​​of an analog signal. That is, the digitized signal provides a continuous signal that can be processed in a similar manner to the original analog signal. This application also relates to the step of processing the digitized output signal as multiple discrete values ​​of an analog signal having multiple discrete values. This latter approach is preferably used when the intensity of the detected light is above a certain threshold (e.g., above 2 million detected photons per second) or, for example, according to the corresponding brightness value based on the quantization depth after digitization. When the photodetector is a silicon photomultiplier tube, at intermediate brightness levels (e.g., the intensity of the detected light is between 2 million and 6 million detected photons per second), the digitized output signal can even be processed as a digitized signal, and the actual number of detected photons can be estimated by discretizing the photon peak area.

[0140] The steps of processing the obtained analog output signal and the subsequently generated digitized output signal preferably include step i) or step ii). Therefore, the digitized output signal is preferably processed into a digitized single photon pulse signal or multiple discrete values ​​of an analog signal. To determine whether to process the digitized output signal according to i) or ii), a field-programmable gate array (FPGA) is preferably used. Using, for example, an FPGA, the switching between photon counting and analog operation can be completed within microseconds. Therefore, the time to determine whether to process the digitized output signal into a digitized signal according to step i) (e.g., using an FPGA) is preferably less than 1 second, more preferably a maximum of 0.05 seconds. The algorithm of the FPGA is preferably run as some kind of "software" on the FPGA. The algorithm (software) used to process the data can be changed, for example, by updating. In other words, it is easy to change the specifications / algorithm of signal processing by software updates. Therefore, no changes to the hardware / detector are required, which provides a significant advantage.

[0141] According to the present invention, the digitized output signal can also be processed simultaneously according to i) and ii). In this way, the decision on whether to process according to step i) or ii) after measurement can be satisfied.

[0142] Figure 3An illustrative example shows the advantage of deciding whether to process the digitized output signal according to i) or ii), depicting a comparison graph of the signal strength of the photon counting signal and the signal strength of an analog signal, where the dashed line represents an extrapolation fit of the linear range. Simply put, a photomultiplier tube is illuminated using a very low-power LED, and the signal is measured within milliseconds. Figure 3 The “analog signal” shown represents the average value of the signal, while the “detected photons / second” signal is obtained by calculating the number of photon peaks in each measurement interval and scaling it to 1 second. Then, the LED power is increased and the measurement is repeated.

[0143] Therefore, as Figure 3 As shown, at a given radiation power, more and more photon peaks overlap and are no longer identified as individual peaks. More specifically, at a certain point, the photon signal no longer increases proportionally with the LED power. Traditional dynamic light scattering instruments measure within the photon count range, therefore, filters are included in the beam path when the signal is too strong (i.e., too many photons are detected).

[0144] Conversely, the dynamic light scattering measurement according to the invention includes analyzing the digitized output signal as a "photon counting signal" with a linear error of less than, for example, 5%, i.e. Figure 3 In the example shown, the average analog signal is higher than approximately 3 × 10⁶ photons per second or 20 counts, while at higher power, the digitized output signal is analyzed as an analog signal. Therefore, using the method and apparatus according to the invention has the advantage of reducing the time required for dynamic light scattering measurements, and thus, increases the processing capacity due to the intensity-dependent signal processing described above.

[0145] Alternatively, the two processing options can be applied in parallel, where the term "parallel" is intended to be understood as having a time offset of less than 1 nanosecond. This is preferably achieved by using, for example, two processors in parallel (preferably synthesized in the same field-programmable gate array). Thus, the digitized output signal can also be processed into both digitized and analog signals, and the resulting data is stored. This has the advantage that both processed digitized output signals can be stored and processed in subsequent stages. Therefore, the step of processing the obtained analog output signal can also include the following steps: storing the processed digitized output signal obtained from steps i) and ii), and further processing one of the stored multiple output signals.

[0146] The steps from emitting light to processing the signal can be briefly summarized as follows. The laser emitted from the optical fiber is collimated parallel by a collimating lens and focused into a container (such as a capillary) by an objective lens. The beam of scattered light, and thus the emitted light from the container, is focused onto the optical fiber of a dynamic light scattering detector (acting as a photodetector) by another collimating lens, using the same or another objective lens (preferably the same objective lens), and can then be detected by a photomultiplier tube (PMT). Since the PMT typically has a current output, while an analog-to-digital converter (ADC) usually requires a voltage signal, an amplifier can be placed between the PMT and the ADC to convert the PMT's current into a voltage signal and amplify it. The ADC can further convert the amplified voltage signal, with a sampling rate of, for example, 40 MS / s (40 million samples per second), into a digital signal with, for example, 16-bit resolution. Preferably, an autocorrelator programmed using a field-programmable gate array (FPGA) can be used in real time to calculate the autocorrelation function of the intensity signal, which can then be evaluated, for example, using a computer.

[0147] In more detail, the steps for processing the obtained analog output signal will refer to... Figure 4A Figures 4a) and 4b) are described by way of example. Thus, the photodetector used for dynamic light scattering measurements, i.e., the dynamic light scattering detector 30, can detect light emitted or scattered from a container containing the sample under study. Preferably, the analog signal obtained by the dynamic light scattering detector is converted into a digital signal, i.e., digitized into a digitized output signal, for example using an analog-to-digital converter (ADC; 31), and further transmitted to a field-programmable gate array (FPGA; 34). The FPGA 34 may include one or more correlators that perform at least one correlation operation, preferably at least one autocorrelation operation. The FPGA 34 may include a correlator 33, as exemplarily shown in Figure 4a). In this case, the FPGA preferably further includes a pulse phase detector 32, and the input signal of the correlator 33 can be switched between photon counting data obtained from the pulse phase detector 32 and digitized data processed as analog data. Alternatively, the FPGA may include two correlators 33 operating in parallel, one in analog mode and the other in photon counting mode based on the signal converted by the pulse phase detector 32, as exemplarily shown in Figure 4b. In all cases, the output of one or more correlators 33, including in the field-programmable gate array 34, is preferably transmitted to a personal computer 35 for further analysis and / or storage on a storage medium such as a hard disk.

[0148] Therefore, the step of processing the obtained analog output signal preferably further includes the following steps: digitizing the analog output signal into a digital output signal, and storing the processed digital output signal obtained from step i) or step ii) on a preferred storage medium (e.g., a hard disk).

[0149] The method according to the invention preferably includes another step: measuring fluorescence. Preferably, the fluorescence of particles in the solution contained in the sample is used for fluorescence measurement, wherein the fluorescence is preferably the autofluorescence of the particles. Measuring the fluorescence of particles in the solution (preferably autofluorescence) can be advantageous. The absence of any autofluorescence indicates that the container is missing or empty, or lacks fluorescence or autofluorescence. If this is the case, any (further) measurements, i.e., dynamic light scattering measurements and preferably further fluorescence measurements, such as nanoscale differential scanning fluorescence measurements, can be omitted. Therefore, using information about the presence or absence of autofluorescence can be beneficial in reducing the time required to characterize particles in the solution using the method according to the invention. Furthermore, information about the signal intensity and / or signal intensity pattern of the detected autofluorescence signal can be obtained. This type of information is particularly advantageous because the information thus obtained can be used, for example, to determine the location of the container containing the solution particles, which may fluoresce upon photoexcitation and / or nanoscale differential scanning fluorescence measurements, and / or to detect samples containing contaminants (e.g., gold particles) that do not fluoresce. Preferably, fluorescence is measured by providing another light source, such as an LED, transmitting light from the other light source into the container containing the sample, thereby exciting the spontaneous fluorescence of the particles, and detecting the light emitted from the container.

[0150] Preferably, another light source provides light with a wavelength of about 280 nanometers to excite the autofluorescence of particles (preferably proteins in the solution).

[0151] In this document, the term "fluorescence" refers to electromagnetic radiation, including the release of energy in the form of emitted electromagnetic radiation, such as when particles are excited by photon absorption. More specifically, particles such as proteins can exhibit autofluorescence, also known as intrinsic fluorescence, if they include, for example, at least one of three specific aromatic amino acids: phenylalanine, tyrosine, and tryptophan. The autofluorescence of proteins is predominantly tryptophan, which has a higher extinction coefficient compared to tyrosine and phenylalanine. The extinction coefficient, also known as the molar absorption coefficient, refers to the attenuation of electromagnetic radiation in a measuring medium, i.e., extinction, and is affected by the path length through the medium and the particle concentration in the solution, where scattering and absorption can lead to attenuation. The maximum absorption wavelengths of tyrosine and tryptophan are approximately 280 nm, and compared to tryptophan, the emission wavelength of tyrosine is less dependent on its position within particles such as proteins. Therefore, the excitation of autofluorescence in particles such as proteins is preferably accomplished using light with wavelengths of approximately 280 nm or 278 nm (preferably from a separate light source). For example, LEDs with a maximum intensity of about 270 to 290 nanometers, but 278 nanometers, 280 nanometers, or 285 nanometers are more suitable for improving efficiency.

[0152] refer to Figure 5 The above figure illustrates the steps for measuring the fluorescence of particles in solution in more detail, by way of example. Figure 5 As illustrated in the example in the figure above, the fluorescence of particles in a solution contained in a sample 12 within a container (e.g., capillary 11) can be measured, for example, using a fluorescence optics element 14 with a predetermined fluorescence focus 16. The position of the container and / or optics element 14 allows the fluorescence focus 16 to be located within the container for measuring the fluorescence of the sample containing particles in the solution 12. After fluorescence measurement, the container can be moved to another location so that the dynamic light scattering focus 17 of the dynamic light scattering optics element 15 is located within the container to perform a dynamic light scattering measurement, wherein the fluorescence focus and the dynamic light scattering focus are located at a predetermined x-distance 18 relative to each other. Alternatively, the position of the container containing the sample can remain unchanged during fluorescence and dynamic light scattering measurements, and the fluorescence optics element 14 and the dynamic light scattering optics element 15 are positioned relative to the container, respectively.

[0153] Therefore, the container (e.g., a capillary) is preferably moved relative to the optical elements 14 and 15. Alternatively, both measurements can be performed simultaneously.

[0154] Because the container needs to be specifically positioned relative to optical elements (such as dynamic light scattering optics) to achieve accurate and repeatable measurements, precise positioning must be ensured even when the container is moved between measurements. Therefore, the method according to the invention preferably includes further steps(s): determining the container position based on measured fluorescence, and optionally positioning the container based on both the measured fluorescence and the determined container position. Thus, the position of the sample can be accurately determined based on measured fluorescence (e.g., based on the fluorescence of particles in the container and / or the solution contained in the sample under study). This is particularly advantageous when measuring multiple samples at high throughput. In this case, manual filling of the device with capillaries is not possible, as the capillaries are typically placed in capillary supports. Furthermore, to obtain reproducible results from high-quality measurements, the measurement position in each capillary must be rapidly located. This can be achieved by determining the capillary position based on fluorescence scanning, which is preferably performed before or in parallel with dynamic light scattering measurements, preferably in parallel with dynamic light scattering measurements. Therefore, for example, 48 capillaries can be measured in approximately 2 seconds using the method according to the invention. Traditional solutions are based on detecting the scattered light signal from the sample container (such as a capillary holder). However, the signal obtained from fluorescence measurements is typically stronger than that obtained from scattered light. Therefore, using this fluorescence scanning is more advantageous because it allows for more accurate localization and, optionally, (re)positioning of the capillary compared to the conventional solutions described above.

[0155] Melting curves can be used to analyze the thermal stability of particles such as proteins. A melting curve represents the change in fluorescence as a function of temperature. More specifically, the melting point Tm, the temperature at which half of the particles denature, can be determined by the first derivative of the melting curve. Therefore, the sample can be heated by applying a temperature ramp (e.g., from 15°C to 95°C, preferably 20°C to 90°C) at a rate between 0.1°C / min and 7°C / min.

[0156] Therefore, the method according to the invention preferably includes the following steps: tempering the container over time at at least a first time point at a first temperature and at a second time point at a second temperature. Preferably, the step of tempering the container over time includes tempering the container at a tempering rate between 0.01°C / min and 30°C / min, preferably between 0.1°C / min and 10°C / min, and / or wherein the first temperature and the second temperature are between -20°C and 160°C.

[0157] The method according to the invention preferably further includes the step of performing nano-differential scanning fluorescence (nano-DSF) measurements. Performing nano-differential scanning fluorescence measurements is beneficial for analyzing conformational changes of particles (preferably proteins). Nano-differential scanning fluorescence is preferably measured by providing another light source (such as an LED), transmitting light from said other light source to a container containing the sample, and detecting the light emitted by the container. Preferably, the other light source provides light with a wavelength of about 280 nanometers for exciting particles (preferably proteins) in the solution. The other light source used to transmit light to the container to perform fluorescence measurements (e.g., for determining the position of the container and performing nano-differential scanning fluorescence measurements, respectively) can be the same other light source or a different other light source.

[0158] Nanoscale differential scanning fluorescence is based on the dependence of protein emission spectra on the location of tryptophan within proteins. Tryptophan is an aromatic amino acid whose native conformation is primarily located within the protein. However, if a protein loses its native conformation due to denaturation or other reasons, the internal portion of the protein is exposed to a more polar environment. Since the emission spectrum of tryptophan depends on its environment, the loss of the protein's native conformation can lead to tryptophan exposure to a more polar environment, causing the emission maximum at approximately 325 nm in the nonpolar environment to shift to a longer wavelength range of approximately 350 nm. Therefore, environment-specific emission spectral information of tryptophan can be used to analyze conformational changes in proteins. Thus, the fluorescence of a protein can be excited at a wavelength of approximately 280 nm, and the resulting emission can be detected at approximately 350 nm and 330 nm. A quotient can be formed based on the fluorescence intensity at approximately 350 nm and 330 nm, which typically increases due to denaturation. Protein denaturation can be caused by chemical and / or thermal denaturing conditions, including elevated temperatures.

[0159] Nanoscale differential scanning fluorometry measurements are preferably performed while exposing particles in solution to different temperatures, for example using a temperature ramp, so that when the container containing the particles in the solution under study is tempered over time, it has a first temperature at least at a first time point and a second temperature at least at a second time point. This is particularly advantageous for obtaining information about conformational changes of the particles in the solution under study, depending on the temperature at which the particles are exposed.

[0160] refer to Figure 5 The incorporation of nanoscale differential scanning fluorescence measurements into the method of this invention is described in more detail and by example. Figure 5As illustrated in the example in the figure above, the fluorescence of particles in a solution contained in a sample 12 within a container (e.g., capillary 11) can be measured, for example, using a fluorescence optics element 14A (e.g., a nanoscale differential scanning fluorescence optics element) with a predetermined fluorescence focus 16. The container (e.g., a heating pad / bed) can be tempered using a tempering element (e.g., a heating pad / bed) that can heat and / or cool the container (e.g., capillary), and the emission fluorescence of the particles in the solution can be measured at a given temperature using dynamic light scattering and fluorescence measurements (e.g., nanoscale differential scanning fluorescence). Therefore, the container, tempered at a first temperature, is positioned so that the fluorescence focus 16 is located within the container for measuring the fluorescence of the sample containing particles in solution 12. After fluorescence measurement, the container can be moved to another location so that the dynamic light scattering focus 17 of the dynamic light scattering optics element 15 is located within the container to perform dynamic light scattering measurements, wherein the fluorescence focus and the dynamic light scattering focus are preferably located at a predetermined x-distance 18 relative to each other. Alternatively, during fluorescence and dynamic light scattering measurements, the position of the container containing the sample can remain unchanged, and the fluorescence optics 14 and dynamic light scattering optics 15 are positioned relative to the container, respectively. As another alternative, both measurements can be performed simultaneously. After dynamic light scattering and fluorescence measurements are performed at a given temperature (e.g. Figure 5 (As shown in the middle and lower figures), for example, the temperature of the sample is increased by increasing the temperature of the tempering element 13 located close to the container.

[0161] Once the sample is tempered at the second temperature, another round of dynamic light scattering and / or fluorescence measurements can be performed as described above. Thus, the particles in the solution contained in the container can be characterized according to temperature by tempering the container containing the sample at a first temperature at at least a first time point and a second temperature at a second time point over time, and performing at least dynamic light scattering and fluorescence (preferably nanoscale differential scanning fluorescence) measurements at each time point.

[0162] Alternatively, or additionally, the method according to the invention preferably includes the step of measuring the back-reflection of a container containing the sample. The back-reflection is preferably measured by providing another light source (such as an LED), transmitting light from said other light source through the container containing the sample, and detecting the reflected light, for example, light reflected by a mirror located below the container (see...). Figure 11Therefore, based on the measured intensity of the backscattered light, the presence of a container and / or sample can be inferred, and / or information about the location of said container and / or sample can be obtained. More specifically, the backscattering intensity of the measured light (e.g., through a mirror below the expected container location) is stronger when no container is provided than when a container is provided. Furthermore, once a container is provided, the intensity of the backscattered light differs between an empty container, a container containing fluid but without the particles under investigation, and / or a container containing contaminants, and a container containing the sample according to the invention. Therefore, measuring backscattering is advantageous for detecting containers and / or samples and / or obtaining information about their precise locations. Moreover, if a sample is provided and at least partially aggregated, the aggregated particles contained in the sample will scatter some light in directions other than the receiving cone of the photodetector. The stronger the aggregation, the weaker the detected signal. Therefore, information about the presence and / or intensity of particle aggregation can be obtained by measuring backscattering, which is beneficial for reducing the time required to perform the method according to the invention and improving its accuracy.

[0163] Preferably, another light source provides light with a wavelength of about 385 nm to excite particles (preferably proteins) in the solution. Preferably, the light with a wavelength of about 385 nm is further detected for backreflection measurements. The preferred LED emitting the desired wavelength is, for example, an LED with a maximum intensity of about 385 nm. The other light source used to transmit light to the container to perform fluorescence measurements, nanometer differential scanning fluorescence measurements, and / or backreflection measurements can be the same or a different light source. Measuring backreflection is advantageous for studying the colloidal stability of particles (such as proteins) in solution.

[0164] For analyses using back reflection, a light beam (e.g., with a wavelength of 385 nm) is used to illuminate a sample containing particles in a solution, held by a sample holder. The beam is reflected by the material of a tempering element beneath the container as the light source passes over the sample again. For example, a tempering element made of silicon can be used for tempering and to provide a reflective surface. Since larger particles (including particle aggregates) scatter light more strongly than smaller particles, the attenuation of reflected light is greater in the case of larger particles. Therefore, the intensity of the reflected light provides qualitative information about the presence of larger particles (including particle aggregates) in the sample under study.

[0165] According to a preferred embodiment, the tempering element is made of silicon (Si), which offers certain advantages, such as a smooth surface, good thermal conductivity, good mechanical and chemical stability, and good reflectivity. Preferably, the container to be tempered is in direct contact with the tempering element. Silicon also does not exhibit autofluorescence, as autofluorescence would interfere with determining the location of the container by fluorescence, as is preferably accomplished in this invention. The silicon tempering element can, for example, be formed from a portion of a silicon wafer. According to another embodiment, the tempering element can be used in a manner in which the surface in contact with the container is modified, for example, by using an interfering coating, to reflect light at wavelengths used for backscattering and / or fluorescence measurements, and not reflect (e.g., absorb and / or transmit) light used for dynamic light scattering measurements. For example, it is preferred to reduce reflectivity at about 405 nm (preferably 400 nm ± 10 nm).

[0166] Furthermore, by measuring changes in reflected light intensity, such as during a temperature ramp, the onset of aggregation (Tagg) can be determined, indicating the temperature at which the size of particles in the solution increases due to the onset of aggregation. Therefore, as previously mentioned, it is preferable to measure back reflection while tempering the container over time to obtain information about colloidal stability and temperature-induced aggregation of particles in the solution.

[0167] Furthermore, as explained in more detail below, there are advantageous effects due to the combination of backscattering and dynamic light scattering. Dynamic light scattering and backscattering can complement each other well and can greatly extend the measurement range of the apparatus and method according to the invention. In particular, dynamic light scattering has limited use for “dirty” samples (e.g., cloudy ones), while backscattering works well for such samples. On the other hand, the sensitivity of backscattering is insufficient to detect small particles with high quality, while high-quality measurements can be obtained from dynamic light scattering for such samples. In addition, sometimes pharmaceutical formulations contain many PEG molecules used to stabilize the actual active substance, but these molecules produce such strong scattering signals that dynamic light scattering becomes saturated and therefore “blind.” Therefore, backscattering measurements can be compared to “turbidity” measurements and are very robust: if the aggregated particles are large and / or frequent (e.g., sample failure and / or appear “milky”), dynamic light scattering optics may saturate and become less useful, while backscattering optics can still perform measurements with high quality. This is impossible with any other apparatus and allows for a very large measurement range.

[0168] As an example: if studying antibodies using capillary tubes and starting at 20°C, dynamic light scattering, nanodiffusion fluorescence, and back reflection can be measured with high quality, even when the sample is heated to the "inflection temperature" (i.e., "melting temperature") of a specific antibody domain at a rate of 1°C per minute. Once the melting temperature is reached, the antibody begins to aggregate. However, up to 5°C above the "inflection temperature," dynamic light scattering, nanodiffusion fluorescence, and back reflection can still be measured with high quality. However, from 5°C above the "inflection temperature," the antibody aggregates so strongly and begins to precipitate that the dynamic light scattering optics may become saturated / blinded, while nanodiffusion fluorescence and back reflection produce interpretable data. However, the back reflection optics may give a scattered light signal due to their lower sensitivity, but can still measure the turbidity of the sample with high quality. Therefore, a combination of these measurements performed on the same sample at essentially the same time can provide enhanced sample characterization.

[0169] The size of particles (such as proteins) in the solution can be further determined using, for example, static scattering light measurement, which has the advantage of a wide measurement range. For static scattering light measurement, light from a monochromatic light source (such as a laser) is transmitted into a container containing the sample under study, and the light scattered by particles in the solution contained in the sample is measured by at least two photodetectors at different angles. Therefore, the method according to the invention preferably further includes the step of providing another photodetector and using said other photodetector to measure the static scattering light of the container containing the sample, preferably at an angle to a longitudinal axis of the container. in Preferably between 10 degrees and 150 degrees, more preferably between 10 degrees and 60 degrees.

[0170] The method according to the invention is preferably applied to multiple samples of a solution containing particles under study to measure the properties of the particles in the solution at high throughput. Therefore, it is preferable to provide multiple containers, each containing a sample of multiple particles in the solution, and to measure multiple properties of the multiple particles in the solution in each container as described herein. Thus, the method according to the invention can be used to characterize multiple particles in a solution at high throughput using multiple containers, wherein, under another experimental condition, such as a different detergent concentration, but each container contains the same solution particles. Alternatively or additionally, the method according to the invention can characterize different particles in the solution at high throughput by providing multiple containers, each containing a sample of different particles in the solution.

[0171] Furthermore, when the method according to the invention, as described above, is applied to multiple containers, preferably i) fluorescence measurement of each container is followed by dynamic light scattering measurement of each container; or ii) dynamic light scattering measurement of each container is followed by fluorescence measurement of each container; or iii) fluorescence measurement and dynamic light scattering measurement are performed on one of the multiple containers, and then fluorescence measurement and dynamic light scattering measurement are performed on another of the multiple containers. Preferably, fluorescence measurement is performed before DLS measurement. This is particularly advantageous because information from fluorescence measurement can be used to accurately locate and optionally (re)locate the individual containers for dynamic light scattering measurement, thereby ensuring high-quality results with reproducible dynamic light scattering measurement. More preferably, fluorescence measurement and dynamic light scattering measurement are performed simultaneously on each of the multiple containers.

[0172] Example of a preferred embodiment

[0173] Preferably, the method according to the invention includes at least the steps described in detail above: detecting a fluorescence signal of a container, determining the exact position of the container, optionally repositioning the container relative to a light source and / or optical elements, performing a dynamic light scattering measurement at a first temperature, preferably further performing a nanometer differential scanning fluorescence measurement, tempering the container, detecting a fluorescence signal of the container, determining the exact position of the container, optionally repositioning the container relative to a light source and / or optical elements, performing a dynamic light scattering measurement at a second temperature, preferably further performing a nanometer differential scanning fluorescence measurement, etc. Therefore, three-dimensional structures, particle size distributions, and particle aggregation, as well as their dependence on parameters such as temperature, can be evaluated in a relatively inexpensive, rapid, reproducible, and accurate manner. Furthermore, the method according to the invention includes at least the aforementioned steps, preferably performed using more than one container. This has the advantage of obtaining accurate measurement results with high throughput.

[0174] The following will refer to Figure 11 A preferred example of the measurement cycle according to the invention is described in more detail. Specifically, taking the use of a capillary as a container as an example, a sample containing the particles under study is drawn into the container by capillary force. The capillary is then placed on a sample carrier / support. According to another embodiment, multiple capillaries are mounted on the support, and the multiple capillaries can be simultaneously filled with different samples by inserting one end of each capillary, for example, by simultaneously inserting the multiple capillaries into corresponding holes of a porous plate. According to a preferred embodiment, 1 to 48 capillaries are provided on the sample carrier / support. Further preferably, these capillaries / containers (see Figure 11The temperature of the capillaries can be controlled very precisely to a specific temperature. For example, a tempering device can be provided so that all capillaries at a temperature of, for example, 75°C are "homogeneous" within a temperature range of 75°C + / - 0.2°C. This means that all, every single capillary, has almost the same temperature, and therefore these capillaries can be compared with each other. This precise temperature control of multiple capillaries / containers allows, for example, isothermal measurements or temperature ramp measurements. For example, in isothermal mode, all samples are maintained at a single temperature with an accuracy of + / - 0.5°C [please specify, for a specific time (e.g., more than 1 minute, more than 2 minutes, more than 1 hour, more than 1 day, up to 7 days) with an accuracy of + / - 0.2°C]. Precise temperature control also allows for temperature ramp modes, for example, tempering samples in multiple capillaries from 20°C to 95°C at a heating rate of 1°C per minute. At ambient temperature, the temperature of all capillaries is substantially the same. However, with this invention, all capillaries can be maintained at a temperature of specified accuracy at all temperatures along the temperature ramp.

[0175] Preferably, the measurement is performed such that the sample carrier moves in the x-direction so that signals from all containers can be continuously detected by corresponding optical elements, for example, for dynamic light scattering and / or nanoscale differential scanning fluorescence measurements. Alternatively, the light source, detector, and / or corresponding optical elements can be moved relative to the sample carrier. According to another embodiment, the carrier and the light source, detector, and corresponding optical elements can be moved. In other words, preferably there is a relative movement between the containers and the detection system. When the sample carrier moves in the positive x-direction, nanoscale differential scanning fluorescence and backscattering measurements can be performed, in which all capillaries are scanned without stopping the sample carrier. When the sample carrier is withdrawn, i.e., moving in the negative x-direction, the corresponding dynamic light scattering measurements can be performed, during which the sample carrier stops for the corresponding measurements. If not all capillaries placed on the sample holder are to be measured, a subset of capillaries can be selected and then considered as optics that can detect the scattering intensity of the individual samples in the capillaries.

[0176] For each measurement, container positioning is crucial for obtaining high-quality results. Furthermore, determining the measurement location within the container can also improve measurement quality and sensitivity. As mentioned above, the container position can be measured using various methods, with the use of autofluorescence signals from the particles and / or the container being preferred. [Reference] Figure 10The preferred method is described in further detail. For example, to determine the thermal stability of particles (e.g., proteins in solution), a sample containing proteins in solution can be heated from 20°C to 95°C (“temperature ramp”). Especially when there is more than one container containing the sample to be analyzed, the sample can be placed in a sample holder on a sample platform that can be moved within each measurement cycle, i.e., measured at at least one predetermined temperature of the temperature ramp, so that all samples can be measured. During such a temperature ramp, the position of the container containing the sample may slightly shift due to the expansion of the sample holder caused by temperature. Therefore, the center of the container in the x-direction is preferably determined and optionally (re)positioned based on the fluorescence signal (e.g., the maximum value of the detected fluorescence signal and / or the maximum value of previous fluorescence measurements) for measurement and / or each measurement cycle (e.g., dynamic scattered light (DSL) and / or nanoscale differential scanning fluorescence measurement). However, the measurement position of the container (preferably a capillary) may vary from measurement to measurement due to inaccurate measurements and / or weakly fluorescent samples. On the other hand, the z-position is preferably set before measurement, and during the temperature rise from 20°C to 90°C, the z-position typically changes by only about ±20 micrometers due to the expansion of the sample holder. If the measurement position in the x-direction deviates from the center of the container by more than a predetermined value, the measurement may be affected by the scattered signal from the container wall. Furthermore, depending on the measurement position, light may refract differently due to the curvature of the container wall; therefore, deviations in the measurement position may affect the measurement angle, thereby affecting the particle characteristics to be measured (e.g., particle attenuation rate). Therefore, measurements can be taken at certain points within the container, the so-called "measurable area of ​​the container" (within...). Figure 10 The green area (represented by the white circle symbolizing the measurement location) is used to correctly determine the sample's attenuation rate. This area depends on the correct focusing of the sample. If light is strongly refracted by the curvature of the container wall, to the point that the detector can no longer detect the illuminated area, different attenuation rates can be observed for the same sample. In this case, the recorded autocorrelation function mainly contains interfering signals (e.g., ambient light or electronic noise). Furthermore, the capillary wall at the top of the capillary strongly affects the scattered signal (purple dots at low z-values), which can cause artifacts, resulting in meaningless data. Therefore, as... Figure 10 As shown, the z-position (low z-value) at the top of the container is most favorable for obtaining accurate results. Therefore, using fluorescence scanning is advantageous for precise localization and, optionally, (re)localization of the container to obtain reproducible and accurate measurement results.

[0177] In addition to the determination mentioned above, it is also preferable to find an optimal location within the container, i.e., a point within the container where the best signal-to-noise ratio can be obtained. For example, measurement locations near the container wall may amplify noise.

[0178] For example, a preferred capillary with an inner diameter of 500 micrometers is used, but the size (e.g., diameter) of the optimal measurement point is preferably only about 50 micrometers. An accuracy of 10 micrometers is achieved through autofluorescence measurement and the use of a strong focusing lens.

[0179] By performing multiple dynamic light scattering measurements at different locations within the capillary, such as using a weakly scattering sample, a preferred location with the optimal signal-to-noise ratio (SNR) can be found. Preferably, the most preferred location is the one where the autocorrelation function has the highest SNR (SNR = (acf amplitude) / (sum of squared fitted residuals)). For example, using a solution containing 2 mg / mL lysozyme, a typical SNR of 50 is obtained over a measurement duration of 200 ms. For example, when using lysozyme, the typical SNR is 50.

[0180] In a preferred embodiment, the method according to the invention includes the following steps: i) providing a container containing a sample of particles in the solution under study; ii) transmitting light from a first provided light source to the container; measuring fluorescence emitted by the container and / or the sample contained therein; determining the position of the container relative to a first optical element containing the first light source based on the detected emitted light; determining the position of the container relative to a measurable region of the first optical element based on the detected emitted light; optionally (re)positioning the container relative to the first optical element; and performing a fluorescence measurement if information obtained from the detected emitted light (e.g., signal intensity) indicates the presence of particles under study; iii) transmitting light from a second provided light source to the container; and performing a dynamic light scattering measurement if information obtained from the detected emitted light (e.g., signal intensity) in step ii) indicates the presence of particles under study; and iv) determining characteristics of the particles in the solution under study, such as particle size distribution and / or particle presence and / or particle aggregation, based on information obtained from the fluorescence and dynamic light scattering measurements performed in steps ii) and iii). The method may also include (e.g., prior to step ii) tempering the container in step iv). Furthermore, the step of measuring fluorescence preferably refers to the step of measuring autofluorescence by performing a nanoscale differential scanning fluorescence method. Additionally, the method can be repeatedly performed by alternately performing steps ii) and iii) and / or steps iv), ii) and iii). Alternatively or additionally, the step of measuring back reflection and / or static light scattering (e.g., using a first optical element) may be further included in the method according to the invention. It is noteworthy that the first and second optical elements can be the same or different.

[0181] In a second aspect, the present invention relates to an apparatus for measuring the properties of particles in a solution, wherein the properties of particles in the solution are preferably measured according to the method of the present invention described above. Therefore, the apparatus according to the present invention is preferably used to perform the method according to the present invention and / or the method according to the present invention is preferably performed using the apparatus of the present invention.

[0182] The apparatus according to the invention includes a means for receiving at least one container containing a sample of the plurality of particles in solution, preferably for receiving 0.1 to 15 μL of the plurality of particles, more preferably for receiving 1 to 15 μL of the plurality of particles, and even more preferably for receiving 8 to 12 μL of the plurality of particles. The apparatus may include, for example, a sample holder, such as a capillary holder or a microplate holder.

[0183] The device according to the invention further includes a monochromatic light source (preferably a laser) and a photodetector (preferably a photomultiplier tube, a silicon photomultiplier tube, or an avalanche photodiode photon counting detector).

[0184] The apparatus according to the invention also includes means for performing dynamic light scattering measurements. The means may include, for example, a field-programmable gate array, a pulse phase detector, a correlator, an analog-to-digital converter, and lenses, such as collimating lenses and / or objectives (or reflecting lenses).

[0185] According to the invention, an apparatus, such as one for performing dynamic light scattering measurements, can be included in a dynamic light scattering optical element. The dynamic light scattering optical element may further include a monochromatic light source, a photodetector, and / or some or all of the control means. Therefore, the apparatus may include the dynamic light scattering optical element exemplarily depicted in FIG6. The dynamic light scattering optical element 15 includes a monochromatic light source 55, such as a laser, a dynamic light scattering detector 30, preferably an objective lens or reflecting objective lens 52, such as... Figure 6A As shown in the diagram. Therefore, the excitation beam 50 can be provided by a monochromatic light source 55, which can be focused by an objective lens 52, preferably such that the resulting fluorescence focal point 17 is located within the sample under study. When analyzing a sample using the apparatus and method according to the invention, particles in the solution contained in the sample scatter light emitted from the dynamic light scattering optical element 15. The emitted / scattered light thus obtained can be detected 51 by a dynamic light scattering detector 50. Specifically, the detected beam 51 can be focused and positioned by the same or another objective lens (or reflecting objective lens) 52 included in the dynamic light scattering optical element 15, so that it can be detected by a dynamic light scattering detector 30 (e.g., a photomultiplier tube or a silicon photomultiplier tube). A portion of the CAD model (cross-sectional view) of the dynamic light scattering optical element 15 is shown in the diagram. Figure 6BAs shown, the dynamic light scattering optical element 15 includes two collimating lenses 56 and one objective lens 52. The collimating lenses 56 can be used to generate light with an approximately parallel beam path from a diverging light source (e.g., scattered or emitted light from a sample under study). The collimating lenses are also advantageous for the detection beam from an excitation beam from a monochromatic light source 55 (not shown) that is focused by the objective lens 52 and / or detected by the dynamic light scattering detector 30 (not shown).

[0186] The apparatus according to the invention further includes a control device adapted to control means for receiving at least one container, control the monochromatic light source for transmitting light from the monochromatic light source to the at least one container, control the photodetector for detecting multiple signals from the at least one container, and control the means for performing the dynamic light scattering measurement.

[0187] Preferably, the apparatus according to the invention further includes means for performing a correlation operation, which is preferably an autocorrelation operation. Furthermore, the autocorrelation operation is preferably an autocorrelation logic implemented in hardware and / or software.

[0188] Preferably, the apparatus according to the invention further includes means for performing data processing operations. The data processing operations preferably include data processing logic for at least performing the following steps: processing the obtained analog output signal according to the method of the invention as described above. Furthermore, the data processing operations are preferably data processing logic implemented in hardware and / or software.

[0189] Preferably, the apparatus according to the invention further includes means for digitizing signals obtained from the photodetector, wherein the control means is adapted to control the apparatus for digitizing a plurality of signals obtained from the photodetector. The means for digitizing signals obtained from the photodetector preferably comprises a field-programmable gate array (FPGA).

[0190] Preferably, the device according to the invention further includes a single-mode optical fiber and means for transmitting monochromatic light from the monochromatic light source through the single-mode optical fiber, wherein the control means is further adapted to control the device to transmit monochromatic light from the monochromatic light source through the single-mode optical fiber.

[0191] Therefore, the device may include dynamic light scattering optical elements and optical fibers, preferably single-mode optical fibers, polarization-maintaining optical fibers, or multimode optical fibers, and more preferably single-mode optical fibers. For example... Figure 7A As shown in the example in ), the monochromatic light source 55 (e.g., a laser) is similar to the one described above. Figure 6ACompared to the dynamic light scattering optical element shown in the example, this one is not directly located in the dynamic light scattering optical element 15 shown, although it is connected to it via an optical fiber 57 that transmits monochromatic light from the monochromatic light source 55 to a collimating lens 56 located in the dynamic light scattering optical element 15. The dynamic light scattering optical element 15 includes one or more (two in this example) additional collimating lenses 56, each for focusing a detection beam 51, which is transmitted via another optical fiber 57 to a photodetector (in this example, not directly located in the dynamic light scattering optical element; 30). Therefore, Figure 7A Exemplary schemes of dynamic light scattering optical elements with one excitation and two photodetectors are described, such as photodiodes with multimode optical fibers and photomultiplier tubes with single-mode optical fibers.

[0192] Figure 7B Another example of the device is shown exemplarily. Figure 7B This shows a confocal version of the dynamic light scattering optics. (Compared to the one described above.) Figure 7A In contrast, the dynamic light scattering optics 15 also includes a beam splitter 58 for splitting the light beam (e.g., into two beams at equal ratios), each of which can be focused by a collimating lens 56. This has the advantage of requiring less space.

[0193] Preferably, the apparatus according to the invention further includes a means for measuring the fluorescence of the plurality of particles in a solution of the sample, wherein the control means is further adapted to control the apparatus for measuring the fluorescence of the plurality of particles in a solution contained in the sample.

[0194] Therefore, in Figure 7C Another example of the device is shown in the illustration. Figure 7C The combined fluorescence optics and dynamic light scattering optics confocal (x distance = 0) are depicted. (This is in contrast to the above.) Figure 7B In contrast, the dynamic light scattering optical element includes a fluorescent optical element 14, and a beam splitter 58 has a predetermined beam splitter wavelength 59, such as a low-pass 620 nm. Therefore, the detected beam 51 is split into two beams, with only light having a wavelength of 620 nm or less passing through the fluorescent optical element 14. The advantage of this device is that it requires less space and allows for simultaneous measurement of fluorescence and scattering using the same sample.

[0195] Preferably, the apparatus according to the invention further includes a positioning device for positioning the sample containing the plurality of particles in the solution, wherein the control device is further adapted to control the positioning of the device for containing the sample.

[0196] Preferably, the apparatus according to the invention further includes a temperature control system for tempering the container over time at at least a first time point at a first temperature and at a second time point at a second temperature, wherein the control device is further adapted to: control the temperature control system for tempering the container over time at at least a first time point at a first temperature and at a second time point at a second temperature.

[0197] Preferably, the apparatus according to the invention further includes a device for performing a nanometer differential scanning fluorescence measurement and / or a device for measuring back reflection, wherein the control device is further adapted to: control the device for performing the nanometer differential scanning fluorescence measurement and / or the device for measuring back reflection.

[0198] Preferably, the apparatus according to the invention further includes another photodetector; and means for performing a static scattered light measurement, wherein the control means is also adapted to: control the apparatus to perform a static scattered light measurement.

[0199] The apparatus according to the invention may further include means for improving the quality of the laser beam. Therefore, the apparatus according to the invention may include optical elements for improving the quality of the laser beam, for example, included in a dynamic light scattering optical element.

[0200] As another example of a device according to the invention Figure 7D The apparatus is illustrated exemplarily in the above-described form. Figure 7A Compared to the device shown, the laser is coupled without single-mode fiber. This has the advantages of reduced cost and power loss when coupled to fiber. Figure 7D In the example shown, a monochromatic light source is located, for example, in the dynamic light scattering optical element 15. Thus, a design example of a dynamic light scattering optical element with a free-space coupled light source is shown.

[0201] The apparatus according to the invention may include another means that improves measurement quality, such as a dichroic filter, a polarizing filter, an aperture for reducing stray light, and / or a cylindrical lens for capillary astigmatism correction. Therefore, in Figure 7E Another example of the apparatus is illustrated in [reference needed], wherein a cylindrical lens 64 for capillary astigmatism correction is positioned between an aperture 63 for reducing stray light and an objective lens 52. Thus, a beam emitted from a monochromatic light source 55 can pass through a collimating lens 56, then through an objective lens 52, a cylindrical lens 64, and then through an aperture 63, for example, before being scattered by the sample. The scattered light, after passing through the dynamic light scattering optics 15, particularly the aperture 63, the cylindrical lens 64, the objective lens 52, and then at least one more, for example, two collimating lenses 56, can be detected by a photodetector 30.

[0202] like Figure 7E As further exemplarily shown in the diagram, the photodetector 30, such as a dynamic light scattering detector, may include a dichroic filter 60, for example, with a bandpass of 405 / 5 nm to block fluorescence, and / or a polarizing filter 61. This is particularly advantageous for weakly scattering samples that exhibit autofluorescence (bandpass) or particles with a high aspect ratio (polarizing filter).

[0203] Figure 7F Another example of the device is described in [reference to a document], showing a design of a dynamic light scattering optics element with two dynamic light scattering arms (detection and excitation) and a confocal fluorescence optics element for simultaneous measurement at a single point. In this design, the dynamic light scattering optics element 15 includes a fluorescence optics element 14 and collimating lenses 56, for example, two collimating lenses 56 in series, without objectives, for guiding light from a monochromatic light source 55 to the sample and guiding emitted light from the sample to the photodetector (3), respectively. In this design, the second collimating lens 56 focuses the excitation in the direction in which the respective beams pass through the two collimating lenses 56 in series. This design of the dynamic light scattering optics element is advantageous because a larger scattering angle, i.e., the angle between the laser and the detector, can be achieved in this way, and light scattering can also be reduced.

[0204] Figure 7G Another preferred example of the device is described in (). Figure 7G This illustrates the design of a dynamic light scattering optical element with two arms (detection and excitation), without the like Figure 5 The objective lens in F) also lacks the fluorescence optical element 14. This is particularly advantageous because fluorescence and scattering measurements of the same sample can be obtained simultaneously.

[0205] The methods according to the invention are equally applicable to containers, samples, particles and their properties, samples, containers, monochromatic light sources, photodetectors, time-varying tempering, (auto)correlation operations, field-programmable gate arrays, and the transmission, propagation, emission and detection of light, as well as measurements including dynamic light scattering measurements, fluorescence measurements, nanoscale differential scanning fluorescence measurements, backscattering measurements and static scattered light measurements, as described above. Furthermore, other features of such measurements may also be described above. Therefore, the advantageous features and characteristics of the first aspect of the invention will be regarded as advantageous features of the second aspect of the invention, and vice versa.

[0206] Other aspects and advantages of the invention will be described in the examples below, which are for illustrative purposes and not for limitation.

[0207] The entire contents of each publication, patent, patent application or other document cited in this application are incorporated herein by reference.

[0208] Example

[0209] This document describes the methods and materials used in this disclosure; other suitable methods and materials known in the art may also be used. The materials, methods, and examples described are illustrative only and are not intended to be limiting.

[0210] The immune system recognizes and defends against pathogens (such as bacteria, viruses, and toxins) through humoral and / or cell-mediated immune responses. Humoral immune responses involve the production of specific proteins, including pathogen-specific antibodies against pathogen-specific antigens. Antibodies circulate as soluble proteins in plasma and lymph and are associated with the recognition, neutralization, agglutination, and precipitation of pathogenic antigens. Due to their specificity to pathogen antigens and their fundamental role in immunity, antibodies are highly relevant in the diagnosis and treatment of diseases.

[0211] However, studying antibodies and developing antibody-based therapies and pharmaceutical compositions is a complex task in terms of their function and stability. For example, antibody instability can be caused by chemical modifications, changes in thermal stability of conformation (especially reductions), and changes in colloidal stability (especially reductions). Any of these aspects can affect the efficiency and / or safety of therapeutic and / or diagnostic antibodies. Therefore, extensive analyses have been conducted to comprehensively characterize and optimize antibody stability.

[0212] In this context, an experiment was conducted to simulate a typical experiment to analyze and optimize the stability of proteins in solution (e.g., a (drug or diagnostic) antibody composition consisting of IgG and buffer).

[0213] The starting material was a commercially available therapeutic antibody formulation of immunoglobulin G (IgG) (HyQvia, Baxalta Innovations GmbH, Vienna, Austria). The IgG antibody was diluted using different buffers (sodium acetate and HEPES). The initial mass concentration of the HyQvia product was 100 mg protein per mL of injection solution. The antibody composition was diluted 50-fold using sodium acetate or HEPES buffer to a final mass concentration of 2 mg protein per mL of buffer. The diluted formulation was centrifuged at 14,000 times the gravitational acceleration for 15 minutes to remove insoluble macroscopic and microscopic particles. Capillaries were loaded with the diluted and centrifuged antibody solution by placing each capillary in the protein solution and allowing it to fill itself with protein solution through capillary force. A thermal deconvolution experiment was performed, including near-simultaneous measurement of fluorescence shift and particle size. The heating rate for inducing thermal deconvolution was 1 °C / min. Dynamic light scattering was measured at 500 ms per capillary.

[0214] Figure 8 illustrates experimental results designed to determine at what temperature IgG exhibits a fluorescence shift (always the upper subplot) and how the distribution or size of the detected particles (always the lower subplot) changes with temperature. By measuring the size or distribution of the protein in solution (always the lower subplot), combined with measurements of protein conformational stability (always the upper subplot) and colloidal stability of the protein particles, these two key parameters can be precisely analyzed. Since the combined assays can also be performed in parallel on up to 48 samples, many different conditions can be tested and efficiently evaluated, such as when the protein in the study is dissolved in different buffers. Therefore, the advantage of combining dynamic light scattering and nanodifference scanning fluorescence measurements is that by combining protein conformation and colloidal stability for combined protein characterization, the development of diagnostic or therapeutic proteins can be accelerated.

[0215] Figure 9 An overview of how dynamic light scattering can determine particle parameters is presented, including the mathematical parameters of the mean cumulative radius, mean cumulative polydispersity index, and autocorrelation function under the particle parameters. Data from the total sample, bovine globulin (BGG), and IgG from the buffer shown in Figure 8 are presented. The temperature was maintained at 25°C during the measurements. Five measurements were recorded per capillary, each for a dynamic light scattering measurement time of 5000 ms. The dots in the figure represent the mean radius determined by the accumulator method. The gray areas represent the particle size distribution detected in the corresponding capillary. It can be seen that differences between certain particle parameters of BGG and IgG can be identified. This experimental setup is beneficial for assessing the homogeneity of a given sample; for example, the narrower the particle size distribution and the smaller the mean cumulative polydispersity index, the more homogeneous the sample under study.

[0216] Figure 15 An application example of antibody buffer screening and antibody candidate selection using NIST mAb reference material RM 8671 is illustrated, designed to evaluate the performance of a method for determining the physicochemical and biophysical properties of monoclonal antibodies. It also provides a representative test molecule for developing novel techniques for the characterization of therapeutic proteins.

[0217] The test sample consisted of 1 mg / mL NISTmAb in saline. The horizontal axis represents temperature, ranging from approximately ambient temperature to above 100°C. The first graph (from top to bottom) shows the first derivative at 350 nm as a function of temperature. The peak between 60°C and 75°C represents the first transition, the peak between 75°C and 90°C represents the second transition, and the peak between 90°C and 100°C represents the third transition (more pronounced in this channel for this antibody). Each transition corresponds to the unfolding of a single protein domain. The second graph shows the turbidity from backscattering, with the starting point marked as a vertical line. The third graph shows the scattering from dynamic light scattering (mean scattering intensity), with the starting point again marked as a line. The fourth graph (bottommost graph) shows the cumulative radius in nanometers, with the starting point also marked as a line.

[0218] IgG / antibodies (represented by NIST mAb) contain three protein domains: CH2, Fab, and CH3. In particular, the "arms" of the Y-shaped IgG molecule contain variable antigen-binding sites and are therefore often referred to as the Fab region (segment, antigen-binding). The "legs" of the Y are responsible for the antibody's immunomodulatory properties and are called the Fc region (segment, crystallizable). The Fc region can be further subdivided into the CH2 and CH3 domains (domains 2 and 3, respectively, of the heavy chain constant portion). In thermal unfolding experiments, three independent unfolding events are typically observed in the unfolding curve, representing the three unfolding events of the CH2, Fab, and CH3 regions. Specifically, the CH2 region unfolds first (at the lowest temperature), followed by Fab and CH3. Depending on the exact molecular structure of IgG, not all three regions will show as separate unfolding events. In some cases, two unfolding events may overlap or even occur simultaneously, making clear separation impossible. Unfolding of the large Fab corresponding to the second transition usually results in an increase in size, which is interpreted as co-unfolding (cumulative radius starts at the lowest point), but without aggregation. The unfolding of CH3, corresponding to the third transformation, leads to aggregation, which is visible as a further increase in size, but also leads to the onset of scattering (from the average intensity of dynamic light scattering) and turbidity (from back reflection). Figure 16 and Figure 15 Similar, but the sample included 1 mg / mL NISTmAb in 25 mM sodium acetate at pH 4.

[0219] The first graph (top) shows the first derivative with temperature at 350 nm. The peak between 50 and 60 °C represents the first transition, and the peak between 70 and 80 °C represents the second transition (this antibody shows a more pronounced transition in this channel).

[0220] Each transition corresponds to the unfolding of a single protein domain. The second plot shows the turbidity from backscattering. The third plot shows the scattering (average scattering intensity) from dynamic light scattering. The fourth plot (bottommost plot) shows the cumulative radius in nanometers, with the starting point marked as a straight line. Fab unfolding leads to an increase in size, which is interpreted as co-unfolding. No aggregation occurs. In particular, this is a good example where the initiation in dynamic light scattering is not the initiation of aggregation. Therefore, in the absence of observed aggregation, this initiation should match the onset temperature (Tonset) (for single-domain proteins), but may be higher for multi-domain proteins, and correspond to the unfolding of the domain that causes the largest change in Rh. In this example, more details about the protein unfolding mechanism can be obtained by monitoring the combination of dynamic light scattering and nDSF.

[0221] Figure 17 Similar to Figure 15 However, the sample contained 1 mg / mL of NIST monoclonal antibody in 25 mM sodium acetate + 130 mM NaCl at pH 4. The first plot (top) shows the first derivative with temperature at 350 nm. The peak between 45 °C and 60 °C represents the first transition, the peak between 60 °C and 75 °C represents the second transition, and the peak between 75 °C and 90 °C represents the third transition (this antibody is more pronounced in this channel). Each transition corresponds to the unfolding of a single protein domain. The second plot shows the turbidity from backscattering. The third plot shows the scattering from dynamic light scattering (mean scattering intensity), where the starting point is marked as a straight line. The fourth plot (lowest plot) shows the cumulative radius.

[0222] The unfolding of Fab, corresponding to the second transformation, results in an increase in size, which is interpreted as co-unfolding. The unfolding of CH3, corresponding to the third transformation, results in slight aggregation, which is visible at the start of scattering and not found in turbidity.

[0223] These three examples demonstrate that the corresponding results can be used to identify the problem domain of candidate molecules and provide guidance on where to begin further structural optimization. Alternatively, these results can be used to identify promising buffer conditions.

[0224] Figure 18 This is an example used to understand aggregation paths. Sample and Figure 15 The sample used in the example is the same; the test sample contains 1 mg / mL NISTmAb in physiological saline. It should be noted that... Figure 15 The upper limit temperature is above 100℃, while Figure 19 The upper limit temperature is approximately 110℃. Therefore, Figure 15 The first graph (from above) shows the first derivative with temperature at 350 nm. Figure 18 The second curve is also similar. Figure 15 The last graph shows the cumulative radius. Figure 18 The third plot shows the size distribution with temperature. In the third plot, after the CH3 domain expands, the random aggregation appears as a speckled pattern. The expansion of the Fab only leads to an increase in size (the initial accumulation radius matches the second transition in nanoscale differential scanning fluorescence).

[0225] Figure 18 This is yet another example for understanding aggregation pathways. The test sample contained 2 mg / mL BSA (Pierce™ ampoules (Bovine Serum Albumin Standard Ampoules, Thermo Fisher Scientific, Rockford, USA)). Ordered aggregation can be deduced from the graphs shown there. In particular, in the second (middle) curve showing the cumulative radius of ta, a steady increase in size distribution can be seen upon unfolding, which can be deduced from the first (top) curve showing the ratio signal. The second curve only shows the average size (cumulative radius) of all particles, while the third curve shows the size distribution (radius distribution). In the third curve, a steady increase in size can be seen upon unfolding. Compared to... Figure 18 In contrast to the speckle pattern in the size distribution, a steady increase in the size distribution indicates aggregation in an ordered manner.

[0226] Therefore, combining fluorescence readings with dynamic light scattering helps to understand aggregation pathways (random or structured aggregation). Similarly, natural and non-natural aggregations can be distinguished by comparing the unfolding transitions and size distributions at different temperatures within the fluorescence signals. The dot pattern preceding the midpoint of the transition will indicate aggregation originating from a natural state.

[0227] Further preferred embodiments of the present invention are described in the following aspects.

[0228] 1. A method for measuring the properties of multiple particles in a solution, characterized in that the method comprises the following steps:

[0229] A container is provided for a sample containing the plurality of particles in a solution, wherein the sample has a volume preferably between 0.1 μL and 15 μL;

[0230] Provides a monochromatic light source and a photodetector; transmits light from the monochromatic light source to the container containing the sample; and

[0231] The photodetector is used to detect light emitted from the container; and a dynamic light scattering measurement is used to determine multiple characteristics of the plurality of particles in the solution contained in the sample.

[0232] For example, a monochromatic light source is preferably provided for dynamic light scattering measurements. A laser is preferably used for dynamic light scattering measurements. Further preferably, an additional light source is provided for fluorescence measurements. For example, an LED emitting light at approximately 280 nanometers is preferably provided. Further preferably, an additional light source is provided for backscattering measurements.

[0233] For example, an LED with a wavelength of 385 nanometers can be used.

[0234] Depending on the provided light source and the measurement performed, dynamic light scattering measurements are preferably performed using a photodetector. It is also preferred to provide an additional photodetector for backscattering measurements, preferably a detector for detecting the wavelength of the light source used for backscattering measurements, for example, for detecting light at approximately 385 nm. It is also preferred to provide at least one, preferably two, additional photodetectors for one or more fluorescence measurements. Preferably, the detection light for fluorescence measurements includes a wavelength longer than the light used to excite fluorescence. For example, one or two photodetectors can be used to detect light at 330 nm and / or 350 nm. Figure 11 The image shows a preferred example of a light source and a photodetector.

[0235] 2. The method of claim 1, wherein the sample has a volume between 0.1 μL and 15 μL, preferably between 1 μL and 15 μL, and more preferably between 8 μL and 12 μL.

[0236] 3. The method as described in aspect 1 or 2, wherein the light from the monochromatic light source is coherent and preferably has a wavelength between 350 nm and 500 nm, more preferably 405 nm, 445 nm or 488 nm.

[0237] 4. The method as described in any one of aspects 1 to 3, wherein the monochromatic light source is a laser, preferably a diode laser, and more preferably a diode laser selected from the group consisting of: frequency-stabilized diode lasers, DPSS lasers, PPLN frequency-doubled diode lasers, frequency-doubled DPSS lasers, diode-pumped fiber lasers, frequency-doubled diode-pumped fiber lasers, and diode-pumped upconversion fiber lasers.

[0238] 5. The method as described in aspect 4, wherein the coherence length of the laser is at least 0.1 mm.

[0239] 6. The method as described in aspect 4 or 5, wherein the power of the laser is between 1 milliwatt and 200 milliwatts, preferably between 10 milliwatts and 180 milliwatts, more preferably between 50 milliwatts and 150 milliwatts, and even more preferably between 70 milliwatts and 120 milliwatts, for example at 100 milliwatts, and the laser is preferably a continuous wave (CW) laser, and preferably not a pulsed laser.

[0240] 7. The method as described in any one of aspects 4 to 6, wherein the monochromatic light is transmitted from the monochromatic light source via a single-mode fiber of the laser wavelength and preferably via a laser wavelength polarization-maintaining single-mode fiber.

[0241] 8. The method as described in any one of aspects 1 to 7, wherein the light emitted by the monochromatic light source is perpendicular to a longitudinal axis of the container. The material is delivered to the container at one angle, wherein... The temperature range is between 0 and 45 degrees Celsius.

[0242] 9. The method of aspect 8, wherein the light detected by the photodetector is at an angle to a longitudinal axis of the container. An angle emanates from the container, wherein The range is between 0 and 45 degrees, where The value is preferably the same as The values ​​are the same.

[0243] 10. The method of aspect 9, wherein an angle exists between the light transmitted from the monochromatic light source to the container and the light emitted from the container and detected using the photodetector. It is between 0 degrees and 150 degrees, preferably between 10 degrees and 150 degrees, and more preferably between 10 degrees and 60 degrees.

[0244] 11. The method of any one of aspects 1 to 10, wherein the transmitted monochromatic light is focused in the container containing the sample using an objective lens, wherein the light emitted from the container is preferably also focused by the objective lens, preferably wherein the objective lens has a focal length between 10 mm and 200 mm, and / or the monochromatic light transmitted therein is focused in the container having a full width at half maximum (FWHM) of a focal spot between 3 μm and 30 μm, preferably resulting in a measurement volume between 0.01 nanoliters and 0.1 nanoliters, preferably between 0.01 nanoliters and 0.02 nanoliters, more preferably about 0.016 nanoliters.

[0245] 12. The method according to any one of aspects 1 to 11, wherein the photodetector is a photomultiplier tube, a silicon photomultiplier tube, or an avalanche photodiode photon counting detector, preferably a photomultiplier tube or a silicon photomultiplier tube.

[0246] 13. The method of any one of aspects 1 to 12, wherein the dynamic light scattering measurement is obtained within 5 seconds, preferably within less than 1 second.

[0247] 14. The method as described in any one of aspects 1 to 13, wherein each sample undergoes only one dynamic light scattering measurement.

[0248] 15. The method of any one of aspects 1 to 14, wherein the dynamic light scattering measurement includes the step of performing at least one correlation operation, preferably at least one autocorrelation operation.

[0249] 16. The method of any one of aspects 1 to 15, wherein the dynamic light scattering measurement comprises the following steps:

[0250] Acquire an analog output signal from the photodetector; and process the acquired analog output signal.

[0251] 17. The method of aspect 16, wherein the step of processing the obtained analog output signal includes the following steps: digitizing the obtained analog output signal to convert it into a digital output signal, preferably by means of an analog-to-digital converter (ADC).

[0252] 18. The method of aspect 17, wherein the digitized output signal is further processed using the following steps:

[0253] i) Processing the digitized output signal into a digitized single-photon pulse signal, preferably when the intensity of the detection light emitted from the container is less than 2 million photons per second; and / or

[0254] ii) Process the digitized output signal into multiple discrete values ​​of an analog signal, preferably when the detected light intensity is higher than 2 million photons per second.

[0255] 19. The method as described in aspect 18, wherein:

[0256] The step of processing the obtained analog output signal includes step i) or step ii), wherein the time for deciding whether to process the digitized output signal into a digitized signal according to step i) or step ii) is less than 1 second, preferably a maximum of 0.05 seconds, and preferably using a field-programmable gate array, or

[0257] - It can process photon counting and discrete output signals simultaneously to satisfy the decision of whether to process according to step i) or step ii) after measurement.

[0258] 20. The method as described in aspect 18 or 19, wherein the step of processing the obtained output signal further comprises the step of:

[0259] Store the processed digitized output signal obtained from step i) or step ii); or

[0260] Store the multiple processed digital output signals obtained from steps i) and ii); and

[0261] One of the stored output signals is further processed.

[0262] 21. The method as described in any one of aspects 1 to 20, the method further comprising the following steps:

[0263] The fluorescence is measured, preferably the fluorescence of the plurality of particles in a solution contained in the material of the sample and / or the container, wherein the fluorescence is preferably the autofluorescence of the plurality of particles and / or the material of the container, and / or the back reflection of the container containing the sample is measured.

[0264] As described above, additional light sources(s) and / or additional photodetectors(s) can be used for the fluorescence and / or back reflection measurements.

[0265] 22. The method of aspect 21, further comprising the steps of: determining the position of the container based on measured fluorescence and / or measured back reflection, and

[0266] The container is selectively located based on the measured fluorescence / back reflection and the determined container position.

[0267] 23. The method as described in any one of aspect 1 or 22, the method further comprising the following steps:

[0268] The container is tempered over time at at least a first time point at a first temperature and at a second time point at a second temperature.

[0269] 24. The method of aspect 23, wherein the step of tempering the container over time at least at a first time point at a first temperature and at a second time point at a second temperature comprises tempering the container at a tempering rate between 0.01°C / min and 30°C / min, preferably between 0.1°C / min and 10°C / min, and / or wherein the first temperature and the second temperature are between -20°C and 160°C.

[0270] 25. The method as described in any one of aspects 1 to 24, the method further comprising the following(s) steps:

[0271] Perform one-nanometer differential scanning fluorescence measurement; and / or

[0272] Measure the back reflection of the container containing the sample.

[0273] 26. The method as described in any one of aspects 1 to 25, the method further comprising the steps of: providing another photodetector, and

[0274] The static scattered light from the container containing the sample is measured using the other photodetector, preferably at an angle to a longitudinal axis of the container. in Preferably between 10 degrees and 150 degrees, more preferably between 10 degrees and 60 degrees.

[0275] 27. The method of any one of aspects 1 to 26, wherein the container is a capillary and / or a porous plate, preferably a glass capillary with a circular cross-section, having an inner diameter between 0.1 mm and 1 mm, preferably between 0.15 mm and 0.5 mm, and preferably having an outer diameter between 0.2 mm and 1.2 mm, preferably between 0.65 mm and 1 mm, and preferably having a length between 5 mm and 70 mm, preferably between 32 mm and 50 mm, more preferably about 50 mm.

[0276] 28. The method of any one of aspects 1 to 27, wherein a plurality of containers are provided, wherein each container contains a sample of a plurality of particles in a solution, and wherein a plurality of properties of the plurality of particles in the solution are measured for each container in accordance with any one of aspects 1 to 27.

[0277] 29. The method as described in aspect 28, wherein:

[0278] A fluorescence measurement for each container is followed by a dynamic light scattering measurement for each container; or

[0279] A dynamic light scattering measurement for each container is followed by a fluorescence measurement for each container; or

[0280] A fluorescence measurement and a dynamic light scattering measurement are performed on one of the multiple containers, and then a fluorescence measurement and a dynamic light scattering measurement are performed on another of the multiple containers.

[0281] 30. The method as described in any one of aspects 1 to 29, wherein said plurality of characteristics are selected from the group consisting of: particle size distribution, aggregation temperature, melting temperature, transition temperature, unfolding temperature, liquid-liquid phase separation temperature (TLLPS), free folding energy, second virial coefficient (B22), particle self-interaction, colloidal stability, hydrodynamic radius, interparticle repulsive or attractive interaction (KD), solubility, long-term protein stability, and critical denaturant concentration.

[0282] 31. An apparatus for detecting multiple properties of multiple particles in a solution, preferably according to any one of aspects 1 to 30, the apparatus comprising:

[0283] An apparatus for containing at least one container, the at least one container containing a sample of the plurality of particles in a solution, preferably the at least one container being used to contain 0.1 to 15 microliters of the plurality of particles;

[0284] A monochromatic light source and a photodetector;

[0285] A device for performing a DSL measurement; and

[0286] One control device is suitable for:

[0287] Control of the means for receiving the at least one container;

[0288] Control the monochromatic light source to transmit light from the monochromatic light source to the at least one container;

[0289] Control the photodetector to detect multiple signals from the at least one container; and

[0290] Control the apparatus used to perform the DSL measurement.

[0291] 32. The apparatus of aspect 31, further comprising:

[0292] A means for performing a correlation operation, wherein the correlation operation is preferably an autocorrelation operation, and wherein the autocorrelation operation is preferably an autocorrelation logic implemented in hardware and / or software; and

[0293] 33. The apparatus as described in aspect 31 or 32, further comprising:

[0294] An apparatus for digitizing a plurality of signals obtained from the photodetector, wherein the apparatus preferably includes a field-programmable gate array, and the control device is further adapted to control the apparatus for digitizing the plurality of signals obtained from the photodetector.

[0295] 34. The apparatus as described in any one of aspects 31 to 33, further comprising:

[0296] An apparatus for measuring the fluorescence of the plurality of particles in a solution of the sample, wherein the control device is further adapted to: control the apparatus for measuring the fluorescence of the plurality of particles in a solution contained in the sample.

[0297] As described above, it is preferable to provide an additional light source for fluorescence measurement, for example, an LED with a (short or shorter) excitation wavelength (e.g., 280 nm). For fluorescence measurement, it is also preferable to provide an additional photodetector, preferably two additional photodetectors for fluorescence measurement, for example, for detecting light with a longer or longer wavelength (relative to the excitation wavelength), for example, one or two detectors for detecting light of about 330 nm and 350 nm.

[0298] 35. The apparatus according to any one of aspects 31 to 34, the apparatus further comprising:

[0299] A positioning device for positioning a sample containing the plurality of particles in a solution, wherein the control device is further adapted to control the positioning of the device for containing the sample.

[0300] 36. The apparatus according to any one of aspects 31 to 35, the apparatus further comprising:

[0301] A temperature control system is configured to temper the container over time at at least a first time point at a first temperature and at a second time point at a second temperature, wherein the control device is further adapted to: control the temperature control system to temper the container over time at at least a first time point at a first temperature and at a second time point at a second temperature.

[0302] 37. The apparatus according to any one of aspects 31 to 36, the apparatus further comprising:

[0303] An apparatus for performing a nanometer differential scanning fluorescence measurement and / or an apparatus for measuring back reflection, wherein the control apparatus is further adapted to: control the apparatus for performing a nanometer differential scanning fluorescence measurement and / or the apparatus for measuring back reflection.

[0304] As described above, it is preferable to provide an additional light source for backreflection measurements, for example, an LED with an excitation wavelength of 385 nm. Further preferably, for backreflection measurements, an additional photodetector is provided, for example, to detect the backreflected light at 385 nm.

[0305] 38. The apparatus according to any one of aspects 31 to 37, the apparatus further comprising:

[0306] Another light detector; and

[0307] An apparatus for performing a static scattered light measurement, wherein the control device is further adapted to: control the apparatus to perform a static scattered light measurement.

[0308] 39. The apparatus according to any one of aspects 31 to 38, the apparatus further comprising:

[0309] One single-mode optical fiber; and

[0310] An apparatus for transmitting monochromatic light from the monochromatic light source via the single-mode optical fiber, wherein the control device is further adapted to: control the apparatus to transmit monochromatic light from the monochromatic light source via the single-mode optical fiber.

[0311] List of reference numerals

[0312] 10: The longitudinal axis of the container, such as the longitudinal axis of a capillary tube.

[0313] 11: Containers, such as capillaries

[0314] 12: Samples containing particles in the studied solution

[0315] 13: Tempering elements, such as heating pads / beds

[0316] 14: Fluorescent optical elements

[0317] 15: Dynamic light scattering optical elements

[0318] 16: Fluorescent Focus

[0319] 17: Dynamic light scattering focus

[0320] 18: x-distance between the fluorescence focal point and the dynamic light scattering focal point

[0321] 19: Reflected beam

[0322] 20: Angle between the capillary axis and the beam being detected

[0323] 30: Photodetectors, such as dynamic light scattering detectors

[0324] 31: ADC (Analog-to-Digital Converter)

[0325] 32: Pulse Phase Detector

[0326] 33: Correlator

[0327] 34: Field Programmable Gate Array (FPGA)

[0328] 35: Personal Computer

[0329] 36: Photon counting threshold

[0330] 38: The digital output signal as a digital photon pulse signal

[0331] 39: Digital output signal of a photomultiplier tube

[0332] 50: Excitation beam

[0333] 51: Detected beam

[0334] 52: Objective lens (or reflecting objective lens)

[0335] 53: Angle between the capillary axis and the excitation beam

[0336] 53': Angle between the capillary axis and the excitation beam Represented as Spend

[0337] 54: Angle between the excitation beam and the detected beam

[0338] 55: Monochromatic light source, such as laser

[0339] 56: Collimating lens

[0340] 57: Optical fiber (single-mode, alternative to ground-maintaining polarization or multimode)

[0341] 58: Beam splitter power, e.g., 50:50

[0342] 59: Beam splitter wavelength, e.g., 620 nm low-pass wavelength.

[0343] 60: Dichroic filter, such as a bandpass filter at 405 / 5 nm, to block fluorescence.

[0344] 61: Polarizing filter

[0345] 62: Optical components for improving laser beam quality

[0346] 63: Reduce the aperture of stray light

[0347] 64: Cylindrical lenses used for capillary astigmatism correction

Claims

1. A method for measuring the properties of multiple particles in a solution, characterized in that, The method includes the following steps: A container for providing a sample containing the plurality of particles in a solution; Provides a monochromatic light source and a photodetector; transmits light from the monochromatic light source to the container containing the sample; and The photodetector is used to detect light emitted from the container; and multiple characteristics of the plurality of particles in the solution contained in the sample are determined based on a dynamic light scattering measurement. The dynamic light scattering measurement includes the following steps: Acquire an analog output signal from the photodetector; and Process the obtained analog output signal, The steps for processing the obtained analog output signal include the following: digitizing the obtained analog output signal to convert it into a digital output signal. The digital output signal is further processed using the following steps: i) Processing the digitized output signal into a digitized single-photon pulse signal when the light intensity detected by the photodetector is less than a predetermined number of photons per second; and / or ii) Processing the digitized output signal into multiple discrete values ​​of an analog signal is performed when the light intensity detected by the photodetector is higher than the predetermined number of photons detected per second.

2. The method as described in claim 1, characterized in that, The container is a capillary tube and / or a porous plate.

3. The method as described in claim 1, characterized in that, The container is a glass capillary tube having a circular cross-section, an inner diameter between 0.1 mm and 1 mm, an outer diameter between 0.2 mm and 1.2 mm, and a length between 5 mm and 70 mm.

4. The method as described in claim 1, characterized in that, The method further includes the following steps: measuring fluorescence, and / or measuring the back reflection of the container containing the sample.

5. The method as described in claim 4, characterized in that, The step of measuring fluorescence includes measuring the fluorescence of the plurality of particles in a solution contained in the material of the sample and / or the container.

6. The method as described in claim 4, characterized in that, The fluorescence is the autofluorescence of the material of the plurality of particles and / or the container.

7. The method as described in claim 1, characterized in that, The monochromatic light is transmitted from the monochromatic light source via a single-mode optical fiber of the laser wavelength.

8. The method as described in claim 7, characterized in that, The monochromatic light is transmitted from the monochromatic light source through a laser wavelength polarization-maintaining single-mode fiber.

9. The method as described in claim 1, characterized in that, The light emitted by the monochromatic light source is transmitted to the container at an angle φL to a longitudinal axis of the container, where φL is between 0 degrees and 45 degrees. The light detected by the photodetector is emitted from the container at an angle φD to a longitudinal axis of the container, where φD is between 0 and 45 degrees.

10. The method as described in claim 9, characterized in that, The value of φL is the same as the value of φD.

11. The method as described in claim 9, characterized in that, An angle φS between the light transmitted from the monochromatic light source to the container and the light emitted from the container and detected by the photodetector is between 0 and 150 degrees.

12. The method as described in claim 11, characterized in that, The angle φS is between 10 degrees and 150 degrees.

13. The method as described in claim 11, characterized in that, The angle φS is between 10 degrees and 60 degrees.

14. The method as described in claim 1, characterized in that, The transmitted monochromatic light is focused into the container containing the sample using an objective lens.

15. The method as described in claim 14, characterized in that, The light emitted from the container is also focused by the objective lens.

16. The method as described in claim 14, characterized in that, The objective lens has a focal length between 10 mm and 200 mm, and / or the monochromatic light transmitted therein is focused in the container, having a full width at half maximum (FWHM) of a focal spot between 3 μm and 30 μm, resulting in a measurement volume between 0.01 nanoliters and 0.1 nanoliters.

17. The method as described in claim 16, characterized in that, The measurement volume is between 0.01 nanoliters and 0.02 nanoliters.

18. The method as described in claim 16, characterized in that, The measured volume is 0.016 nanoliters.

19. The method as described in claim 1, characterized in that, The dynamic light scattering measurement was obtained in less than 5 seconds.

20. The method as described in claim 19, characterized in that, The dynamic light scattering measurement is obtained in less than 1 second.

21. The method as described in claim 1, characterized in that, The container has a volume between 0.1 microliters and 15 microliters.

22. The method as described in claim 21, characterized in that, The container mentioned is a glass capillary tube.

23. The method as described in claim 1, characterized in that, The method further includes the following steps: The container is tempered over time at at least a first time point at a first temperature and at a second time point at a second temperature. The step of tempering the container over time at at least at a first time point at a first temperature and at a second time point at a second temperature includes tempering the container at a tempering rate between 0.01°C / min and 30°C / min, and / or wherein the first temperature and the second temperature are between -20°C and 160°C.

24. The method as described in claim 23, characterized in that, The step of tempering the container at at least at a first time point at a first temperature and at a second time point at a second temperature over time includes tempering the container at a tempering rate between 0.1°C / min and 10°C / min.

25. The method as described in claim 1, characterized in that, The aforementioned properties are selected from the group consisting of: particle size distribution, aggregation temperature, melting temperature, transition temperature, unfolding temperature, liquid-liquid phase separation temperature, free folding energy, second virial coefficient, particle self-interaction, colloidal stability, hydrodynamic radius, repulsive or attractive interactions between particles, solubility, long-term protein stability, and critical denaturant concentration.

26. An apparatus for detecting multiple properties of multiple particles in a solution, comprising the method according to any one of claims 1 to 25, characterized in that, The device includes: An apparatus for containing at least one container, the at least one container containing a sample of the plurality of particles in a solution; A monochromatic light source and a photodetector; A device for performing a DSL measurement; and One control device is suitable for: Control of the means for receiving the at least one container; Control the monochromatic light source to transmit light from the monochromatic light source to the at least one container; Control the photodetector to detect multiple signals from the at least one container; and Control the apparatus used to perform the DSL measurement.

27. The apparatus as claimed in claim 26, characterized in that, The apparatus for containing at least one container of a sample comprising the plurality of particles in solution is configured to contain 0.1 to 15 microliters of the plurality of particles.

28. The apparatus as claimed in claim 26, characterized in that, The device further includes: An apparatus for digitizing a plurality of signals obtained from the photodetector, wherein the control device is further adapted to control the apparatus for digitizing the plurality of signals obtained from the photodetector.

29. The apparatus as claimed in claim 26, characterized in that, The device further includes: An apparatus for performing a nanometer differential scanning fluorescence measurement and / or an apparatus for measuring back reflection, wherein the control apparatus is further adapted to: control the apparatus for performing a nanometer differential scanning fluorescence measurement and / or the apparatus for measuring back reflection.

30. The apparatus as claimed in claim 26, characterized in that, The device further includes: Another light detector; and An apparatus for performing a static scattered light measurement, wherein the control device is further adapted to: control the apparatus to perform a static scattered light measurement.

31. The apparatus as claimed in claim 26, characterized in that, The device further includes: One single-mode optical fiber; and An apparatus for transmitting monochromatic light from the monochromatic light source via the single-mode optical fiber, wherein the control device is further adapted to: control the apparatus to transmit monochromatic light from the monochromatic light source via the single-mode optical fiber.

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