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US-12618848-B2 - Characterization of particles in solution

US12618848B2US 12618848 B2US12618848 B2US 12618848B2US-12618848-B2

Abstract

A method for measuring characteristics of particles in solution and to a device for performing the same, wherein the method includes the steps of providing a vessel containing a sample of the particles in solution, wherein the sample has preferably a volume between 0.1 μL and 15 μL, providing a monochromatic light source and a light detector, transmitting light from the monochromatic light source to the vessel containing the sample, detecting light emitted from the vessel with the light detector, and determining characteristics of the particles in solution in the sample based on a dynamic light scattering (DLS) measurement.

Inventors

  • Philipp Baaske
  • Jonathan Derix
  • Robert Haslinger

Assignees

  • NANOTEMPER TECHNOLOGIES GMBH

Dates

Publication Date
20260505
Application Date
20201106
Priority Date
20191108

Claims (20)

  1. 1 . Method to measure characteristics of particles in solution, said method comprising the steps of: providing a vessel comprising a sample of said particles in solution; providing a monochromatic light source and a light detector; transmitting light from the monochromatic light source to the vessel comprising the sample; detecting light emitted from the vessel with the light detector; and determining characteristics of said particles in solution comprised in the sample based on a dynamic light scattering (DLS) measurement, wherein the DLS measurement comprises the steps of obtaining an analog output signal obtained from the light detector; and processing the obtained analog output signal, wherein the step of processing the obtained analog output signal comprises the step of digitalizing the obtained analog output signal into a digitalized output signal, wherein the digitalized output signal is further processed with the step(s) of i) processing the digitalized output signal as a digitalized single photon pulse signal, in case an intensity of the light detected by the light detector is below a predetermined number of detected photons per second; and/or ii) processing the digitalized output signal as discrete values of an analog signal, in case the intensity of the light detected by the light detector is above said predetermined number of detected photons per second.
  2. 2 . Method of claim 1 , wherein the vessel is a capillary and/or multi-well plate.
  3. 3 . Method of claim 1 , the method further comprising the step of measuring fluorescence, and/or measuring back-reflection of the vessel comprising the sample.
  4. 4 . Method of claim 3 , the method further comprising the steps of determining a position of the vessel based on the measured fluorescence and/or based on the measured back-reflection, and optionally positioning the vessel based on the measured fluorescence and/or back-reflection and the determined vessel position.
  5. 5 . Method of claim 1 , wherein the light from the monochromatic light source is coherent.
  6. 6 . Method of claim 1 , wherein the monochromatic light source is a laser.
  7. 7 . Method of claim 6 , wherein the laser has a coherence length of at least 0.1 mm.
  8. 8 . Method of claim 1 , wherein the DLS measurement is obtained in less than 5 sec.
  9. 9 . Method of claim 1 , wherein the vessel has a volume between 0.1 μL and 15 μL.
  10. 10 . Method of claim 1 , wherein light from the monochromatic light source is transmitted to the vessel with an angle φL to a longitudinal axis of the vessel, wherein φL is between 0 degrees and 45 degrees.
  11. 11 . Method of claim 10 , wherein light detected with the light detector is emitted from the vessel with an angle φD to a longitudinal axis of the vessel, wherein φD is between 0 degrees and 45 degree.
  12. 12 . Method of claim 11 , wherein an angle φS between the light that is transmitted from the monochromatic light source to the vessel and the light emitted from the vessel that is detected with the light detector is between 0 degrees and 150 degrees.
  13. 13 . Method of claim 1 , wherein the transmitted monochromatic light is focused in the vessel comprising the sample using an objective lens.
  14. 14 . Method of claim 1 , wherein the light detector is a photomultiplier tube (PMT), a silicon photomultiplier (SiPM), or an Avalanche photodiode (APD) photon counting detector.
  15. 15 . Method of claim 1 , wherein the DLS measurement is performed only once per sample.
  16. 16 . Method of claim 1 , wherein the DLS measurement comprises the step of performing at least one correlation operation.
  17. 17 . Method of claim 1 , wherein the digitalized output signal is further processed with the step(s) of i) processing the digitalized output signal as a digitalized single photon pulse signal in case the intensity of the detected light emitted from the vessel is below 2 million detected photons per second; and/or ii) processing the digitalized output signal as discrete values of an analog signal in case the intensity of the detected light is above 2 million detected photons per second.
  18. 18 . Method of claim 17 , wherein the step of processing the digitalized output signal comprises either step i) or step ii), and wherein the time to decide whether to process the digitalized output signal as a digitalized output signal according to step i) or ii) is less than 1 sec or a photon counting and analog output signal can be processed simultaneously such that the decision whether to process according to step i) or step ii) can be met after the DLS measurement.
  19. 19 . Method of claim 17 , wherein the step of processing the obtained analog output signal further comprises the step(s) of storing the processed digitalized output signal obtained from step i) or step ii); or storing the processed digitalized output signals obtained from step i) and step ii); and further processing one of the stored output signals.
  20. 20 . Method of claim 1 , the method further comprising the step of tempering the vessel over time at least with a first temperature at a first time point and a second temperature at a second time point.

Description

CROSS-REFERENCE TO RELATED APPLICATION This Application is a Section 371 National Stage Application of International Application No. PCT/EP2020/081370, filed 6 Nov. 2020 and published as WO 2021/089834 A1, on 14 May 2021, in German, which claims priority to EP 19208187.5, filed on 8 Nov. 2019, and EP 20197910.1, filed on 23 Sep. 2020, the contents of which are hereby incorporated by reference in their entireties. FIELD OF INVENTION In general, the present invention relates to a method to characterize particles in a solution on the basis of dynamic light scattering. In particular, the present invention relates to a method which allows characterization of the three-dimensional structure of proteins and the change of the three-dimensional structure of proteins in a solution, preferably including their stability for example in dependence of temperature, especially in case of small sample volumes using dynamic light scattering (DLS), preferably measured for less than 1 sec, for example in combination with differential scanning fluorimetry (DSF); and to a device for performing the same. The present invention preferably provides a method and a system which provide enhanced accurate measurements of aggregation and intrinsic properties, for example folding of a particle, e.g., a protein, within a short time and a single system. BACKGROUND Determining characteristics of products in a fast and accurate manner is crucial for many applications. For example, in pharmaceutical or biotechnological contexts as well as in food industry and material science, it is important to ensure a high degree of purity and/or a stable quality of a product during its production and potential storage. Quality controls are routinely performed based on a sample of the product and/or a sample comprising the product such as particles, e.g. proteins, in solution. These controls are typically time consuming and can cause a considerable delay until results are available. Hence, for reducing costs arising from impurities and/or undesired product properties, it would be desirable to have at hand solutions for assessing characteristics of a product, e.g. of particles in solution, with high accuracy and preferably in real-time. One example of such particles are proteins. Proteins are involved in almost all cellular processes and thus, crucial for the function of cellular organisms including humans. Depending on their function, proteins can be classified for example as structural proteins determining the structure of cells and tissues; proteins with catalytic functions, i.e. enzymes; ion channels regulating cellular ion concentrations and thus osmotic homeostasis and signal transduction; transport proteins; regulatory proteins including hormones; and proteins involved in immune reactions such as antibodies. The amount and/or activity of a protein can be affected for example in case of exposure to physiological stress conditions including high temperatures and/or in case of hereditary diseases. Due to their prevalence and their impact on cellular processes, changes in the amount and/or activity of a protein can have a significant impact on an organism's survival and health. Due to their relevance, proteins have been intensively investigated in view of their structure, function, distribution, level, and potential use in various applications including medicine. Proteins are macromolecular compounds consisting of amino acids that are linked by peptide bonds. The specific amino acid sequence, also referred to as the primary structure of a protein, is genetically determined. The amino acid sequence can be folded because of hydrogen bonds between amino acids residues of a protein, which results in a conformation that is also referred to as secondary structure, wherein the spatial arrangement of the amino acid sequence is referred to as tertiary structure. The tertiary structure, and thus the three-dimensional folding of a protein, is of special interest as its investigation provides not only information about the molecular structure of a protein. It can further provide detailed information about the spatial arrangement of reactive amino acid residues, e.g. in the catalytically active center of enzymes or in the antigen binding site of antibodies, and thus, about its activity. Furthermore, some proteins have a quaternary structure, which refers to an aggregation and/or association of proteins thus forming a stable (oligo)protein with the individual proteins being referred to as subunits of the (oligomeric) protein. As a deviation of a protein's native conformation(s) is usually associated with a reduction in its efficacy, especially the three-dimensional structure of a protein can be considered as being essential for its biological effect. Optimizing the availability of a protein, especially in a biologically active conformation, represents a promising approach in therapeutic contexts for example. In case a subject exhibits a reduced level of a protein compar