Methods for determining the identity of biomolecules

Abstract

The present invention relates to improved methods for determining the identity of biomolecules using interferometric light scattering instruments, and in particular for distinguishing between variants of biomolecules, such as viral serotypes, etc. Such methods have the potential to simplify testing for biological production, as well as for diagnosing disease, monitoring environmental problems and determining contaminants.

Description

[Technical field]

[0001] The present invention relates to improved methods for determining the identity of biomolecules, and in particular for distinguishing between variants of biomolecules, such as viral serotypes. Such methods have the potential to simplify testing for biological production, as well as for diagnosing disease, monitoring environmental problems, and determining contaminants. [Background technology]

[0002] Biological medicines, or biologics, can be described as treatments that contain complex active substances made by biological processes or derived from biological sources. "Biologics" is a broad term that encompasses a wide range of products, such as cells, tissues, blood-derived products, nucleic acids, recombinant proteins, antibodies, vaccines, gene therapy viruses, hormones, and enzymes. Biologic drugs are generally administered by injection or infusion and are already available to treat many diseases.

[0003] The production of many biologics involves the use of living cells, such as human cells, animal cells or microorganisms, commonly using gene manipulation methods. For example, monoclonal antibodies can be produced in Chinese Hamster Ovary cells (CHO).

[0004] Biologics testing is an essential process during manufacturing because these complex molecules can be altered by changes in their production process and they can be susceptible to factors that can cause aggregation and degradation that affect the activity of the biologics, the safety and quality of the product. Unintended or unaccounted for changes in the production process can lead to different product attributes (i.e., drift). Determination of the exact nature of the product is therefore essential.

[0005] Quality, safety, and efficacy must be constantly monitored to meet stringent regulatory requirements, such as determining the presence, identity, and concentration of contaminants during manufacture.

[0006] In biologics manufacturing, the identity and relative concentration of contaminants as well as the exact nature of the product may change over time due to increasing heterogeneity of the producing cells, the metabolic state of the cells as well as other process conditions in the bioreactor or downstream purification. Such drift is an analytical challenge, since numerous possible changes (oligomerization and protein assembly, post-translational modifications, changes in purification efficiency or failure of processing steps) can lead to very different changes in the composition of the final product that can only be analyzed by combining various analytical techniques that explore different aspects of the product quality. This results in long and expensive release testing procedures (Ramanan et al., BioDrugs 28, 363-372 (2014)). This is particularly important when biosimilars are launched, which are biologics that are highly similar to another already approved biological drug (the "reference drug"). It may therefore happen that similar biologics are produced by multiple producers with different quality systems. Different patterns of product drift can result in clinically meaningful differences among biologics.

[0007] One class of biologics is the monoclonal antibody (MAb), which has become one of the most powerful therapeutic and diagnostic tools available. Recent advances in molecular biology, immunology, and the development of robust manufacturing platforms will drive the development of more antibodies suitable for treating other diseases and conditions. This is expected to lead to the development of larger industrial facilities to increase production to meet the increasing demand. Those skilled in the art recognize the essential quality control attributes characteristic of antibodies, and the scale-up considerations that need to be made (Gaughan, CL, Molecular Diversity, Vol. 20, pp. 255-270 (2016)).

[0008] Currently, drift and shipping or finished product testing can involve a number of testing protocols including, but not limited to, appearance, osmolality, pH, cell-based bioassays, endotoxin testing, various chromatographic techniques: size exclusion chromatography, reverse phase chromatography, ion exchange chromatography, amino acid analysis, protein concentration determination, peptide mapping, glycan profiling, capillary gel electrophoresis, capillary isoelectric focusing, and host cell assays including sugar, vitamin, metabolite, protein (commonly ELISA) and DNA determination.

[0009] There is an incentive to provide more real-time release testing for biologics. Real-time release testing (RTRT) is defined as "the ability to assess and ensure the quality of in-process and / or final drug product based on process data, typically including a reasonable combination of measured material attributes and process controls" (Jiang et al., Biotech & Bioeng 2017, https: / / doi.org / 10.1002 / bit.26383). However, despite the potential improvements that RTRT could provide, producers are still exploring the practicality of implementing this approach and therefore have yet to fully benefit from its promise. Many questions remain, including which equipment should be used and when and where the assay should be performed during production.

[0010] One specific method of interferometric light scattering microscopy is mass photometry, which uses specific spatial filters to allow for higher sensitivity. Mass photometry provides an efficient and effective method for measuring the mass and relative concentration of biomolecules in a sample. This technique measures the mass of individual biomolecules by capturing the light scattered by each molecule at the interface. Extraction of the scattering contrast provides an indirect measurement of mass. Mass photometry builds on the principles of interference reflection microscopy and interferometric scattering microscopy. Through carefully controlled illumination, novel spatial filtering strategies in the detection beam path and careful image analysis, it has been demonstrated that minute amounts of light scattered by single molecules can be reliably detected and, more importantly, that it is directly proportional to molecular mass (Young et al., Science 2018, 360(6387), pp. 423-7). However, currently, mass photometry and interferometric light scattering techniques generally cannot distinguish between particles of very similar molecular mass. Particles may have very similar masses, for example, because they are variants of the same biomolecule. Such variants may be differently glycosylated proteins. Sometimes the resolution is insufficient to distinguish between individual species. Therefore, current methods of interferometric light scattering microscopy, and especially mass photometry, cannot alone accelerate techniques such as release testing, since additional testing is required. Summary of the Invention

[0011] It is therefore an object of the present invention to provide a method for distinguishing between biomolecules of similar mass. Biomolecules of similar mass may be related to each other, i.e. isoforms of the same biomolecule. Biomolecules of similar mass may be variants of the same biomolecule. Additionally, it is an object of the present invention to provide improved methods for determining the identity of a biomolecule in a sample, for example allowing discrimination between possible variants, such as isoforms, glycoforms, lipoforms, species, serotypes or mutants. Such a determination makes it possible to carry out techniques, such as release testing, where it is known that the sample contains a biomolecule. Thus, the method may be used to determine certain characteristics of a molecule with a certain mass. In release testing, the expected mass of the biomolecule may be known, and therefore the determination of further characteristics of the molecule with the appropriate mass may be determined.

[0012] Since biological molecules play an essential role in biological processes, including disease development, accurate detection and / or identification of biological molecules is essential for disease diagnosis and treatment.Furthermore, considering that biological environments and industrial samples, etc. may also contain particles of similar mass, the method for identifying biological molecules in samples may have further applications.

[0013] Biological molecules are undoubtedly complex. Biological molecules produced in cells are processed according to the cellular pathways of the cell. Differential processing can result in the production of isoforms even in the natural context. The host cell may use different pathways for processing when compared to the source cell for the biological molecule. This can result in differential processing, such as changes in protein folding, changes in nucleic acid methylation, changes in glycosylation patterns, and changes in surface charge. The physical characteristics of biological molecules can therefore be altered by processing in different cell types. Biological molecules can be classified into four general classes, proteins, carbohydrates, lipids, and nucleic acids, depending on the monomeric units used. These monomeric units are varied and contain different chemical groups that can have different attributes, for example amino acids (the building blocks of peptides and proteins) can be polar, non-polar, positively charged, or negatively charged. Biological molecules may be composed of more than one type of monomer, for example, glycosylated proteins, glycosylated RNA, lipoproteins, lipid-modified oligonucleotides and glycolipids. Generally, biological molecules are folded to some extent to place some of the monomers in the core and expose other monomers at the surface. Biological molecules therefore have numerous chemical groups on their surface, all of which help determine the physical characteristics of the biological molecule. Changes to the way biological molecules are manufactured can change the surface chemistry, which leads to changes in characteristics, especially surface characteristics. Similarly, when molecules change conformation and parts that were previously internal form parts of the external surface, they also have certain characteristics.

[0014] Any biomolecule, whether protein, carbohydrate, or nucleic acid, can be distinguished from other molecules based on differences in some physical characteristics. Physical characteristics include, but are not limited to, size, defined as either length or mass, charge, binding affinity (e.g., ability to form weak non-covalent bonds, i.e., hydrogen bonds, ionic bonds, and van der Waals forces, as well as favorable hydrophobic interactions), and hydrophobicity. Biomolecules may thus be defined, for example, by their molecular weight (mass) and by their isoelectric point (pI). The isoelectric or isoionic point of a biomolecule is the pH at which the biomolecule has no net charge and is therefore considered neutral. The pI of a biomolecule depends primarily on the dissociation constant (Kd) of any ionizable group. Arginine, aspartic acid, cysteine, histidine, glutamic acid, lysine, and glutamic acid are all exemplary ionizable groups in proteins, and they play a large role in determining the pI of a protein. However, co- and post-translational modifications (including phosphorylation, methylation, alkylation) can play a significant role in determining the pI of a biomolecule. All biomolecules, including without limitation viruses, nucleic acids, polysaccharides, and lipids, have an isoelectric point.

[0015] The present invention relies on the interaction of biomolecules with a surface. How a biomolecule interacts with a surface is determined by the properties or characteristics of the biomolecule. This can be exploited by selecting an appropriate surface and determining the specific properties or characteristics with or without changing one or more conditions, such as temperature, in which the biomolecule is brought into contact with the surface. The interaction of the biomolecule with the surface may be determined using interferometric light scattering microscopy. Such techniques examine the interface between the surface and the sample and detect biomolecules that interact with the surface. Each biomolecule may be tracked by making repeated measurements, which gives a picture of the interaction of the biomolecule with the surface. This allows the determination of the mobility of the biomolecule on the surface.

[0016] The interactions between the surface and the biomolecules may be due to any type of interaction, ranging from hydrophobic to covalent bond formation. The interactions between the surface and the biomolecules may be due to ionic / electrostatic interactions. These interactions are due to the physical characteristics of the biomolecules.

[0017] The present invention therefore provides a method for determining the identity of a biomolecule in a sample, the method comprising the step of determining the interaction of the biomolecule with a surface. 1. A method for identifying a biomolecule in a sample using an interferometric light scattering device, comprising: (i) contacting the sample with a first surface and taking a plurality of measurements of the biomolecules on the first surface and using the measurements to determine mobility of the biomolecules on the surface; (ii) repeating step (i) using a second surface and determining the mobility of the biomolecule on said surface; (iii) constructing a profile for a biomolecule using the mobilities of the biomolecule determined in step (i) and step (ii); (iv) comparing the profile obtained in step (iii) with at least one reference profile to identify the biomolecule; A method is provided that includes:

[0018] The method of the present invention therefore includes a number of steps which may be carried out in any suitable order. The surface may be selected such that the surface chemistry allows for the determination of at least one physical characteristic of the biomolecule. The surface may be selected to determine the interaction of the biomolecule with the surface, most particularly the mobility of the biomolecule on the surface.

[0019] The surface is contacted with a sample that may contain biomolecules.The one or more conditions under which the surface is contacted with the sample may be selected to allow determination of at least one physical characteristic of the biomolecule.The surface may be selected to determine the interaction of the biomolecule with the surface, most particularly the mobility of the biomolecule on the surface.

[0020] The identity of a biomolecule in a sample may be determined by determining the mobility of the biomolecule on a surface by creating or preparing a profile, the profile comprising data obtained from at least the first surface and the second surface (e.g. steps (i) and (ii)).

[0021] The first and second surfaces may be different surfaces. The first and second surfaces may be the same surface. In this scenario, the second surface is contacted with the sample under at least one altered condition (when compared to the first surface). The altered condition may be selected from any one of temperature, pH, ionic strength, redox potential, potential applied between the surface and the solution, and the presence of a reagent. The reagent may be any reagent that alters the interaction of the biomolecule with the surface, such as any one or more of a surfactant, a chaotropic agent, a reducing agent, a buffer, an ion, a salt, a solvent, a denaturant, and / or a cross-linking agent. Multiple conditions may be altered as required.

[0022] Once a biomolecule is detected on the surface, repeated measurements may be made of the biomolecule while it remains in contact with the surface. This allows for tracking of the biomolecule as it moves across the surface accordingly. These repeated measurements may be described as making "multiple measurements" and may also be described as measurements made over time or over a time period. The rate at which these measurements are made may be appropriately selected depending on the expected mobility of the biomolecule. The rate may be in the range of 10 Hz to 10 kHz, optionally 50 Hz to 5 KHz, preferably 100 Hz to 1 kHz. Any suitable rate may be selected within or from these ranges. Multiple measurements are therefore made of the biomolecule over time to monitor the mobility of the biomolecule on the surface.

[0023] Taking multiple measurements of the interaction of a biomolecule with a surface allows for tracking of the biomolecule. Detection and / or tracking can be performed at the level of a single biomolecule. Any measurement may be used to determine the mass of the biomolecule, optionally a measurement made in step (i) or step (ii) may be used, or an independent measurement may be made to determine the mass.

[0024] A reference profile may be prepared by choosing a known biomolecule and performing the method with at least the first and second surfaces. This reference profile may be a single combined profile or a set of profiles obtained from each surface. A summary of the biomolecule's interactions (e.g., mobility) with the surface may be used to construct the profile. A profile may be defined as a set of features that identify or are believed to identify a particular biomarker. A profile may be a summary or analysis of data in any suitable form, such as a graph or table, that represents the distinguishing features.

[0025] According to an alternative aspect of the invention, there is provided a method of identifying a biomolecule in a sample comprising the steps of contacting the sample with a first surface and taking a plurality of measurements of the biomolecule over time using an interferometric scattering device and using the plurality of measurements to determine the mass and mobility of the biomolecule on the first surface to determine at least one physical characteristic of the biomolecule.

[0026] The following features may apply to any aspect of the invention. The first surface may be selected such that the surface chemistry allows for the determination of at least one physical characteristic of a biomolecule.

[0027] The first surface is contacted with a sample that may contain a biomolecule. The conditions under which the first surface is contacted with the sample may be selected to allow for the determination of at least one physical characteristic of the biomolecule.

[0028] The identity of the biomolecule in the sample may be determined by determining the mass and mobility of the biomolecule on the first surface without further measurements being required. Alternatively, further measurements may be required, for example, in more complex samples or when potential variants of the biomolecule have similar characteristics.

[0029] Further measurements include: contacting the sample with a second surface and taking a plurality of measurements of the biomolecule over time using an interferometric scattering device and determining the mass and mobility of the biomolecule on the second surface using the plurality of measurements to determine at least one physical characteristic of the biomolecule. may include:

[0030] Alternatively or additionally, the further measurement may include: contacting the sample with a first surface and varying one or more conditions of the sample to determine at least one physical characteristic of the biomolecule; The conditions may be defined as conditions that affect the binding of biomolecules to the surface and therefore affect reactions between the surface and the biomolecules.

[0031] Sample conditions that may be altered include, but are not limited to, temperature, pH, ionic strength, the presence of chemical entities such as detergents, chaotropic agents, reducing agents, oxidizing agents, redox reagents, buffers, ions, solvents, denaturants, and / or cross-linking agents.

[0032] If further measurements are required, the measurements obtained using the different surface and / or altered conditions can be combined or compared with the measurements obtained using the first surface to provide sufficient information to identify the biomolecule. This can result in the determination of the mass and two or more physical characteristics of the biomolecule.

[0033] Multiple measurements can be applied on one or more surfaces or under one or more conditions, so that a profile that matches the biomolecule can be determined. This profile can be sufficient alone to determine the identity of the biomolecule, or the profile can be compared to a reference profile previously obtained for a known biomolecule.

[0034] According to one embodiment, the method of the present invention is a method for identifying a biomolecule in a sample, comprising the steps of: contacting the sample with a first surface and taking a plurality of measurements of the biomolecule over time using an interferometric scattering device and using the plurality of measurements to determine a mass and mobility of the biomolecule on the first surface, and using the mass and mobility to define a profile for the biomolecule which may be compared to a reference profile. The present invention provides a method comprising:

[0035] The method may be repeated with a second surface and / or with a second set of conditions to replenish the profile.The method may be repeated with additional surfaces and / or with additional sets of conditions to replenish the profile.

[0036] According to one embodiment, the method of the present invention is a method for identifying a biomolecule in a sample, comprising the steps of: contacting the sample with a first surface and taking a plurality of measurements of the biomolecules over time using an interferometric scattering device and determining the mass and mobility of the biomolecules on the first surface using the plurality of measurements; contacting the sample with a second surface and taking a plurality of measurements of the biomolecules over time using an interferometric scattering device and using the plurality of measurements to determine the mass and mobility of the biomolecules on the second surface; and combining or comparing the measurements on the first and second surfaces to determine the identity of the biomolecule. The present invention provides a method comprising:

[0037] According to one embodiment, the method of the present invention is a method for identifying a biomolecule in a sample, comprising the steps of: contacting the sample with a first surface under a first set of conditions and taking a plurality of measurements of the biomolecules over time using an interferometric scattering device and using the plurality of measurements to determine the mass and mobility of the biomolecules on the first surface under the first conditions; contacting the sample with a first surface under a second set of conditions and taking a plurality of measurements of the biomolecules over time using an interferometric scattering device and using the plurality of measurements to determine the mass and mobility of the biomolecules on the first surface under the second conditions; and combining or comparing the measurements under the first and second conditions to determine the identity of the biomolecule. The present invention provides a method comprising:

[0038] The following features may apply to any aspect of the invention. Multiple measurements over time may be used to determine the time of interaction of each biomolecule with the surface and / or the movement of the biomolecule on the surface. Such measurements may be considered to be the "mobility" of the biomolecule on the surface. Mobility may therefore be defined as the distance traveled on the surface and / or the length of time the biomolecule interacts with the surface. Either or both may be measured. It is possible to measure the distance traveled by the biomolecule on the surface, which may be described as a lateral or horizontal movement at the surface. Alternatively or additionally, the time of interaction of the biomolecule with the surface may be measured. Such interaction time indicates the movement away from the surface such that the biomolecule is no longer interacting with the surface and moves or diffuses into the sample solution. This form of mobility depends on the strength of the interaction of the biomolecule with the surface. The interaction between the surface and the biomolecule may be indicative of the characteristics of the biomolecule and therefore provide information about it. [Brief description of the drawings]

[0039] [Figure 1]FIG. 1 is a diagram showing an interferometric light scattering (iSCAT) microscope. [Diagram 2] FIG. 2 is an image captured by the iSCAT microscope. [Diagram 3] FIG. 3 is a diagram showing the distance traveled by AAV5 and AAV9 in the samples, with the inset box showing the trajectory of viral particle movement on the surface. [Figure 4] Figure 4A shows the distance traveled by an IgG molecule on glass, and Figure 4B shows the distance traveled by an IgG molecule on a trimethylchlorosilane-modified surface, showing reduced migration on the trimethylchlorosilane-modified surface. [Diagram 5] FIG. 5 shows a typical antibody molecule with possible groups available for interaction with a surface highlighted. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] All biological molecules (biomolecules or biologics) have surfaces with exposed chemical entities, and thus the characteristics of biomolecules depend on their chemical entities. Exemplary entities include the amino acid side chains in proteins, the nucleotide bases in single-stranded nucleic acids, the sugar-phosphate backbones in nucleic acids, the chemistry and organization of the monosaccharide units in glycosylated biomolecules, the hydrophobic side chains and polar head groups of some lipids, and any interactive metal ions or solvents with any of such groups.

[0041] Simple variations in how these biomolecules are produced, including in disease states or in transgenic animals or ex vivo production, alter the surface characteristics of the biomolecules, for example through changes in protein folding, glycosylation or lipidation. It is an object of the present invention to provide a method for determining the characteristics of biomolecules. When characteristics can be determined, they can then be compared to "wild type" or expected characteristics determined using known samples of the biomolecule. This is particularly true for the production of biologics.

[0042] As used herein, biomolecules may be described as objects, particles or species. The invention involves contacting a surface with a biomolecule. The biomolecule may interact with this surface and it is this interaction that the invention seeks to determine based on the mobility of the biomolecule (contact / non-contact and / or lateral mobility). The particle is tracked and imaged over time so that the time of the particle's interaction with the surface and / or the distance traveled along the surface can be determined. Such parameters are indicative of the strength of the interaction between the biomolecule and the surface, which is an indirect determination of the physical characteristics of the biomolecule. The physical characteristics are generally characteristics of the outer surface of the biomolecule, although the sample may be placed under conditions in which the internal parts of the biomolecule are exposed (denatured or unfolded).

[0043] To determine the characteristics of a biomolecule, the surfaces (first, second and / or further) may be selected to interact with a particular chemical entity. As such, the material that constitutes the surface may be selected based on its ability to interact with a chemical entity present on or in the biomolecule. For example, the surface may be glass, and many biomolecules are known to have a tendency to bind non-specifically to glass (e.g., via protein adsorption). For example, Figure 4A depicts the mobility of IgG on a glass surface.

[0044] For example, proteins and lipids are sometimes described as amphiphilic macromolecules because they have both hydrophobic and hydrophilic groups that are attracted to non-polar and polar groups, respectively. Hydrophilic and charged groups tend to be located on the outer surface of the particle. However, hydrophobic groups are also present on the outer surface. Such groups can interact electrostatically with the surface. Particle binding occurs when the free energy between the particle and the surface is lower than the free energy between the surface and water (or other solvent).

[0045] Surfaces for use in the present invention may be any suitable surface compatible with interferometric detection methods. Suitable surfaces may have the ability to be functionalized or derivatized, or otherwise coated to modify the surface chemistry.

[0046] The surface is preferably solid (i.e. not a gel or liquid). Preferably, the surface is transparent to ultraviolet light (which may be defined herein as having a wavelength in the range of 10 nm to 380 nm), visible light (which may be defined herein as having a wavelength in the range of 380 nm to 740 nm), and / or infrared light (which may be defined herein as having a wavelength in the range of 740 nm to 300 μm). Preferably, the surface allows the transmission of light in the visible light spectrum. Preferably, the surface is substantially transparent. The surface may be smooth or textured. A smooth surface is preferred. The surface may be patterned.

[0047] The surface may be of any suitable material, including, for example, without limitation, glass, diamond, plastic, polymeric materials (e.g., cyclic olefin copolymers, polyvinyls, e.g., polyethylene (PE), including polyethylene terephthalate (PET) and high density polyethylene (HDPE) and low density polyethylene (LDPE), polyacrylates (acrylics), polystyrenes (PS), including high impact polystyrene (HIPS), silicones, polyesters (e.g., polylactic acid (PLA) or polylactic-co-glycolic acid (PGLA)), polyurethanes, polypropylenes (PP), polyamides (nylons), acrylonitrile butadiene styrene (ABS), polyethylene / acrylonitrile butadiene styrene (PE / ABS), bakelite, rubber, latex, polycarbonate (PC), polycarbonate / acrylonitrile butadiene styrene (PC / ABS), and polyvinyl chlorides, including, for example, polyvinylidene chloride (PVDC), and sapphire.

[0048] The surface may include a lipid membrane, such as a lipid bilayer, lipid micelles, and lipid nanodiscs. The lipid bilayer may be any suitable bilayer, such as a supported lipid bilayer. The bilayer may be supported, for example, on a glass coverslip. Such supported lipid bilayers as surfaces for iSCAT are discussed in Foley, E. D. B. et al., Mass photometry enables label-free tracking and mass measurement of single proteins on lipid bilayers. Nat Methods 18, pp. 1247-1252 (2021) and Vala, M., Piliarik, M. Weighing single protein complexes on the go. Nat Methods 18, pp. 1159-1160 (2021).

[0049] The lipid membrane may be provided in any suitable format, with any suitable lipid and / or protein combination as required. The lipid membrane may have any suitable geometric shape.

[0050] The surface may have any suitable geometry.For example, the surface may comprise a flat plate, such as a cover slip, or may be a well, a plate, a bead, a channel, a container, a flow cell, a flow chamber, a microfluidic cell or chamber, or a slide.The surface may be part of a larger geometry or device.Suitable surfaces may be glass cover slips or plastic cover slips, where plastic is as described above for the surface.

[0051] The surface preferably forms part of a sample holder. The sample holder may be an element of a light scattering microscope. The sample holder may be a high surface to volume ratio chamber.

[0052] The surface may be functionalized. The surface may be passivated, activated, coated, treated or derivatized. The surface may be a passivated surface. Passivation is the process of treating or coating a surface to enhance or reduce chemical reactivity and thus increase or decrease the number of interaction events. Those skilled in the art will recognize suitable passivation agents, examples of which include BSA, PVPA, DDS and / or PEG. Alternatively, the surface is not passivated.

[0053] Surfaces may be activated, coated, treated and / or derivatized to modify the chemistry of the surface. Surface functionalization allows for the determination of specific features of biomolecules. Surface functionalization may be to allow for various interactions, e.g. chemical interactions (e.g. covalent bonds) or physical interactions (e.g. adsorption). When surfaces are coated, derivatized or modified, a thin surface modification may be desired. Modification of too thick a surface layer may change the light scattering properties of the surface. Modification of only the outermost few molecular layers (3-10 nm) may be desired.

[0054] Any suitable surface coating may be applied to change the chemistry of the surface. The surface may be modified to increase hydrophobicity or increase hydrophilicity. Suitable methods and coatings are known and available to those skilled in the art, including, for example, without limitation, functionalized PEG, silanes, carboxy-silanes, amines, aminosilanes, trimethylchlorosilanes, aldehyde modifications using amine-modifying reagents, such as APTES, epoxy modifications using, for example, GOPTS, carboxylic acid modifications using, for example, EDC, NHS, HOBt, TBTU, PAMAM, diazo using, for example, NaNO2, and supramolecules, such as calixarenes.

[0055] Preferably, altering the surface chemistry of the surface does not substantially affect the optical transmission of the surface. The nature of the surface, e.g., chemistry, may have an influence on the biomolecule. For example, a hydrophobic coating may result in partial unfolding of the protein to expose a hydrophobic "interior" compartment, so that the determination of characteristics may go beyond the evaluation of the external surface of the biomolecule. Alternatively, different binding characteristics may be tested by applying different surface modifications, e.g., the introduction of different surface charges by coating amino or carboxyl groups. At neutral pH, amino groups are protonated and carboxyl groups are deprotonated.

[0056] Biomolecules may interact with surfaces in many different ways. Biomolecules may be absorbed onto surfaces, which may be favored by hydrophobic interactions and therefore require a hydrophobic surface. Biomolecules may chemically react with functional groups of surfaces via free reactive groups, such as amine or carboxyl groups. Such covalent bonds limit movement / mobility, both lateral and in / out of contact with the surface. Hydrophobic, hydrogen, van der Waals, ionic and coordinate bond types may also occur between biomolecules and surfaces, along with electrostatic interactions. These types of interactions may be particularly affected by conditions, so that changes in temperature and pH may alter the interaction of biomolecules with surfaces.

[0057] The sample is contacted with a surface (first, second and / or further) and this may take place inside any device for detecting light scattering, however the step may take place before the surface is placed in the device.

[0058] It will be understood by those skilled in the art that if a sample is to be tested repeatedly using different surfaces and / or different conditions, it is preferable to use a fresh (not previously used) sample to contact each surface. Thus, the methods of the invention may be described as being performed on portions or aliquots of the sample such that multiple portions or aliquots may be contacted in parallel with multiple surfaces.

[0059] A biomolecule may be any suitable biological molecule, including but not limited to proteins, polypeptides, peptides, glycoproteins, glycosaminoglycans, glycosphingolipids, lipoproteins, nucleic acids, oligomers, oligonucleotides, glycosylated nucleic acids, lipid-nucleic acid complexes, lipids, glycolipids, triglycerides, phospholipids, steroids, carbohydrates, oligosaccharides and polysaccharides. As used herein, a biomolecule may be an oligomer of various units and may form larger structures, such as virus particles. Viruses may be identified in exactly the same way as biomolecules of lower complexity, by assessing characteristics including mass and / or mobility on the surface. The external part of the virion may be proteinaceous or associated with a lipid membrane. The biomolecule may thus be a virus or a virus-like particle (VLP). The biomolecule may be a lipoplex, a lipid shell or a lipid nanoparticle (LNP), such as an LNP designed to deliver pharmaceuticals, such as RNA therapeutics.

[0060] Biomolecules may exist in variant forms, such as isoforms, glycoforms, lipoforms, species, serotypes or mutant forms.It is the objective of some embodiments of the present invention to determine which variant biomolecules are present in a sample.For example, it has been shown that the method of the present invention can distinguish virions of serotypes AAV5 and AAV9 due to their differential mobility on the surface.This mobility-based profile can be used to distinguish these AAV serotypes in unknown samples.

[0061] Isoforms may be encoded by the same gene but may involve differential processing. The resulting biomolecules may have similar functions and similar masses but may have different characteristics. Serotypes (or serovars) are distinct variations within a species of virus. Glycoforms are isoforms of biomolecules that differ only with respect to the number or type of glycans attached. Glycoproteins often consist of many different glycoforms, with alterations in the attached saccharides or oligosaccharides. Variations in glycosylation are particularly relevant for immunoglobulins. Different isoforms of lipids, called lipoforms, may exist.

[0062] Since variants of biomolecules generally have similar masses, simply determining the mass may not provide enough information to determine the identity of a biomolecule more precisely. The present invention therefore seeks to determine further information on the characteristics of a biomolecule, such as charge, pI, or the presence of a specific chemical group, so that variants can be more closely identified. These can be described as structural and physicochemical characteristics of a biomolecule. For example, in the case of a monoclonal antibody, important characteristics include mass, N / C terminus, charge, glycosylation pattern, aggregation, and FcgR binding. In the production of biological molecules, the expected mass of the product may be known, but the method of the present invention seeks to provide further information on the product with a certain mass. This method allows the determination of further characteristics of a biomolecule beyond the determination of mass. Such is particularly important when the process may result in incorrect glycosylation of the protein product.

[0063] Proteins are composed of amino acid monomers, which provide the variety of side chains that provide many of the protein characteristics. The amino acid side chains vary in size, charge, shape, hydrogen-bonding ability, hydrophobic character, and chemical reactivity.

[0064] To enable this, biomolecules are contacted with surfaces, which may be prepared to provide different binding characteristics. Additionally or alternatively, conditions may be altered to examine specific effects. Conditions may be altered in any suitable manner, including but not limited to, changes in pH, salt concentration, ionic strength, addition of buffers, detergents, chaotropic agents, etc. Illustratively, altering the pH may alter the net charge on the biomolecule (due to protonation / deprotonation), which has an impact on charge-mediated surface binding. Altering the salt concentration may also be used to affect charge-mediated binding. Altering the ionic strength may, for example, allow denaturation of double-stranded nucleic acids. Detergents, denaturants, chaotropic agents may be used to partially unfold or dissociate biomolecules to examine features that are not surface exposed. Crosslinkers may stabilize molecules to allow closer examination of surface chemistry. Alternatively or additionally, redox potentials may be altered, for example, by altering the concentration of oxidizing and / or reducing agents in the sample. Another example of an altered condition is the application of an electrical potential between the surface and the solution, which alters the attraction of biomolecules to the surface.

[0065] Conditions of the sample that may be altered include, but are not limited to, temperature, pH, ionic strength, the presence of chemical entities / reagents such as detergents, chaotropic agents, reducing agents, buffers, ions, solvents, denaturants, and / or cross-linking agents. Further conditions include redox potential and the application of a potential between the surface and the sample.

[0066] The mass of the biomolecule may also be determined in the method of the present invention. The mass may be determined at any suitable step, including either step (i) and / or (ii). A separate measurement step for determining the mass may be performed.

[0067] Interferometric light scattering precisely measures molecular mass by quantifying light scattering from individual biomolecules in solution. Such techniques have been previously described, for example, in Young and Kukura, Annual Review of Physical Chemistry 2019 70:1, pp. 301-322; Young et al., Science 27 Apr 2018: vol. 360, no. 6387, pp. 423-427, and Strack, R. Weighing single proteins with light. Nat Methods 15, 477 (2018), all of which are incorporated herein by reference.

[0068] The sample may be any suitable sample, but is preferably taken from a biomolecule production where known biomolecules are present, and the method is used to determine whether the correct variant is present and / or the presence of undesired contaminants.The sample is therefore preferably not a complex sample where multiple different unknown biomolecules are present, because the presence of many biomolecules of similar mass can be confusing.The sample is preferably a liquid, such as a solution or suspension.

[0069] The sample may be a biological sample, such as a medical or veterinary sample. Such a biological sample may be any suitable type of body fluid or any suitable type of tissue suspension. The sample may be an environmental sample. Such an environmental sample may be a water sample or a sample taken from the environment and suspended in a solvent.

[0070] If the sample is suspected to be concentrated, it may be appropriately diluted prior to contacting the sample with the surface.Dilution may use any suitable solvent, for example water.In examples 1 and 2, the sample is appropriately diluted to ensure that the number of biomolecule / surface interaction events measured is within a suitable level to allow device detection and tracking.

[0071] Any suitable device may be used in the methods described herein. A standard iScat device or an interference reflection microscope may be suitable for some embodiments. A mass spectrometer available from Refeyn, UK may be suitable. Generally, labeling of biomolecules is not required. Generally, biomolecules are not tethered to a surface.

[0072] The invention involves the use of an interferometric light scattering instrument, such as a microscope or mass spectrometer. Such a device examines the interface between a sample and a surface. Such an instrument may perform multiple measurements. Biomolecules on the surface scatter light to generate contrast in the measurement. It is possible to localize each scattering event (and therefore each biomolecule) by fitting the contrast signal to a profile (e.g. a Gaussian profile) and locating the event to the peak of the fit. Repeated measurements may be taken at a suitable rate, depending on the expected speed of movement of the biomolecules. Suitable rates at which measurements are taken range from 10 Hz to 10 kHz, optionally 50 Hz to 5 KHz, preferably 100 Hz to 1 kHz. Any suitable rate may be selected within or from these ranges. Individual biomolecules may then be tracked by using repeated fitting and repeated localization of the contrast signal, which may create a "track" of the biomolecule on the surface. If the contrast signal disappears, this is an indication that the biomolecule has left the surface. Measurements that may be taken to indicate mobility therefore include, but are not limited to, the time associated with the surface, the distance traveled on the surface, the rate of movement on the surface and / or the frequency of interaction with the surface (measurements of association / dissociation from the surface).

[0073] The measurements made can be used to construct a profile for the biomolecule. For example, the normalized number for the distance traveled can be plotted, or the speed at which the biomolecule travels can be plotted. Alternatively, the trajectory of the biomolecule can be plotted (see the inset of FIG. 3), where the trajectory is the path taken by the biomolecule on the surface. The constructed profile can thus reflect various characteristics or properties of the biomolecule according to its interaction with the surface.

[0074] A reference profile may be prepared by choosing a known biomolecule and carrying out the method described with at least the first and second surfaces. This reference profile may be a single combined profile based on multiple data sets for two or more surfaces, or a set of profiles obtained from each surface individually. A summary of the biomolecule's interactions with the surface (e.g., mobility) may be used to construct the profile. A profile may be defined as a set of features that identify or are believed to identify a particular biomarker. A profile may be a summary or analysis of data in any suitable form, such as a graph or table, that represents the distinguishing features. The reference profile may be stored in a database that allows the comparison to be made by computer-implemented means. Figures 3 and 4 contain representations of collected data that may be used to construct a profile for a biomarker.

[0075] A profile constructed using the method of the invention (e.g., using steps (i)-(iii) of the method) may be compared to a reference profile. This involves examining the biomarker profile and the reference profile to find similarities. Similarities in profile attributes allow for identification of the biomolecule. It will be understood by those skilled in the art that for an unknown sample to be compared to a reference sample, the comparison needs to be meaningful such that the same features are determined for both the reference biomarkers and the biomarkers in the sample. Generally, this means that the first and second (and any further) surfaces are the same for both the reference and sample biomarkers. However, there may be some cases where one or more surfaces are interchangeable when determining features, such that the surfaces should be of the same class for the reference and sample profiles. For example, surfaces that may be used to determine hydrophobicity may have exposed methyl (-CH3), methylene (-CH2-), methoxy (-OCH3), or trifluoromethyl ester (-OCF3) groups, or alternatively may include or be coated with polymeric biomaterials, such as PTFE, PDMS, PET, polyethylene, and polypropylene. Thus, the surface may be selected from a class of surfaces designed to interrogate particular properties or characteristics of biomolecules. The comparison step may thus involve comparing an unknown biomarker profile on a "hydrophobic" surface and comparing it to a reference biomarker profile that involved the use of an alternative "hydrophobic" surface.

[0076] In the devices, microscopes and methods described herein, the light used may be ultraviolet light (which may be defined herein as having a wavelength in the range of 10 nm to 380 nm), visible light (which may be defined herein as having a wavelength in the range of 380 nm to 740 nm), infrared light (which may be defined herein as having a wavelength in the range of 740 nm to 300 μm). The light may be a mixture of wavelengths. In this specification, the terms "optical" and "optics" are used generally to refer to the light to which the methods are applied.

[0077] FIG. 1 illustrates an iSCAT microscope 1 that is configured as follows. Microscope 1 includes the following components, which have a configuration conventional in the art of microscopy, except for a spatial filter, which will be described in more detail below.

[0078] The microscope 1 includes a sample holder 2 for holding a sample 3 at a sample position. The sample 3 may be a liquid sample containing the object to be imaged, as described in more detail below. The sample holder 2 may take any form suitable for holding the sample 3. Typically, the sample holder 2 holds the sample 3 on a surface, which forms an interface between the sample holder 2 and the sample 3. For example, the sample holder 2 may be a coverslip and / or may be made of glass. The sample 3 may be provided on the sample holder 2 in a direct manner, for example using a micropipette.

[0079] The microscope 1 further comprises an illumination source 4 and a detector 5 . The illumination source 4 is configured to provide illumination light. The illumination light may be coherent light. For example, the illumination source 4 may be a laser. The wavelength of the illumination light may be selected depending on the nature of the sample 3 and / or the properties to be investigated. In one example, the illumination light has a wavelength of 405 nm.

[0080] Optionally, the illumination light may be spatially modulated to eliminate speckle patterns arising from the coherent nature of the illumination and laser noise, as detailed, for example, in Kukura et al., "High-speed nanoscopic tracking of the position and orientation of a single virus," Nature Methods 2009 6:923-935.

[0081] The detector 5 receives the output light in reflection from the sample location. Typically, the microscope 1 may operate in a wide field mode, in which case the detector 5 may be an image sensor that captures an image of the sample 3. The microscope 1 may alternatively operate in a confocal mode, in which case the detector 5 may be an image sensor or may be a point-like detector, such as a photodiode, in which case a scanning arrangement may be used to scan an area of ​​the sample 3 to build up an image. Examples of image sensors that may be used as the detector 5 include CMOS (complementary metal oxide semiconductor) image sensors or CCDs (charge-coupled devices).

[0082] Microscope 1 further includes an optical system 10 configured between sample holder 2, illumination source 4 and detector 5. Optical system 10 is configured as follows for directing illumination light to a sample position for illuminating sample 3, and for collecting output light on reflection from the sample position and directing the output light to detector 5.

[0083] The optical system 10 includes an objective lens 11, which is a lens system arranged in front of the sample holder 2. The optical system 10 also includes a condenser lens 12 and a tube lens 13.

[0084] Condenser lens 12 condenses illumination light (shown by a solid line in FIG. 1) from light source 11 through objective lens 11 onto sample 3 at the sample position. The objective lens 11 collects output light that includes both (a) illumination light reflected from the sample position (indicated by a solid line in FIG. 1 ) and (b) light scattered from the sample 3 at the sample position (indicated by a dotted line in FIG. 1 ). The reflected light is mainly reflected from the interface between the sample holder 2 and the sample 3. Typically, this is a relatively weak reflection, e.g., a glass-water reflection. For example, the intensity of the reflected illumination light may be on the order of 0.5% of the intensity of the incident illumination light. The scattered light is scattered by objects in the sample 3.

[0085] In a manner similar to conventional iSCAT, light scattered from objects at or near the surface of the sample actively interferes with the reflected light and is therefore visible in the image captured by detector 5. This effect differs from microscopes operating in transmission, where the illumination light that reaches the detector is transmitted throughout the depth of the sample, leading to much smaller imaging contrast.

[0086] 1, the reflected illumination light and the scattered light have different directionality. In particular, the reflected illumination light has a numerical aperture that results from the geometry of the beam of light output by the light source 4 and the optical system 6. The scattered light fills a larger numerical aperture than the reflected illumination light because it is scattered over a wider range of angles.

[0087] Tube lens 13 focuses the output light from objective lens 11 onto detector 5 . The optical system 6 also includes a beam splitter 14 configured to split the light path for the illumination light from the light source 4 and the output light directed to the detector 5. Except for the provision of a spatial filter as described below, the beam splitter 14 may have a conventional configuration that provides partial reflection and partial transmission of the light incident on it. For example, the beam splitter 14 may be a plate, typically equipped with a film, which may be metallic or dielectric, configured at 45° to the light path. Alternatively, the beam splitter 14 may be a cube beam splitter formed by a matched pair of prisms with a partially reflective film at the interface between the prisms. Alternatively, the beam splitter 14 may be a polarizing beam splitter used in combination with a quarter wave plate between the beam splitter 14 and the sample 3.

[0088] In the example shown in FIG. 1, the light source 4 is offset from the optical path of the objective lens 11, such that illumination light from the light source 4 is reflected into the objective lens 11 by the beam splitter 14, and conversely, the detector 5 is aligned with the optical path of the objective lens 11, such that output light from the sample position is transmitted through the beam splitter 14 towards the detector 5.

[0089] In addition to the above-mentioned components, which may be of conventional configuration, the microscope 1 includes a spatial filter 20. In the example shown in FIG. 1, the spatial filter 20 is formed on the beam splitter 14, thereby being positioned behind the rear aperture of the objective lens 11, and therefore immediately behind the rear focal plane 15 of the objective lens 11. The spatial filter 20 may therefore be constructed without entering the objective lens, as in phase contrast microscopy. Placing the spatial filter just behind the entrance aperture of the objective lens, rather than in a conjugate plane (as described below for example), has the distinct advantage of strongly suppressing back reflections originating from the multiple lenses in high numerical aperture microscope objectives. This in turn reduces imaging noise, lowers incoherent background, and reduces the complexity of the experiment, the number of optical components and the optical path length leading to an increase in the stability of the optical setup and therefore image quality.

[0090] However, this location is not essential and a spatial filter having equivalent functionality may be provided elsewhere, as described below. Spatial filter 20 is thereby positioned to filter the output light passing to detector 5. In the example shown in Figure 1, where detector 5 is aligned with the optical path of objective lens 11, spatial filter 20 is therefore transmissive.

[0091] The spatial filter 20 is partially transparent and therefore passes the output light, including the reflected illumination light, but with a reduction in intensity. The spatial filter 20 is also aligned with the optical axis and has a predetermined numerical aperture, so that it provides a reduction in intensity within the predetermined numerical aperture. Numerical aperture is defined herein in its usual manner as being a dimensionless quantity that characterizes the range of angles relative to the sample position from which the output light originates. In particular, the numerical aperture NA may be defined by the formula NA=n·sin(θ), where θ is the half angle of collection and n is the refractive index of the material (e.g., the material of a component of the optical system 6) through which the output light passes.

[0092] Spatial filter 20 does not provide an intensity reduction outside the predetermined numerical aperture. In principle, spatial filter 20 could alternatively provide a reduction in intensity outside the predetermined numerical aperture, but with a smaller reduction in intensity than the reduction in intensity within the predetermined numerical aperture, although this would be less desirable.

[0093] The spatial filter 20 may be formed in any suitable manner, typically comprising a layer of a deposited material. The material may be, for example, a metal, such as silver. The deposition may be carried out using any suitable technique.

[0094] Because sub-diffraction sized objects near the interface preferentially scatter light to larger numerical apertures than to the reflected illumination light, the reduction in intensity provided by spatial filter 20 preferentially reduces the intensity in the detection of reflected illumination light relative to scattered light. Thus, the reduction in intensity by spatial filter 20 at low numerical apertures primarily affects the reflected illumination light and has minimal effect on the scattered light, thereby maximizing contrast in the captured image. The enhanced imaging contrast allows for high contrast detection of objects that are weak scatterers.

[0095] Contrast enhancement can be understood as follows: When the spatial filter 20 passes a portion of the output light at a predetermined numerical aperture (i.e., is partially transmissive in this example), a portion of the illumination light and scattered light field reaches the detector and interferes with the fully coherent illumination source. The light intensity I reaching the detector det Next, I det =|E inc | 2 {r 2 t 2 +|s| 2 +2rt|s|cosΦ}, and E inc is the incident light field, and r 2 is the reflectance of the interface, and t 2is the transmittance of the spatial filter 20, s is the scattering amplitude of the object, and Φ is the phase difference between the transmitted illumination light and the scattered light. Thus, the scattering contrast is enhanced, but at the expense of the total number of photons detected.

[0096] Thus, contrast is provided in a manner similar to conventional iSCAT, but is additionally controlled by the spatial filter transmittance, t 2 In the case where the spatial filter 20 is a layer of deposited material, the transmittance t 2 may be selected by material choice and / or layer thickness. Such tuning may be performed, for example, according to the scattering object of interest, the full well capacity of the camera, and the magnification.

[0097] To maximize these beneficial effects for iSCAT, the predetermined numerical aperture may be, but is not required to be, the numerical aperture of the reflected illumination light in the output beam. For example, benefits of a similar nature may be achieved if the predetermined numerical aperture is slightly smaller or larger than the numerical aperture of the reflected illumination light.

[0098] The use of spatial filter 20 does not fundamentally change the achievable sensitivity limit or SNR (signal to noise ratio) for scattering with a given object, incident light intensity and exposure time. However, by improving the contrast and reducing the overall detected photon flux, it does dramatically simplify the implementation of iSCAT to achieve a given sensitivity or SNR. Existing iSCAT microscopes have complex and expensive components that require, for example, an optical table, and expensive and complex optics, electronics, as well as specialist operation. Such requirements are greatly alleviated by the use of spatial filter 20. Performance equivalent to existing iSCAT microscopes can be achieved, for example, by simply adding spatial filter 20 to existing commercial microscopes that do not have the complex and expensive components mentioned above. The spatial filter 20 itself is of course a simple and inexpensive component. Additionally, the spatial filter 20 allows the use of standard CMOS or CCD cameras with low full well capacity without loss of imaging sensitivity.

[0099] An image acquired using the example microscope 1 is shown in FIG. 2. In this example, coherent bright field illumination light is provided and spatial filter 20 is 1×10 -2 It consisted of a 180 nm thick layer of silver deposited on fused silica with a diameter of 3.5 mm to transmit a reflected light intensity of 10 kW / cm at an image capture rate of 10 frames / s. 2 ) resulting in a scattering contrast of 1% and an SNR of 10 for a single 395 kDa protein. FIG. 2 is an image captured using a low-cost CMOS camera as detector 5. As can be seen, a high-contrast image is achieved. Furthermore, bright-field illumination ensures that the strongest unwanted back reflections, usually originating from the objective lens, are directed away from detector 5, minimizing imaging background and allowing a large field of view without complex scanning of the illumination light beam.

[0100] The advantage of enhanced contrast allows imaging of very weakly light scattering objects that are difficult to image using other techniques. For example, the present invention may be applied with advantage to samples containing objects having a mass of 5000 kDa or less. Typically, the present invention may be applied to samples containing objects having a mass of 10 kDa or more, for example objects having a mass in the range of 10 kDa to 5000 kDa.

[0101] Alternatively or additionally, the present invention relates to a method for producing a 10 -12 m 2 or less, or more preferably 10 -17 m 2 The present invention may be applied to a sample that includes an object having a scattering cross section with respect to the illumination light of 10 or less. Typically, such an object may also have a scattering cross section with respect to the illumination light of 10 or less. -20 m 2 , or more preferably 10 -26 m 2 or larger, e.g. 10 -17 m 2 ~10 -26 m 2 The scattering cross section may have a scattering cross section for the illuminating light in the range of 1 / 2 to 1 / 2. The scattering cross section is a fundamental, measurable property related to the effective size of an object for incident light of a particular wavelength, independent of the technique used for the measurement. The scattering cross section may be measured, for example, by dark field microscopy. Examples of objects to which the present invention may be applied include proteins or small aggregates thereof, or their binding partners.

[0102] To image objects that are relatively weak scatterers, the spatial filter 20 is 10 -2 ~10 -4 The spatial filter 20 may be configured to pass the reflected illumination light with a reduction in intensity within a predetermined numerical aperture to an intensity within a range of the incident intensity (in this context, the intensity of the output light that is incident on the spatial filter 20).

[0103] In other cases, the microscope 1 may be designed and operated without the spatial filter 20. For example, the field of view can be adjusted by changing the focal condition of the illumination light. Similarly, multicolor imaging does not require more than coupling an additional laser source into a single mode fiber when such a fiber is used to deliver the illumination light. In general, the microscope 1 may be adapted to use other components and techniques known for iSCAT, as disclosed, for example, in Kukura et al., "High-speed nanoscopic tracking of the position and orientation of a single virus," Nature Methods 2009 6:923-935, and Ortega-Arroyo et al., "Interferometric scattering microscopy (iSCAT): new frontiers in ultrafast and ultrasensitive optical microscopy," Physical Chemistry Chemical Physics 2012 14:15625-15636.

[0104] First Method A sample suspected or known to contain biomolecules is placed on a sample holder with a glass surface. The microscope 1 performs multiple measurements of particles in the sample on the surface at a rate of 100 Hz to 1 kHz. Individual particles in contact with the surface scatter the measurement light, generating contrast in the measurement. Each scattering event is localized by fitting the contrast signature to a profile (e.g. a Gaussian profile) and localizing the event to the peak of the fit. Individual particles are tracked using repeated fitting and repeated localization of the contrast signal to create a trajectory of each particle on the surface. This allows "lateral" mobility to be monitored and therefore measured. However, if the contrast signal disappears, the particle is away from the surface, which is indicative of mobility in contact / out of contact with the surface (pointing to a specific feature in terms of interaction with the surface). From these measurements, the distance each biomolecule travels during its interaction with the surface can be calculated, or the length of time the biomolecule interacts with the surface can be measured to determine mobility, and the molecular mass of each biomolecule can be calculated. The normalized counts for distance traveled can be plotted as shown in FIG. 3. How far the biomolecule travels during interaction with the surface can indicate the strength of the interaction with the surface, and therefore can indicate different characteristics about the biomolecule. How often the biomolecule travels away from the surface and / or how long the biomolecule interacts with the surface can also indicate the strength of the interaction with the surface, and can indicate different characteristics about the biomolecule. Either or both measurements represent the mobility of the molecule. As can be seen, the two samples used in FIG. 3 each contained a biomolecule - an adeno-associated virus (AAV): AAV5 and AAV9 viral particle. It can be seen that AAV5 and AAV9 have different mobility on the surface, with AAV9 having a more significant range of mobility ranging from 0 to 10 nm. Different AAV serotypes have variations on the surface of their capsid that may be involved in receptor binding, host tropism, and antigenic determinants.Therefore, the method can determine between AAV5 and AAV9 based on different capsid chemistry because different capsid chemistry changes the mobility on the surface. Therefore, this data can be used to construct a reference profile for AAV5 and / or AAV9 that can be used to determine the serotype of an unknown AAV sample by constructing a profile using the method of the present invention for the unknown AAV sample.

[0105] The surface of the sample holder and / or the coating on the surface of the sample holder may be selected to determine unique features of the biomolecule, either the external surface features or the internal features of the biomolecule may be determined, i.e. by exposing the protein to a hydrophobic surface that can cause unfolding, or by adding a denaturing agent to the sample to intentionally unfold the biomolecule.

[0106] The methods described above use distance traveled as an indicator of mobility, however, time of interaction could equally be used, or alternatively, a combination of time of interaction and distance traveled, e.g., speed of movement.

[0107] Second Method A sample containing immunoglobulin IgG is placed on a sample holder containing a surface, either glass or a glass surface coated with TMCS (a trimethylchlorosilane modified surface), and the mobility is measured (similar to the first method). The microscope 1 performs multiple measurements of particles in the sample on the surface at a rate of 100 Hz to 1 kHz. From these measurements, the distance each biomolecule moves during its interaction with the surface can be calculated to determine the mobility, and the molecular mass of each biomolecule can also be calculated. Normalized counts for the distance traveled can be plotted as shown in Figure 4a and Figure 4b. The frequency, speed and distance of movement of a biomolecule during interaction with the surface can indicate the strength of the interaction with the surface and thus indicate different features for the biomolecule. How often a biomolecule moves away from the surface and / or how long the biomolecule interacts with the surface can also indicate the strength of the interaction with the surface and thus indicate different features for the biomolecule. Either or both measurements represent the mobility of the molecule.

[0108] Immunoglobulin G (IgG) antibodies are large globular proteins with a molecular weight of about 150 kDa that are composed of two different types of polypeptide chains. The approximately 50 kDa polypeptide chain is called the heavy chain, and the other 25 kDa polypeptide chain is called the light chain. Each IgG molecule consists of two heavy chains and two light chains. The two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. IgG may further be glycosylated. Figure 5 illustrates a typical IgG molecule with two heavy chains (dotted bars) and two light chains (striped bars). Each chain has carboxyl (-COOH) and amine (-NH2) groups provided by terminal and internal amino acids. The heavy chains are linked by two disulfide bridges in the hinge region. Each light chain is linked to a respective heavy chain through a disulfide bridge. The amine and carboxyl groups are reactive functional groups present in physiological conditions. Sulfhydryl groups can be generated after reducing disulfide bonds (e.g., by changing conditions to include reducing agents including DTT or TCEP). Carbohydrates can be activated by periodate treatment (an oxidizing agent). A net negative charge is achieved at a pH higher than its pI, which may be achieved using a buffer. Immunoglobulins can then be less mobile on positively charged surfaces because they are "immobilized" by ionic / electrostatic interactions.

[0109] Silanes are silicon chemicals that have a hydrolytically sensitive center with organic substitutions that can react with inorganic substrates (e.g., glass) to form stable covalent bonds and modify the physical interactions of the treated substrate. Some of the properties of surfaces that can be modified with silanes include hydrophobicity, adhesion, release, dielectric, absorption, orientation, hydrophilicity, and / or charge conductivity. Silanization of surfaces involves liquid or vapor phase chemical reactions, is straightforward, and low cost. Silane reactions are most often used to modify hydroxylated surfaces. Glass is rich in hydroxyl groups, making silanes particularly useful for modifying this material. A large number of silane compounds are commercially available, allowing a wide range of chemical functionalities to be incorporated onto surfaces. The advantages of silane reactions are the simplicity and stability that are attributed to the covalent crosslinked structure.

[0110] As can be seen, IgG molecules are relatively mobile on glass, with some IgG molecules moving as far as 400 nm while being monitored. However, in comparison on modified surfaces, the amount of mobility is greatly restricted. This represents a different type of interaction between the IgG molecules and the surface. It is inferred that the IgG on the modified surface forms a covalent bond that restricts mobility on the surface and may further restrict the immunoglobulin from leaving the surface, or that the IgG is partially unfolded, restricting mobility. This mobility of IgG on glass and modified surfaces may be used to construct a reference profile for IgG that can be used as a comparison when data on unknown immunoglobulins are obtained.

[0111] Various further aspects and embodiments of the invention will be apparent to those of skill in the art in view of the present disclosure. Unless the context dictates otherwise, the descriptions and definitions of features set forth above are not limited to any particular aspect or embodiment of the invention, but apply equally to all aspects and embodiments described.

[0112] While the present invention has been described by way of example with reference to certain embodiments, it will be further understood by those skilled in the art that the invention is not limited to the disclosed embodiments, and that alternative embodiments may be constructed without departing from the scope of the present invention as defined in the appended claims.

[0113] "And / or," as used herein, is to be construed as a specific disclosure of each of the two specified features or components, with or without the other. For example, "A and / or B" is to be construed as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each of the following were individually set forth herein.

[0114] All references to publications are incorporated herein by reference. The present invention will now be described with reference to some specific embodiments, which are not intended to limit the scope of the invention. EXAMPLES

[0115] Example 1 Adeno-associated virus (AAV) is a non-pathogenic virus composed of a protein shell that surrounds and protects a small, single-stranded DNA genome. The predicted molecular weight (MW) of an empty AAV capsid is approximately 3700 kDa. For all examples described herein, a mass spectrometer from Refeyn (Oxford, UK) was utilized.

[0116] Approximately 1 × 10 known AAV5 or AAV9 samples 11 pp / ml or 1×10 11The samples were diluted to a final concentration of vg / ml. The aim is to obtain 500-2000 events in the AAV mass range using the mass spectrometer. The sample was placed on a sample holder with a glass slide (in this case an unfunctionalized surface with mixed native charge). The mass spectrometer performs multiple measurements of AAV in the sample on the surface at a rate of 100Hz-1kHz, typically 1kHz. Individual AAV particles in contact with the surface scatter the measurement light, generating contrast in the measurement. Each scattering event is localized by fitting the contrast signature to a profile (e.g. a Gaussian profile) and localizing the event to the peak of the fit. Individual particles are tracked using repeated fitting and repeated localization of the contrast signal to create a trajectory of each AAV particle on the surface. This allows "lateral" mobility to be monitored and therefore measured. However, if the contrast signal disappears, the particle is away from the surface, which is indicative of mobility in contact / out of contact with the surface (pointing to a specific feature in terms of interaction with the surface). From these measurements, the distance each AAV particle travels during its interaction with the surface can be calculated, or the length of time that AAV particles interact with the surface can be measured to determine mobility, and the molecular mass of each AAV particle can be calculated. From these measurements, the distance each AAV particle travels during its interaction with the surface can be calculated to determine mobility, and the molecular mass of each AAV can be calculated (data not shown). As shown in Figure 3, the normalized counts for the distance traveled are plotted. Experimental studies were performed for AAV5 and AAV9 individually, and the results were plotted together to show the difference in mobility. Such data on mobility on glass surfaces can be used to construct reference profiles for AAV5 and AAV9 to allow comparison when samples containing unknown serotypes of AAV are examined on the same surface. Therefore, these AAV serotypes can be distinguished by their mobility on surfaces due to their different capsid chemistry.

[0117] Example 2 A sample of IgG (Sigma Aldrich, UK) was diluted so that 500-2000 events in the IgG mass range were achievable using the mass spectrometer and placed in either a sample holder containing a glass surface or a surface derivatized with trimethylchlorosilane (Gelest, US). As in Example 1, the mass spectrometer performs multiple measurements of IgG in the sample on the surface at rates of 100 Hz to 1 kHz. From these measurements, the distance traveled by each IgG molecule during its interaction with the surface was calculated to determine mobility, along with the molecular mass (data not shown). Normalized counts were plotted against the distance traveled, as shown in Figures 4a and 4b. IgG has substantially reduced mobility on derivatized surfaces compared to glass, consistent with a different mechanism of interaction with the derivatized surfaces. The class and mobility of IgG on derivatized surfaces may be used to construct a reference profile for this IgG molecule. Such a reference profile serves as a comparison with profiles from other immunoglobulins to determine the identity of the unknown immunoglobulin.

[0118] Terms: A. A method for identifying a biomolecule in a sample comprising the steps of contacting the sample with a first surface and taking a plurality of measurements of the biomolecule over time using an interferometric scattering device and using the plurality of measurements to determine the mass and mobility of the biomolecule on the first surface to determine at least one physical characteristic of the biomolecule. B. The method of claim A, further comprising the steps of contacting the sample with a second surface and taking a plurality of measurements of the biomolecule over time using an interferometric scattering device and using the plurality of measurements to determine the mass and mobility of the biomolecule on the second surface to determine at least one physical characteristic of the biomolecule. C. The method of clause B, wherein the first and second surfaces are different. D. The method of any one of clauses A-C, further comprising contacting the sample with a first surface and altering one or more conditions of the sample to determine at least one physical characteristic of the biomolecule. E. The method of clause c, wherein the condition is one that affects binding of the biomolecule to the surface, such as temperature, pH, ionic strength, the presence of a chemical entity such as a detergent, chaotropic agent, reducing agent, buffer, ion, solvent, denaturant, and / or crosslinker. F. The method of any preceding clause, wherein the mass and mobility are used to define a profile for the biomolecule, which may be compared to a reference profile. G. contacting the sample with a first surface and taking a plurality of measurements of the biomolecule over time using an interferometric scattering device and using the plurality of measurements to determine the mass and mobility of the biomolecule on the first surface; contacting the sample with a second surface and taking a plurality of measurements of the biomolecule over time using an interferometric scattering device and using the plurality of measurements to determine the mass and mobility of the biomolecule on the second surface; combining or comparing the measurements on the first and second surfaces to determine the identity of the biomolecule; A method for identifying a biomolecule in a sample according to clause A, comprising: H. contacting the sample with a first surface under a first set of conditions and taking a plurality of measurements of the biomolecule over time using an interferometric scattering device and using the plurality of measurements to determine the mass and mobility of the biomolecule on the first surface under the first conditions; contacting the sample with a first surface under a second set of conditions and taking a plurality of measurements of the biomolecule over time using an interferometric scattering device and using the plurality of measurements to determine the mass and mobility of the biomolecule on the first surface under the second set of conditions; combining or comparing said measurements under said first and second conditions to determine the identity of said biomolecule; A method for identifying a biomolecule in a sample according to clause A, comprising: I. The method of any preceding clause, wherein the surface is selected from any one of the following: glass, diamond, plastic, polymeric material (e.g., cyclic olefin copolymers, polyvinyls, e.g., polyethylene (PE), including polyethylene terephthalate (PET) and high density polyethylene (HDPE) and low density polyethylene (LDPE), polyacrylates (acrylics), polystyrene (PS), including high impact polystyrene (HIPS), silicone, polyester (e.g., polylactic acid (PLA) or polylactic-co-glycolic acid (PGLA)), polyurethane, polypropylene (PP), polyamide (nylon), acrylonitrile butadiene styrene (ABS), polyethylene / acrylonitrile butadiene styrene (PE / ABS), bakelite, rubber, latex, polycarbonate (PC), polycarbonate / acrylonitrile butadiene styrene (PC / ABS), and polyvinyl chloride, including e.g., polyvinylidene chloride (PVDC), and sapphire. J. The method of any preceding clause, wherein the surface is activated, coated, treated and / or derivatized. K. The method of claim J, wherein the surface is activated, coated, treated and / or derivatized with any one or more of functionalized PEG, silane, carboxy-silane, amine, aminosilane, trimethylchlorosilane, aldehyde modification with amine modifying reagents, epoxy modification, carboxylic acid modification, NHS, HOBt, TBTU, PAMAM, diazo, and supramolecules. L. The method of any one of the preceding clauses, wherein the step of determining the mobility of the biomolecule using the plurality of measurements includes determining how long a surface interaction between the biomolecule in the sample and the surface lasts, how far a species in the sample moves along the surface during the surface interaction, or any one or more of these combinations. M. The method of any preceding clause, wherein the physical characteristic is selected from any one or more of charge, pI, size, binding affinity and / or hydrophobicity. N. A method for release testing a biological molecule during production, comprising the step of using a method defined in any one of clauses A through M. O. The method of any preceding clause, which is capable of distinguishing between different variants of a biomolecule.

Claims

1. 1. A method for identifying a biomolecule in a sample using an interferometric light scattering device, comprising: (i) contacting the sample with a first surface and taking a plurality of measurements of the biomolecules on the first surface and using the measurements to determine mobility of the biomolecules on the surface; (ii) repeating step (i) using a second surface to determine the mobility of the biomolecule on said surface; (iii) constructing a profile for the biomolecule using the mobilities of the biomolecule determined in step (i) and step (ii); (iv) comparing the profile obtained in step (iii) with at least one reference profile to identify the biomolecule; The method includes:

2. The method of claim 1 , wherein the first and second surfaces are different.

3. 2. The method of claim 1, wherein the second surface is the same as the first surface, but the sample is contacted with the second surface under at least one altered condition compared to the first surface.

4. the altered conditions are selected from any one of temperature, pH, ionic strength, redox potential, a potential applied between the surface and the solution, and the presence of a reagent, and optionally the reagent is selected from any one or more of a detergent, a chaotropic agent, a reducing agent, a buffer, an ion, a salt, a solvent, a denaturant, and / or a cross-linking agent; The method according to claim 3.

5. The mobility of at least one single biomolecule is tracked using an interferometric light scattering device; and / or The mobility of the biomolecule is (a) the distance traveled by the biomolecule on the surface; (b) the rate of migration of the biomolecule on the surface; (c) the length of time that the biomolecule interacts with the surface; and / or (d) the frequency of interactions of the biomolecule with the surface. is defined by The method of claim 1.

6. 2. The method of claim 1, wherein step (i) may be repeated one or more times with a further surface, the mobility of the biomolecules on which is used to construct the profile in step (iii), and wherein the further surface may be a different surface than the first and / or second surface or may be the same surface, but the sample is contacted under at least one altered condition.

7. 2. The method of claim 1, wherein any one of the surfaces may be selected from any one of the following: glass, diamond, plastic, polymeric materials (e.g. cyclic olefin copolymers, polyvinyls, e.g. polyethylene (PE), including polyethylene terephthalate (PET) and high density polyethylene (HDPE) and low density polyethylene (LDPE), polyacrylates (acrylics), polystyrene (PS), including high impact polystyrene (HIPS), silicones, polyesters (e.g. polylactic acid (PLA) or polylactic-co-glycolic acid (PGLA)), polyurethanes, polypropylene (PP), polyamides (nylons), acrylonitrile butadiene styrene (ABS), polyethylene / acrylonitrile butadiene styrene (PE / ABS), bakelite, rubber, latex, polycarbonate (PC), polycarbonate / acrylonitrile butadiene styrene (PC / ABS), and polyvinyl chloride, including e.g. polyvinylidene chloride (PVDC), and sapphire.

8. Any one of said surfaces may be activated, coated, treated and / or derivatized, and optionally the surface is activated, coated, treated and / or derivatized with any one or more of functionalized PEG, silane, carboxy-silane, amine, aminosilane, trimethylchlorosilane, aldehyde modification with amine modifying reagents, epoxy modification, carboxylic acid modification, NHS, HOBt, TBTU, PAMAM, diazo, and supramolecules; The method of claim 1.

9. The method of claim 1 , wherein any one of the surfaces is a lipid membrane, optionally a lipid bilayer.

10. 2. The method of claim 1, wherein at least one measurement from step (i) or step (ii) is used to determine the mass of the biomolecule.

11. selecting said surface and / or the conditions applied to said surface to determine the physical characteristics of said biomolecules, and optionally The physical characteristic is selected from any one or more of charge, pI, size, binding affinity, lipophilicity and / or hydrophobicity; The method according to any one of claims 1 to 10.

12. 13. A method for production testing of biomolecules during production comprising the step of using a method as defined in claim 1.

13. 13. The method of claim 1 or 12, wherein the biomolecule is a protein, carbohydrate, lipid, nucleic acid, virus or virus-like particle, lipid nanoparticle or lipoplex.

14. Optionally, distinguish between different variants of a biomolecule; The variant is a seroform, an isoform, a glycoform, a lipoform, or a mutant form.

13. The method of claim 1 or 12.

15. 15. The method of claim 14, wherein the variants of the biomolecule have the same mass as measured by an interferometric light scattering instrument.

16. a) the interferometric light scattering device is a mass spectrometer; and / or b) the measurement is performed at an interface between the surface and the sample; and / or c) the biomolecule is unlabeled, and / or d) a fresh sample is used for each surface, and / or e) the reference profile is prepared using known biomolecules; 13. The method of claim 1 or 12.

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