Method for measuring interaction kinetics

Abstract

A method for measuring binding interactions between a first object and a second object using mass photometry comprising detecting the reversible binding of the first object, the second object and / or an entity derived from the interaction of said first and second objects to a modified surface using mass photometry.

Description

[0001] Method for Measuring Interaction Kinetics

[0002] This patent application claims priority from GB patent application no.: 2418668.6 filed on 19 December 2024, the contents of which are hereby incorporated by reference.

[0003] Field of the Invention

[0004] The invention relates to measuring the binding kinetics between objects, for example binding kinetics between biomolecules and / or chemical molecules. Accordingly, the invention provides a method for measuring binding interactions, suitably measuring real-time binding interactions, between objects in solution using mass photometry. Suitably, although not exclusively, the method may be used to measure dynamic binding interactions between objects in solution. Suitably, the method may be used to determine kinetic parameter(s) of binding interactions, suitably kinetic parameter(s) of dynamic binding interactions, for example, rate of association (ka), rate of dissociation (kd), or equilibrium dissociation constant, “affinity”, (KD), suitably such kinetic parameter(s) of binding interactions in real-time, between objects in solution.

[0005] Background of the Invention

[0006] Binding kinetics relates to the binding interactions between two objects, e.g. biomolecules and / or chemical molecules. The accurate and efficient measurement of intermolecular interactions and binding events is a crucial element for many research programmes, e.g. drug discovery programmes. Binding interactions between molecules are typically facilitated by intermolecular forces which enable intermolecular connections, for example, Van der Waals forces, hydrogen bonding, electrostatic forces. Typically, many binding interactions may be non-covalent and reversible. Suitably, some of the binding interactions may be based on covalent bonds. Accordingly, binding interactions between two molecules are typically not static, i.e. dynamic binding interactions occur until an equilibrium is reached.

[0007] Suitably, it is desirable to measure binding interactions between molecules and determine kinetic parameter(s) of the binding interactions. For example, the rate of association (ka), rate of dissociation (kd), and / or equilibrium dissociation constant, “affinity”, (KD), may be used to draw conclusions on selectivity and potency of a potential drug candidate to its chemical structure. It can be more desirable to measure dynamic binding interactions in real-time, rather than determining kinetic parameters of interaction at equilibrium, as dynamic interactions may be more representative of interactions which occur in-vivo and the acquisition of data under these conditions is generally faster as the system does not have to first reach equilibrium.

[0008] Traditional methods for studying the binding interactions of chemical systems (e.g. associations of multiple proteins into complexes and dissociation thereof) include biolayer interferometry and surface plasmon resonance. However, these procedures typically require immobilisation of at least one component of the chemical system on a chip. The immobilisation of a component on a chip may change its kinetics of interaction with other components in the original system. Further, such procedures typically require a bespoke optimised chip for each system being studied. A further shortcoming with such procedures is that they typically do not readily identify multiple interaction paths, the formation of multiple species, or rare binding events.

[0009] More recently, interferometric scattering microscopy (iSCAT), most notably mass photometry, has been used to study binding interactions of biochemical / chemical systems. For example, Soltermann, et al. Angew. Chem. (2020) 59(27) 10774-10779 have studied the kinetics of an IgG-FcRn receptor interaction (glycosylated and deglycosylated IgG) using mass photometry. However, the use of mass photometry as a technique to measure binding interactions between molecules generally remains under-developed. For example, current mass photometry procedures are not suitable for measuring binding interactions, or providing kinetic parameters thereon, at the zero-time point of interaction (i.e. at t = 0 when interaction between two molecules is first initiated). Suitably, current mass photometry procedures may not be suitable for measuring binding interactions, or providing kinetic parameters thereon, at the zero-time point of interaction and for a time immediately thereafter. During this initial time frame of interaction (i.e. from the zero-time point of interaction to a period immediately thereafter) dynamic binding interactions between the molecules typically occurs and the system is typically in a non-equilibrated state. Accordingly, the procedure is typically not suitable for measuring dynamic binding interactions between molecules or providing real-time kinetic parameters of these dynamic binding interactions, during an initial time frame starting at the zero-time point of interaction whilst the system is in a non-equilibrated state. Furthermore, as mass photometry procedures require the components of the chemical system interact with a surface, the concentration of components in the original system may change over time which may affect the validity of the measurements.

[0010] Accordingly, the invention aims to provide an improved mass photometry procedure to measure more accurately and more easily binding interactions, suitably dynamic binding interactions, of multiple objects in solution. Suitably, the procedure may be used to measure binding interactions, such as dynamic binding interactions, of multiple objects in solution in real-time. Suitably, the procedure may be used to measure binding interactions of multiple objects in solution at the zero-time point of interaction (i.e. at t = 0 when interaction between two molecules is first initiated). Suitably, the procedure may be used to measure binding interactions of multiple objects in solution at the zero-time point of interaction and for a time immediately thereafter, suitably a time immediately thereafter when dynamic binding interactions occur between the objects (i.e. when the system is in a non-equilibrated state). The changes to a system are most pronounced immediately following the zero-time point therefore measuring in this time window produces more accurate association and dissociation rates.

[0011] Suitably, the procedure may be used to provide kinetic parameter(s) of binding interactions, suitably kinetic parameter(s) of dynamic binding interactions, for example, rate of association (ka), rate of dissociation (kd), or equilibrium dissociation constant, “affinity”, (KD), suitably such kinetic parameter(s) in real-time, between objects in solution. Suitably, the procedure may be used to provide real-time kinetic parameter(s) of dynamic binding interactions between objects in solution from the zero-time point of interaction to a time immediately thereafter when dynamic binding interactions occur between the objects (i.e. for a time frame starting at the zero-time point of interaction whilst the system is in a non-equilibrated state).

[0012] Summary of the Invention

[0013] According to a first aspect of the invention, there is provided a method for measuring binding interactions between a first object and a second object using mass photometry, the method comprising:

[0014] (i) providing a first solution comprising a first object;

[0015] (ii) providing a second solution comprising a second object;

[0016] (iii) contacting the first solution with the second solution on a modified surface to form a mixture of the first and second solutions on the modified surface, said modified surface comprising a surface which is modified to permit the reversible binding thereto of the first object, the second object or an entity derived from the interaction of said first and second objects, or combinations thereof; and,

[0017] (iv) detecting the reversible binding of the first object, the second object and / or an entity derived from the interaction of said first and second objects to the modified surface using mass photometry; and, wherein at least one of said first object, said second object or an entity derived from the interaction of said first and second objects is detectable by mass photometry.

[0018] Suitably, at least one of the first object or second object has a mass (i.e. molecular mass) that is detectable by mass photometry, such as a mass (i.e. molecular mass) of greater than 10 kDa, such as greater than or equal to 15 kDa, such as greater than or equal to 20 kDa, such as greater than or equal to 25 kDa.

[0019] In an embodiment, either the first object or the second object has a mass (i.e. molecular mass) that is detectable by mass photometry, such as a mass (i.e. molecular mass) of greater than 10 kDa, such as greater than or equal to 15 kDa, such as greater than or equal to 20 kDa, such as greater than or equal to 25 kDa, and the other said object has a mass (i.e. molecular mass) which is itself not detectable by mass photometry but which significantly affects the mass of the first object through interaction, such as a mass (i.e. molecular mass) of greater than or equal to 100 Da, such as greater than or equal to 150 Da, such as greater than or equal to 200 Da, such as greater than or equal to 250 Da, such as greater than or equal to 300 Da, such as greater than or equal to 350 Da and a mass (i.e. molecular mass) of less than 10 kDa, such as less than 7.5 kDa, such as less than 5 kDa, such as less than 2.5 kDa, such as less than 2 kDa. Suitably, the mass (i.e. molecular mass) of the other said object is from 100 Da to of less than 10 kDa, such as 100 Da to 7.5 kDa, such as 100 Da to 5 kDa, such as 100 Da to 2.5 kDa, such as 100 Da to 2 k Da, such as 200 Da to of less than 10 kDa, such as 200 Da to 7.5 kDa, such as 200 Da to 5 kDa, such as 200 Da to 2.5 kDa, such as 200 Da to 2 k Da, such as 250 Da to of less than 10 kDa, such as 250 Da to 7.5 kDa, such as 250 Da to 5 kDa, such as 250 Da to 2.5 kDa, such as 250 Da to 2 k Da, such as 300 Da to of less than 10 kDa, such as 300 Da to 7.5 kDa, such as 300 Da to 5 kDa, such as 300 Da to 2.5 kDa, such as 300 Da to 2 k Da.

[0020] In an embodiment, both the first object and the second object each independently has a mass (i.e. molecular mass) that is detectable by mass photometry. Suitably, the first object, as defined herein, may have a mass (i.e. molecular mass) of greater than 10 kDa, such as greater than or equal to 15 kDa, such as greater than or equal to 20 kDa, such as greater than or equal to 25 kDa. Suitably, the second object, as defined herein, may have a mass (i.e. molecular mass) of greater than 10 kDa, such as greater than or equal to 15 kDa, such as greater than or equal to 20 kDa, such as greater than or equal to 25 kDa. Suitably, both the first object and the second object each independently has a mass (i.e. molecular mass) of greater than 10 kDa, such as greater than or equal to 15 kDa, such as greater than or equal to 20 kDa, such as greater than or equal to 25 kDa.

[0021] Suitably, by contacting the first solution with the second solution on the modified surface provides a mixture of said first and second solutions on the modified surface at the zero-time point of interaction. Thus, the mass photometry procedure may be used to measure binding interactions of multiple objects in solution at the zero-time point of interaction (i.e. at t = 0 when interaction between two molecules is first initiated), thereby gaining insight into the initial stages of the interaction between the objects. Accordingly, the method permits measurement of binding interactions between objects at the zero-time point of interaction.

[0022] Suitably, the method may be used to measure binding interactions, such as dynamic binding interactions, of the first and second objects in real time.

[0023] Suitably, step (iv) of the method is performed in a time-resolved manner (i.e. a series of measurements are made at known time points / delays, e.g. the time between each measurement may be from between 1 millisecond (ms) to 100 seconds (s), thereby providing insight into the binding interactions of an evolving system.

[0024] Suitably, step (iv) of the method is performed when dynamic binding interactions occur between the objects (i.e. when the system is in a non-equilibrated state).

[0025] Suitably, step (iv) of the method is performed in a time-resolved manner and when dynamic binding interactions occur between the objects (i.e. when the system is in a non-equilibrated state).

[0026] Suitably, the method may be used to measure binding interactions of multiple objects in solution at the zero-time point of interaction and for a time immediately thereafter, suitably a time immediately thereafter when dynamic binding interactions occur between the objects (i.e. when the system is in a non-equilibrated state). Suitably, the measurements of binding interactions continue until the system reaches an equilibrium state.

[0027] Suitably, step (iv) is performed essentially immediately after the first solution is contacted with the second solution on the modified surface in step (iii), thereby measuring the binding interactions between the first and second objects at the zero-time point of interaction.

[0028] Suitably, step (iv) of the method is performed essentially immediately after the first solution is contacted with the second solution on the modified surface and then step (iv) is repeated in a time-resolved manner, as disclosed herein.

[0029] Optionally the method may further comprise contacting the first solution, the second solution, or the mixture of the first and second solutions with a third solution comprising a third object on the modified surface and detecting the reversible binding of the first object, the second object, the third object and / or an entity derived from an interaction of said first, second, and / or third objects to the modified surface using mass photometry.

[0030] Suitably, the method includes optional, albeit preferred, step (v) of determining a kinetic parameter of binding interaction, suitably a kinetic parameter of dynamic binding interaction (i.e. when the system is in a non-equilibrated state), between said first and second objects and / or an entity formed by interaction of said first and second objects. Suitably, step (v) involves determining a rate of association (ka), rate of dissociation (kd), or equilibrium dissociation constant (KD), also known as “affinity”, or combination thereof, between said first and second objects and / or an entity formed by interaction of said first and second objects. Suitably, said kinetic parameter(s) of interaction may be determined in real-time and in a time- resolved manner, as disclosed herein. According to an embodiment, step (iii) of the method comprises: a first step where the first solution only is added to the modified surface, and a second subsequent step where the second solution is added to the first solution on the modified surface to form said mixture of first and second solutions on the modified surface. Suitably, the method may include a further optional step of performing a mass photometry measurement of the first solution on the modified surface before the second subsequent step of adding the second solution to the first solution.

[0031] According to an alternative embodiment, step (iii) of the method comprises a single step of simultaneously adding both the first and second solutions to the modified surface to form said mixture of first and second solutions on the modified surface.

[0032] Typical surfaces used for mass photometry measurements, such as glass, tend to bind to objects irreversibly. This means that once an object binds to the surface it does not disassociate therefrom or at least disassociates very slowly. As a result, the surface typically becomes coated with objects and the concentration of objects in solution is depleted. Suitably, the use of a modified surface that permits reversible binding of the first object, second object and / or an entity derived from interaction of said first and second objects thereto reduces and / or mitigates the irreversible binding of said objects and entities derived therefrom to the surface.

[0033] Reversible binding of objects to a surface, as used herein, means that the objects interact with the modified surface transiently and do not irreversibly bind to the surface, for example, to produce a stable coating on the modified surface. Of course, there may be a population of objects bound to the modified surface at any given time, but this population is not static and is in exchange with the population of objects in the bulk solution. Suitably, the concentration of objects in solution may remain essentially constant. The release of bound objects from the modified surface is spontaneous, i.e. a change in conditions or addition of a further entity is not required to trigger the release of bound objects from the modified surface.

[0034] Suitably, the validity of the measurement(s) made, data collected, and determined kinetic parameter(s) of interaction are typically not compromised by significant fluctuations in the concentration of components present in the mixture due to their binding / sequestering on the modified surface.

[0035] Suitably, the modified surface comprises a surface which comprises, is formed from, a glass, diamond, sapphire, cubic zirconia or a niobate. Suitably, the modified surface comprises a surface which includes, is formed from, a glass, such as borosilicate glass.

[0036] Suitably, modification of a surface to permit the reversible binding thereto of the first object, the second object or an entity derived from the interaction of said first and second objects, may be achieved by modifying the physical structure of the surface or by modifying the chemical structure of the surface.

[0037] Suitably, the modified surface may comprise a surface, as defined herein, wherein the physical structure of the surface has been modified by physical and / or chemical means. For example, the physical structure of the surface may be modified by using: a cleaning process, a polishing process, an ion exchange process, a basic washing process, an acidic washing process, a washing process comprising use of Piranha solution, a washing process comprising use of a peroxide solution, a sonication process, a plasma treatment process, a corona treatment process, a flame treatment process, a blasting process, an ablation process, or an etching process.

[0038] Suitably, the modified surface may comprise a surface, as defined herein, wherein the chemical structure of the surface has been modified. For example, the surface may be functionalised with a chemical moiety or the surface may include a coating including a chemical moiety, wherein the chemical moiety reduces and / or mitigates the irreversible binding of said objects and entities derived therefrom to the surface. Suitable chemical moieties which may be used to functionalise the surface and / or included in a surface coating include a chemical moiety selected from: organosilyl moiety, such as a fluorinated organo silyl, a poly(alkenyl) moiety, a polymeric silyl moiety, poly(oxyalkylene)aminosilyl moiety, poly(aminoalkylene)silyl moiety, (polyalkylenyl ether)alkylenyl moiety, (polyvinylpyrrolidone)aminosilyl moiety, poly(ethylene glycol) moiety, poly(ethylene oxide) moiety, zwitterionic silyl moiety, hydroxyl silyl moiety, poly(acrylamide) moiety, poly(L-lysine)- graft-poly(ethylene)glycol polymer moiety, sulfobetaine moiety, poly(sulfobetaine) moiety, carboxybetaine moiety, poly(carboxybetaine) moiety, poly(oxazoline) moiety, poly(2-methyl-2- oxazoline) moiety, poly(2-ethyl-2-oxazoline) moiety, poly(2-isopropenyl-2-oxazoline) moiety, poly(glycerol) moiety, hyperbranched poly(glycerol) moiety, phosphorylcholine moiety, poly(phosphorylcholine) moiety, or poly(2-methacryloyloxyethyl phosphorylcholine) moiety. Suitably, the chemical moiety includes a silyl / silicon containing moiety, such as a moiety selected from organosilyl, such as a fluorinated organo silyl, a polymeric silyl moiety, poly(oxyalkylene)aminosilyl, poly(aminoalkylene)silyl, (polyvinylpyrrolidone)aminosilyl, zwitterionic silyl, hydroxyl silyl. Suitably, the chemical moiety comprises a poly(oxyalkylene)aminosilyl moiety, such as / V, / V-bis(2-hydroxyethyl)-3- aminopropyltriethoxysilane.

[0039] Suitably, the modified surface comprises a surface which is modified such that the release rate of the first object, the second object and / or an entity derived from interaction of said first and second objects from the modified surface is at least 4 times greater than the respective binding rate of the first object, the second object and / or an entity derived from said first and / or second objects to the modified surface. Suitably, said release rate and said binding rate may be determined by mass photometry.

[0040] Suitably, the modified surface comprises a surface which is modified such that less than 20%, suitably less than 10%, suitably less than 5 %, by number of the total number of the first object, the second object and / or an entity derived from interaction of said first and second objects in the mixture is irreversibly bound to the modified surface. For example, when mass photometry detection is performed in step (iv).

[0041] Suitably, the modified surface comprises a surface which is modified such that the residency time of the first object, the second object and / or an entity derived from interaction of said first and second objects in the mixture on the modified surface is less than 30 seconds, such as less than 20 seconds, such as less than 10 seconds. Suitably, the residency time may be determined by mass photometry.

[0042] Suitably, the first object is a molecule, such as a biomolecule or a chemical molecule, suitably a biomolecule, as defined herein.

[0043] Suitably, the second object is a biomolecule or a chemical molecule, suitably a biomolecule, as defined herein.

[0044] Suitably, both the first and second object is a biomolecule, as defined herein.

[0045] Suitably, the second object is a known or putative reaction partner of the first object (e.g. first biomolecule). Suitably, the second object may or may not be a biomolecule (e.g. a synthetic compound which interacts with a protein).

[0046] Suitable biomolecules include a polypeptide, a protein or a fragment thereof, a lipoprotein, a glycoprotein, a protein complex, an antibody or an antibody fragment thereof, an enzyme, a nucleic acid, a protein-nucleic acid complex, a virus or portion thereof, a virus-like particle, a viral vector such, an exosome, a cell, a nanoparticle, a quantum dot, a liposome, a vesicle, a micelle, a lipid, a carbohydrate, a polysaccharide.

[0047] Suitable chemical molecules may include any chemical which binds, or may bind, to another object wherein the binding causes a change in mass detectable by mass photometry. Suitable chemical molecules include drugs or drug candidates, cofactors and enzyme substrates.

[0048] Where the first and / or second object are chemical molecules they are not: a known buffering compound present in solution for the purpose of buffering, optionally selected from phosphate, acetate, citrate, Tris, Bis-Tris, Trizma, TEA, EPPS, AMPD, AMPSO, CHES, CAPSO, AMP, CAPS, CABS, imidazole, carbonate, or a Good’s buffer selected from MES, ADA, PIPES, ACES, MPSO, cholamine chloride, MOPS, BES, TES, HEPES, DIPSO, TAPSO, acetamidoglycine, POPSO, HEPPSO, HEPBS, tricine, glycinamide, glycylglycine, bicine, or TAPS; a known simple salt present in solution for regulation of osmotic or ionic strength, optionally selected from NaCI, KCI, NH4CI, Na2SO4, (NH4)2SO4; an additive present in solution for regulation of the solution’s polarity or viscosity e.g. a simple alcohol, such as ethanol or isopropanol, or glycerol; or a detergent present in solution to maintain an otherwise unstable molecule in solution.

[0049] Suitably, said first solution comprising first said object is an aqueous solution of the first object.

[0050] Suitably, said second solution comprising second said object is an aqueous solution of the second object.

[0051] Suitably, both of said first and second solution is an aqueous solution.

[0052] In an embodiment, there is provided a method for measuring binding interactions between a biomolecule, as defined herein, and a second object, as defined herein, using mass photometry, the method comprising:

[0053] (i) providing a first aqueous solution comprising a biomolecule, as defined herein, wherein the biomolecule is detectable using mass photometry;

[0054] (ii) providing a second aqueous solution comprising a second object, wherein the second object is a known or putative reaction partner of the biomolecule;

[0055] (iii) contacting the first solution with the second solution on a modified surface, as defined herein, to form a mixture of the first and second solutions on the modified surface, said modified surface comprising a surface which is modified to permit the reversible binding thereto of the biomolecule, the second said object or an entity derived from the interaction of the biomolecule and the second said object, or combinations thereof;

[0056] (iv) detecting the reversible binding of the biomolecule, the second object and / or an entity derived from the interaction of the biomolecule and the second said object to the modified surface using mass photometry; and, optionally,

[0057] (v) determining a kinetic parameter of binding interaction, as defined herein, between said first and second biomolecules.

[0058] According to a second aspect, there is provided the use of a modified surface, as defined herein, to measure binding interactions, such as dynamic binding interactions, between a first object, as defined herein, and a second object, as defined herein, in solution using mass photometry, wherein the solution comprising the first and second objects is formed by contacting a first solution of the first object with a second solution of the second object on the modified surface.

[0059] According to a third aspect, there is provided the use of a modified surface, as defined herein, to measure binding interactions between a first object, as defined herein, and a second object, as defined herein, in solution at the zero-time point of interaction using mass photometry, wherein the solution comprising the first and second objects is formed by contacting a first solution of the first object with a second solution of the second object on the modified surface.

[0060] According to a fourth aspect, there is provided a kit of parts including a modified surface suitable for use in mass photometry to measure binding interactions between a first object and a second object in solution, wherein:

[0061] (i) the modified surface comprises, or is formed from, glass, glass derivatives, diamond, sapphire, cubic zirconia or a niobate; and,

[0062] (ii) the modified surface is functionalised with a chemical moiety or comprises a surface coating including a chemical moiety which reduces or mitigates irreversible binding of first said object, second said object and an entity derived from interaction of first and second said objects thereto, and wherein the chemical moiety is selected from: an organosilyl, a fluorinated organosilyl, polyalkylenyl, polymeric silyl, poly(oxyalkylene)aminosilyl, poly(aminoalkylene)silyl, (polyalkylenyl etherjalkylenyl, (polyvinylpyrrolidone)aminosilyl, poly(ethylene glycol), poly(ethylene oxide), zwitterionic silyl, hydroxylic silyl moiety, poly(acrylamide), poly(L-lysine)-graft-poly(ethylene)glycol polymer, sulfobetaine, poly(sulfobetaine), carboxybetaine, poly(carboxybetaine), poly(oxazoline), poly(2-methyl-2- oxazoline), poly(2-ethyl-2-oxazoline), poly(2-isopropenyl-2-oxazoline), poly(glycerol), hyperbranched poly(glycerol), phosphorylcholine, poly(phosphorylcholine), or poly(2-methacryloyloxyethyl phosphorylcholine).

[0063] It will be understood that features of each aspect of the invention may be regarded as features of every other aspect of the invention. Accordingly, any preferred and more preferred features of one aspect of the invention may be independently combined with other preferred and / or more preferred features of the same or different aspects of the invention.

[0064] Brief Description of Figures

[0065] Figure 1 shows data in raw [left-hand panels, measured over 600 s (top) and 575 s (bottom)], cumulative counts of species (right hand panel, top) and counts of species during a 30 s sliding window applied across the measurement time (right hand panel, bottom), for the association of protein A with human IgG. Formation of the resulting human IgG-protein A complex is almost immediate, but larger species take longer to form. At longer times the proportion of human IgG-protein A complex appears to decrease more than the human IgG does, suggesting it is important in forming higher order oligomers. Thus, the present invention determines the kinetics of association and dissociation which informs on the mechanism.

[0066] Figure 2 shows data in raw (left-hand panels, measured over 330 s), cumulative counts of species (right-hand panel, top) and counts of species during a 60 s sliding window applied across the measurement time (right-hand panel, bottom), for the dissociation of human IgG- protein A complex into protein A with human IgG after initial dilution. Relatively little change is observed in the proportions of species.

[0067] Figure 3 shows data in raw [left-hand panels, measured over 300 s (top) and 270s (bottom)], cumulative counts of species (right-hand panel, top) and counts of species during a 60 s sliding window applied across the measurement time (right-hand panel, bottom) for the dissociation of human IgG-protein A complex into protein A with human IgG after further dilution over the dilution described in Figure 2. A large reduction in the amount of species is observed as lots of protein A is released from larger species, showing the effect of dilution on breakdown of complexes.

[0068] Figure 4 shows counts of species over 30 s sliding window for association of protein A and human IgG and fits to data using a kinetic model to determine kaand kd for formation of individual species.

[0069] Detailed Description of the Invention

[0070] The present disclosure relates to a method for measuring the interaction kinetics of a first object with a second object, the method comprising contacting a first solution comprising the first object and a second solution comprising the second object to form a third solution comprising the first and second objects; wherein the first and second solutions are contacted on a modified surface; and wherein the interaction kinetics of the first object with the second object are determined by detecting the reversible binding of the first object, the second object and / or any entities derived therefrom to the modified surface using mass photometry.

[0071] Interaction kinetics which can be measured by this method include associations where objects or entities bind to form larger entities, and dissociations where objects or entities come apart to form smaller entities.

[0072] The First Object

[0073] The first object may be any object which has a mass that can be reliably detected and quantified by mass photometry or an object which forms part of, or is hypothesised to form part of, an entity which has a mass that can be reliably detected and quantified by mass photometry. Suitably, the first object should therefore have a mass (i.e. molecular mass) greater than 10 kDa, such as greater than or equal to 15 kDa, such as greater than or equal to 20 kDa. Suitably, the first object may have a mass (i.e. molecular mass) of less than or equal to 500 kDa, such as less than or equal to 450 kDa, less than or equal to 400 kDa.

[0074] Suitably, the first object may be selected from a biomolecule, a polypeptide, a protein or a fragment thereof, a lipoprotein, a glycoprotein, a protein complex, an antibody or an antibody fragment thereof, an enzyme, a nucleic acid, a protein-nucleic acid complex, a virus or portion thereof, a virus-like particle, a viral vector such, an exosome, a cell, a nanoparticle, a quantum dot, a liposome, a vesicle, a micelle, a lipid, a carbohydrate, a polysaccharide, an organic polymer, a chemical molecule or compound.

[0075] In some embodiments, the first object may be selected from the group consisting of a single molecule, a macromolecule, a supermolecule, or a complex of molecules, macromolecules (such as polymers) and supermolecules. Examples of suitable macromolecules may include, but are not limited to, nucleic acid molecules, either natural nucleic acids such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or artificial nucleic acids such as peptide nucleic acid (PNA), morpholino and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Complexes can include assemblies such as viruslike particles where envelope or capsid proteins are associated.

[0076] Suitably, the first object may be selected from the group consisting of a protein, nucleic acid, polysaccharide, PEG-based polymer, pullulan-based nanogel or polymer, glycopolymer, conjugated polymer, polymer having either positive or negative charge, polyampholyte, zwitterionic polymer, vesicle, virus, virus-like particle, particulate polymer conjugate, polymeric nanoparticle, liposome, micelle, dendrimer, carbon nanotube and gold nanoparticle.

[0077] When the first object is a protein (or peptide), the protein may be selected from the group consisting of: a protein which is found in nature (i.e. is naturally occurring), such as from a plant, animal, virus, bacterium or fungus (i.e. catabolised, anabolised or existing in a wild-type or diseased state thereof), a protein which has been altered from that which is naturally occurring, as defined above, a post-translationally modified form of said naturally occurring or altered protein, and a de novo designed protein.

[0078] When a protein is a protein which is found in nature, it may be selected from the group consisting of an enzyme, antibody, structural protein, messenger protein, peptide hormone, receptor, signal transduction protein, transport protein, motor protein, glycoprotein, lipopeptide, antimicrobial peptide, peptide hormone, opiate peptide, neurotrophic factor, antibiotic peptide, bacteriocin, microcin and peptide amphiphile, or a fragment or prepropeptide of any of the above proteins or peptides.

[0079] When a protein is a protein which is altered from that which is naturally occurring, said altered protein typically shares >80% sequence homology with a naturally occurring protein, such as >90% sequence homology, such as >95% sequence homology with a naturally occurring protein, yet comprises at least one alteration ( / .e. a chemical alteration) to the primary structure over said naturally occurring protein.

[0080] When a protein is a protein which is a post-translationally modified form of a protein which is found in nature or of a protein which is altered from that which is naturally occurring, as defined above, said post-translational modification comprises at least one a chemical or physical alteration selected from the group consisting of: denaturation of a naturally occurring protein or of a protein which is altered from that which is naturally occurring, as defined above; formation of a complex with a naturally occurring protein or of a protein which is altered from that which is naturally occurring, as defined above; phosphorylation, hydroxylation, sulfonation, palmitoylation or glycosylation of a naturally occurring protein or of a protein which is altered from that which is naturally occurring, as defined above; formation of a disulfide bridge within a naturally occurring protein or of a protein which is altered from that which is naturally occurring, as defined above; and formation of a tag on a naturally occurring protein or of a protein which is altered from that which is naturally occurring, as defined above. Suitably, a protein which is post-translationally modified may be a denatured, complexed, phosphorylated, hydroxylated, sulfonated, palmitoylated, glycosylated, disulfidated or tagged form of a protein which is found in nature or of a protein which is altered from that which is naturally occurring, as defined above.

[0081] Denaturation of a naturally occurring protein or of a protein which is altered from that which is naturally occurring, as defined above, typically results in at least one change to the secondary, tertiary and / or quaternary structure of said protein and may take place in the presence of an acid (pH < 7 at 20 °C), a base (pH > 7 at 20 °C), a concentrated inorganic salt (e.g. sodium chloride), an organic solvent (e.g. methanol, ethanol or chloroform), agitation (e.g. ultrasonication), and / or radiation (e.g. heat > 40 °C).

[0082] When a protein is a complexed form of a protein which is found in nature or a complexed form of a protein which is altered from that which is naturally occurring, as defined above, said protein is comprised in a complex formed by complexing said protein with at least one compound.

[0083] Complexing of the protein with said at least one compound ( / .e. formation of a protein-ligand complex) may be via a non-covalent or a covalent bonding interaction. In particular, binding of the compound to the protein may be via intermolecular forces such as an ionic bond, hydrogen bond and van der Waals force, or by a o-bond, TT-bond, metal-to- metal bond, agostic interaction or three-center two-electron bond. Binding of the compound to the protein may be reversible or irreversible. Suitably, the compound is bound to the protein via a reversible bond.

[0084] Suitably, each compound with which the protein may be complexed is independently selected from the group consisting of: a protein or peptide, a lipid, a nucleic acid, a nucleoside, a nucleotide, a polysaccharide, a monosaccharide, a metabolite, a drug, a drug-fragment, a potential drug, a metal and a moiety comprising a metal. Suitably, each compound is independently selected from the group consisting of: a protein or peptide, a nucleic acid, a lipid, a polysaccharide, a metabolite, a drug, a drug candidate, a drug-fragment, a metal and a moiety comprising a metal.

[0085] When the protein is comprised in a complex formed by complexing it with at least one other protein ( / .e. at least one compound complexed with said protein is a protein or peptide), each protein may be independently selected from a naturally occurring protein or a protein which has been altered from that which is naturally occurring, as defined above. Suitably, each protein in said complex is the same protein ( / .e. the complex is an n-mer of said protein, wherein n represents the number of units of said protein in said complex). For example, when the complex comprises 4 units of protein it is a tetramer. Even more preferably, each protein comprised in the complex is a native protein.

[0086] When the protein is comprised in a complex formed by complexing it with at least one nucleic acid ( / .e. at least one compound complexed with said protein is a nucleic acid), each nucleic acid may be independently selected from a deoxyribonucleic acid or ribonucleic acid, suitably a deoxyribonucleic acid or ribonucleic acid encoding a naturally occurring protein or a protein which has been altered from that which is naturally occurring, as defined above.

[0087] When the protein is comprised in a complex formed by complexing it with at least one nucleoside ( / .e. at least one compound complexed with said protein is a nucleoside), each nucleoside may be independently selected from the group consisting of: a deoxyribonucleoside and a ribonucleoside. Suitably, each nucleoside is independently selected from the group consisting of: adenosine, deoxyadenosine, guanosine, deoxyguanosine, 5-methyluridine, thymidine, uridine, deoxyuridine, cytidine and deoxycytidine.

[0088] When the protein is comprised in a complex formed by complexing it with at least one nucleotide ( / .e. at least one compound complexed with said protein is a nucleotide), each nucleotide may be independently selected from the group consisting of: a nucleoside monophosphate, a nucleoside diphosphate and a nucleoside triphosphate, wherein said nucleoside is as defined above.

[0089] When the protein is comprised in a complex formed by complexing it with at least one lipid ( / .e. at least one compound complexed with said protein is a lipid), each lipid may be independently selected from the group consisting of: a fatty acid, glycerolipid, glycerophospholipid, sphingolipid, saccharolipid, polyketide, sterol lipid and prenol lipid, or a salt thereof. Suitably, each lipid is independently selected from the group consisting of: a monoglyceride, diglyceride, triglyceride, formic acid, acetic acid, propionic acid, butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, decandioic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, oleic acid, linoleic acid, a-linolenic acid, y-linolenic acid, stearidonic acid, nonadecanoic acid, icosanoic acid, (5Z,8Z, 77Z)-eicosa-5,8, 11 -trienoic acid, arachidonic acid, eicosapentaenoic acid, heneicosanoic acid, docosanoic acid, phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine.

[0090] When the protein is comprised in a complex formed by complexing it with at least one polysaccharide or monosaccharide ( / .e. at least one compound complexed with said protein is a polysaccharide or monosaccharide), each polysaccharide may be independently selected from the group consisting of: a homopolysaccharide, heteropolysaccharide, glycoprotein and glycolipid and each monosaccharide is preferably independently selected from the group consisting of: an aldose and a ketose. Suitably, each polysaccharide is independently selected from the group consisting of a starch, glycogen, galactogen, inulin, an arabinoxylan, cellulose, chitin, a pectin, an acidic polysaccharide, a bacterial polysaccharide and a lipopolysaccharide, and each monosaccharide is independently selected from the group consisting of: glucose, fructose, mannose, galactulose, ribose, deoxyribose, erythrulose, xylose, rhamnose, furanose, lyxose, altrose, galactoseamine, glucosamine, sialic acis, N- acetylglucosamine, sulfoquinovose, ascorbic acid, mannitol, glucuronic acid and glyceraldehyde, wherein said monosaccharides and the monosaccharide subunits of said polysaccharides are each independently selected from D- or L-monosaccharides.

[0091] For the purposes of the present invention, a drug may equate with a therapeutic agent, while a drug candidate may equate with a potential therapeutic agent or drug candidate and a drugfragment may equate with a fragment of a therapeutic agents such as an ion, salt or metabolite of a drug or drug candidate. When the protein is comprised in a complex formed by complexing it with at least one drug, drug candidate or drug-fragment ( / .e. at least one compound complexed with said protein is a drug, drug candidate or drug-fragment), each respective drug, drug candidate or drug-fragment may be a molecule that interacts with (e.g. binds to) said protein. Suitably, said drug, drug candidate or drug-fragment is a substrate, agonist, antagonist, inverse agonist or neutral agonist of said protein, or is a cofactor to said protein. Said drug may be in clinical use or clinical trials.

[0092] The drug, drug candidate or drug-fragment may be an active compound or ion, salt or metabolite of a drug or drug candidate, which, when administered to an organism (human or non-human animal), induces a desired pharmacologic, immunogenic, and / or physiologic effect by local and / or systemic action. Examples of drugs, drug candidates or drug-fragments include, without limitation, vaccines and biopharmaceutical agents. Thus, drug, drug candidate or drug-fragment may include small-molecule therapeutic agents, therapeutic proteins, peptides and fragments thereof (whether naturally occurring, chemically synthesised or recombinantly produced), nucleic acid molecules (including both double-and single-stranded molecules, gene constructs, expression vectors, antisense molecules and the like) and neurotransmitters. The drugs, drug candidate or drug-fragment may be a synthetic or naturally occurring compound.

[0093] Particular examples of drugs, drug candidates and drug-fragments include, but are not limited to, anti-cancer agents, anti- infective agents (e.g. antibiotics and antiviral agents), analgesic agents, anorexic agents, anti-inflammatory agents, antiepileptic agents, anaesthetic agents, hypnotic agents, sedatives, antipsychotic agents, neuroleptic agents, antidepressants, anxiolytics, antagonists, neuron blocking agents, anticholinergic and cholinomimetic agents, antimuscarinic and muscarinic agents, antiadrenergics agents, hormones, nutrients, antiarthritics agents, antiasthmatic agents, anticonvulsants, antihistamines, antinauseants agents, antineoplastic agents, antipruritics agents, antipyretic agents; antispasmodic agents, cardiovascular agents (e.g. calcium channel blockers, beta-blockers, beta-agonists, antiarrhythmic agents, antihypertensive agents, diuretics and vasodilators), central nervous system stimulants; decongestants, hormones, bone growth stimulants, bone resorption inhibitors, immunosuppressive agents, muscle relaxants, psychostimulants, sedatives and tranquilisers. It will be appreciated that this list of drugs, drug candidates and drug-fragments is merely illustrative and should not be considered to be limiting. Many other drugs, drug candidates and drug-fragments are known in the art and may be utilised in a method of the present invention. A detailed description of various drugs may be found in e.g. Remington's Pharmaceutical Sciences (21st edition, 2005, Mack Publishing Company).

[0094] The drug, drug candidate or drug-fragment may exhibit optical isomerism and / or diastereoisomerism. Accordingly, the drug, drug candidate or drug-fragment may be in the form of a single enantiomer or diastereoisomer, or a mixture (e.g. a racemic mixture) thereof. In an embodiment, the drug, drug candidate or drug-fragment has a molecular weight of less than 2000 Daltons, e.g. less than 1500 Daltons, e.g. less than 1000 Daltons, e.g. less than 500 Daltons. In an embodiment, the drug, drug candidate or drug-fragment is a non-polymeric organic compound having a molecular weight of less than 1000 Daltons, e.g. less than 800 Daltons, e.g. less than 500 Daltons.

[0095] When the protein is comprised in a complex formed by complexing it with at least one metabolite ( / .e. at least one compound complexed with said protein is a metabolite), each metabolite may be a molecule that interacts with (e.g. binds to) said protein and results from anabolism or catabolism of a protein or peptide, a nucleic acid, a lipid, a polysaccharide, or a drug, drug candidate or drug-fragment, which is respectively defined as per the above definitions of a protein or peptide, nucleic acid, lipid, polysaccharide, or drug, drug candidate or drug-fragment capable of forming a complex with said protein.

[0096] When the protein is comprised in a complex formed by complexing it with at least one metal or a moiety comprising a metal ( / .e. said protein is comprised in a metalloprotein complex), said metal or moiety comprising a metal may be or comprises, respectively, a metal or metal ion (e.g. iron, sodium, potassium, calcium, aluminium, cobalt, manganese, chromium, copper).

[0097] When a protein is a tagged form of a protein which is found in nature or a tagged form of a protein which is altered from that which is naturally occurring, as defined above, said protein is post-translationally modified by appending an affinity tag, solubilisation tag, chromatography tag, epitope tag, fluorescence tag or protein tag thereto.

[0098] When the first and / or second object is a nucleic acid, each nucleic acid may be independently selected from a deoxyribonucleic acid or ribonucleic acid, such as a deoxyribonucleic acid or ribonucleic acid encoding a naturally occurring protein or a protein which has been altered from that which is naturally occurring, as defined above. The deoxyribonucleic acid or ribonucleic acid may be single stranded, partially double stranded or double stranded.

[0099] When the first object is a polysaccharide, the polysaccharide may be selected from the group consisting of a homopolysaccharide, heteropolysaccharide, glycoprotein and glycolipid. Suitably, the polysaccharide may be independently selected from the group consisting of starch, glycogen, galactogen, inulin, an arabinoxylan, cellulose, chitin, a pectin, an acidic polysaccharide, a bacterial polysaccharide and a lipopolysaccharide.

[0100] When the first object is a PEG-based polymer, pullulan-based nanogel or polymer, glycopolymer, conjugated polymer, polymer having either positive or negative charge, polyampholyte or zwitterionic polymer, the PEG-based polymer, pullulan-based nanogel or polymer, glycopolymer, conjugated polymer, polymer having either positive or negative charge, polyampholyte or zwitterionic polymer may be selected from the types disclosed in Rajan, R. et al. Mater. Adv. (2021) 2, 1139-1176.

[0101] When the first object is a vesicle, the vesicle is may be selected from the group consisting of a bilayer liposome, a unilamellar liposome, a micelle and a reverse micelle. Suitably, said vesicle comprises phospholipids and is between 20 nm and 200 pm in diameter.

[0102] When the first object is a virus, the virus may be selected from the group consisting of a DNA virus, RNA virus and a reverse transcription virus.

[0103] When the first object is a virus-like particle, the virus-like particle may comprise viral proteins from one of the aforementioned types of virus, but may lack any genetic material.

[0104] When a first object is a particulate polymer conjugate, polymeric nanoparticle, liposome, micelle, dendrimer, carbon nanotube or gold nanoparticle, the particulate polymer conjugate, polymeric nanoparticle, liposome, micelle, dendrimer, carbon nanotube or gold nanoparticle is a nanoparticle, suitably a synthetic (man-made) nanoparticle, such as a nanoparticle or nanocage of the type disclosed in Zaman, M. et al. Int. J. Nanomed. (2014) 9, 899-912.

[0105] The Second Object

[0106] The second object may be any object which significantly affects, or is hypothesised to significantly effect, the mass of the first object through an interaction. In this context, “significantly effects” means the mass of the first object is changed to a degree which is detectable by mass photometry.

[0107] Optionally, like the first object, the second object has a mass that can be reliably detected and quantified by mass photometry. Therefore, the second object may have a mass (i.e. molecular mass) of greater than 10 kDa, such as greater than or equal to 15 kDa, such as greater than or equal to 20 kDa. Suitably, the second object may have a mass (i.e. molecular mass) of less than or equal to 500 kDa, such as less than or equal to 450 kDa, less than or equal to 400 kDa.

[0108] Optionally, and alternatively, the second object, for example a chemical molecule such as a drug candidate, may have a mass that is not readily detectable per se by mass photometry, for example less than 5 kDa, but the second object does have a mass which significantly affects the mass of the first object through interaction. Suitably, the second object may have a mass (i.e. molecular mass) of greater than or equal to 100 Da, such as greater than or equal to 150 Da, such as greater than or equal to 200 Da, such as greater than or equal to 250 Da, such as greater than or equal to 300 Da, such as greater than or equal to 350 Da and a mass (i.e. molecular mass) of less than 10 kDa, such as less than 7.5 kDa, such as less than 5 kDa, such as less than 2.5 kDa, such as less than 2 kDa.

[0109] Suitably, at least one of the first object or second object may have a mass (i.e. molecular mass), as defined herein, that is detectable by mass photometry.

[0110] The second object may be independently selected from any of the lists from which the first object is selected. Alternatively, the second object may be selected from: a drug, drug candidate, drug-fragment, substrate acid, base or a biochemically reactive agent.

[0111] As used to define the second object a drug or drug candidate or drug-fragment may be a substrate, an agonist, an antagonist, an inverse agonist or a neutral agonist of the first object or is a cofactor to said first object. Said drug may be in clinical use.

[0112] As used to define the second object, a substrate may be an enzyme substrate.

[0113] As used to define the second object an acid may be an organic acid, a mineral acid or a Lewis acid. Said organic acid may be selected from the group consisting of an amino acid, a fatty acid, formic acid, acetic acid, propionic acid, butanoic acid, 2-methyl propanoic acid, pentanoic acid, 3-methylbutanoic acid, 2-methylbutanoic acid, hexanoic acid, 2,3-dimethylbutanoic acid, 3,3-dimethylbutanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, decandioic acid, undecanoic acid, dodecanoic acid, tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, oleic acid, linoleic acid, a-linolenic acid, y-linolenic acid, stearidonic acid, nonadecanoic acid, icosanoic acid, (5Z,8Z,11Z)-eicosa-5,8,11-trienoic acid, arachidonic acid, eicosapentaenoic acid, heneicosanoic acid, docosanoic acid, cervonic acid, lactic acid, formic acid, citric acid, c / s- aconitic acid, frans-aconitic acid, isocitric acid, oxalic acid, uric acid, glucuronic acid, glycolic acid, glyoxylic acid, glyceric acid, pyruvic acid, malonic acid, methylmalonic acid, tartronic acid, crotonic acid, D-2-hydroxybutyric acid, L-2-hydroxybutanic acid, D-3-hydroxybutyric acid, L-3-hydroxybutyric acid, 4-hydroxybutanoic acid, L-DOPA, 2-oxobutanoic acid, 3-oxobutanoic acid, succinic acid, succinic semialdehyde, fumaric acid, maleic acid, malic acid, tartaric acid, oxaloacetic acid, dioxosuccinic acid, 3-hydroxypentanoic acid, 4-hydroxypentanoic acid, p- hydroxy p-methylbutyric acid, glutaric acid, 2-oxopentanedioic acid, 3-oxopentanedioic acid, furan-2-carboxylic acid, hexanedioic acid, sorbic acid, pimelic acid, benzoic acid, salicylic acid and a beta-lactam antibiotic. Said mineral acid may be selected from the group consisting of hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid and boric acid. Said Lewis acid is an electrophile, such as selected from the group consisting of a H+ion, a Li+ion, a Na+ion, a K+ion, a Mg2+ion, a Ca2+ion, a Mn2+ion, a Co2+ion, a Ni2+ion, a Fe3+ion, a Fe4S4 cluster, a FesS4 cluster, or a complex comprising any of said ions or clusters such as chlorophyll A, ferredoxins, haemoglobin, vitamin B12, and NADH dehydrogenase. Said amino acid may be an alpha-amino acid or a beta-amino acid, such as a proteinogenic amino acid. Said fatty acid may be (i) a short chain fatty acid selected from the group consisting of formic acid, acetic acid, propionic acid, butanoic acid, 2-methyl propanoic acid, pentanoic acid, 3-methylbutanoic acid and 2-methylbutanoic acid, (ii) a medium chain fatty acid selected from the group consisting of hexanoic acid, 2,3-dimethylbutanoic acid, 3,3-dimethylbutanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, decandioic acid, undecanoic acid and dodecanoic acid, or (iii) a long chain fatty acid selected from the group consisting of tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, heptadecanoic acid, octadecanoic acid, oleic acid, linoleic acid, a-linolenic acid, y-linolenic acid, stearidonic acid, nonadecanoic acid, icosanoic acid, (5Z,8Z,11Z)-eicosa-5,8,11-trienoic acid, arachidonic acid, eicosapentaenoic acid, heneicosanoic acid, docosanoic acid, cervonic acid, tetracosanoic acid and hexacosanoic acid.

[0114] As used to define the second object a base may be a Lewis base, a hydroxide of a quaternary ammonium compound, or a conjugate base of any acid defined above. Said Lewis base is a compound capable of sharing a lone pair of electrons with a Lewis acid, as defined hereinabove, to form a Lewis adduct, and may be selected from the group consisting of an amine of the formula NH(3-X>AXwhere A = alkyl or aryl and x is a whole number selected from between 0 and 3, pyridine, a phosphine of the formula P (3-X)A”X’, where A’ = alkyl, A” = aryl and x’ is a whole number selected from between 0 and 3, a compound comprising O, S, Se or Te in a -2 oxidation state such as water, ethers and ketones, an OH- ion, a SH_ion, a Cl- ion, a Bn ion, an I- ion, pyridine, imidazole, histidine, urea and guanidine. Said hydroxide of a quaternary ammonium compound may be selected from the group consisting of tetramethylammonium hydroxide, tetraethylammonium hydroxide, tetrapropylammonium hydroxide, tetrabutylammonium hydroxide and choline hydroxide.

[0115] As used to define the second object, a biochemically reactive agent may be selected from: a cross-linking reagent, a succinimide based label or other similarly active labels.

[0116] Suitably, the second object is a known or putative interaction partner of the first object.

[0117] In preferred embodiments, the first object is a protein and the second object is a protein; or the first object is a protein and the second object is a nucleic acid, polysaccharide, PEG-based polymer, pullulan-based nanogel or polymer, glycopolymer, conjugated polymer, polymer having either positive or negative charge, polyampholyte, zwitterionic polymer, vesicle, virus, virus-like particle, particulate polymer conjugate, polymeric nanoparticle, liposome, micelle, dendrimer, carbon nanotube or gold nanoparticle; or the first object is a nucleic acid, polysaccharide, PEG-based polymer, pullulan-based nanogel or polymer, glycopolymer, conjugated polymer, polymer having either positive or negative charge, polyampholyte, zwitterionic polymer, vesicle, virus, virus-like particle, particulate polymer conjugate, polymeric nanoparticle, liposome, micelle, dendrimer, carbon nanotube or gold nanoparticle and the second object is a protein; or the first object is a nucleic acid and the second object is a virus or virus-like particle; or the first object is a virus or virus-like particle and the second object is a nucleic acid.

[0118] The effect of the second object on the mass of the first object may be direct, i.e. the second object binds to the first object to form another entity which has a mass sufficiently distinct from the mass of the first object. An example of a direct effect is where the first (01) and second (02) objects are different medium sized proteins which form a heterodimer (E1) which has significantly higher mass.

[0119] 01 + 02-> E1

[0120] The effect of the second object may alternatively be indirect, i.e. the second object binds to the first object to form an entity, which may or may not be distinct from the first object, and wherein the entity then undergoes a change such as a dissociation or further assembly with other objects. An example of an indirect effect is where the first object (01) is a protein and the second object (02) is a cross-coupling reagent. The entity (E1‘) formed by the reaction of the protein with the cross-coupling reagent may not be distinguishable from the first object using mass photometry, however the interaction of the entity with another object or entity (which in this example is another first object) may form a cross-coupled entity (E2) which is able to be distinguished from the first object.

[0121] 01 + 02 -> E1*

[0122] ET + 01 -> E2

[0123] Another example of an indirect effect is where the first object is a multimer (01) and the interaction of the second object (02) with the first object forms an entity (E1‘) which then dissociates into at least 2 entities (E3 & E4).

[0124] 01 + 02 -> E1*

[0125] E1* -> E3 + E4

[0126] Where the effect of the second object is indirect, other kinetic information may need to be known in order to extract the kinetics of interaction of the first and second objects. For example, with the dissociation example above, the rate of dissociation of the E1* entity into Entities E3 and E4 must be known independently in order to extract the rate of association of 01 and 02.

[0127] The interactions above are shown here as irreversible reactions however they may be, and in practise are likely to be, reversible interactions for example:

[0128] 01 + 02 <-> E1

[0129] In this case there is a forward reaction and a reverse reaction. The rate constant of the forward reaction is the rate constant of association (ka). The rate constant of the reverse reaction is the rate constant of dissociation (kd). Given enough time this system will reach an equilibrium where the concentrations of each object and entity are stable. The equilibrium dissociation rate constant Kd is calculated by the following equation for the system at equilibrium:

[0130] _ kd_

[0001]

[0002] D" ka" [E1]

[0131] The method of the claimed invention may be used to determine any one or more of ka, kd, KD, the half-life of association or the half-life of dissociation. Equilibrium kinetics can mask a much more complex set of interactions which are only able to be determined under non-equilibrium conditions.

[0132] Non-equilibrium conditions are described by coupled differential equations which, for a simple association are: d

[0001] / dt = -a

[0001]

[0002] + E1] d

[0002] / dt = -a

[0001]

[0002] +d[E1] d[E1] / dt =a

[0001]

[0002] - E1]

[0133] The association and dissociation constants and other kinetic parameters may be derived by fitting to time-resolved evolutions of the concentrations of the objects and entities.

[0134] The third object

[0135] The third object, when present, is an object meeting any of the definitions of the first or second object, i.e. the third object may be independently selected from any of the lists from which the first object is selected. Alternatively, the third object may be any object which significantly affects, or is hypothesised to significantly effect, the mass of the first object, the second object, or an entity derived therefrom through an interaction. In this context, “significantly effects” means the mass of the first object, the second object, or an entity derived therefrom is changed to a degree which is detectable by mass photometry. Optionally, like the first object, the third object has a mass that can be reliably detected and quantified by mass photometry. Therefore, the third object may have a mass greater than 10 kDa.

[0136] In an embodiment the first object is a bispecific antibody and the second and third objects are antigens.

[0137] The Solutions

[0138] Before the start of the method of the present invention, the first and second objects are comprised in first and second separate solutions respectively. The solutions comprise a liquid medium which can solublise or suspend the first and / or second object. The mediums of the first and second solutions may be the same or different. The mediums of the first and second solution may be independently selected from water, an organic solvent or a solution of water and an organic solvent, wherein said water is regular water or a solution comprising regular water and deuterated water or tritiated water, preferably at a temperature of between 1 and 50 °C. Said organic solvent may be selected from the group consisting of ethanol, ethanal, ethanoic acid, methanol, terf-butanol, ethyl acetate, acetone, dimethylsulfoxide, ethylene glycol and dimethyl formamide. Suitably, both the first and second solutions comprise an aqueous solution, such as a solution formed from water, such as regular water.

[0139] The mediums of the first and second solutions may be aqueous buffer solutions comprising water and a biologically suitable buffering component. Suitably, said aqueous buffer solution has a pH in the range of 2 to 12. Said aqueous buffer solutions may be selected from the group consisting of an aqueous phosphate buffer solution, an aqueous HEPES [2[4-( hydroxyethyl)piperazin-1ethanesulphonic acid] buffer solution, an aqueous carbonate buffer solution, an aqueous acetate buffer solution, an aqueous citrate buffer solution, an aqueous citrate-phosphate buffer solution, an aqueous phosphate-buffered saline solution (e.g. Dulbecco’s phosphate buffered saline solution) an aqueous phosphate-albumin buffered saline solution, an aqueous tris buffer solution, an aqueous tris-acetate buffer solution and an aqueous tris-chloride buffer solution.

[0140] Suitably, the aqueous buffer solution may further comprise at least one salt selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, magnesium chloride, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium hydrogen carbonate and sodium citrate, wherein the salt may be present in a concentration of from 1 nM to 2M.

[0141] The aqueous buffer solution optionally may comprise D-glucose, citric acid and / or tris(hydroxymethyl)aminomethane (tris). The first and second solutions may comprise small molecules which may or may not be involved in the interaction of the first and second objects.

[0142] The contacting of the first and second solutions may be achieved using a pipette, autopipette, syringe (and needle), capillary, or flow cell channel or any other suitable means available to the skilled person.

[0143] Mass Photometry

[0144] The method of the invention uses mass photometry which is an application of interferometric scattering microscopy (iSCAT). iSCAT is a powerful analytical technology for single-molecule detection, offering a simple and cost-efficient alternative to assays such as ELISA. iSCAT provides information about the relative distribution of particles of different masses in solution, without the requirement to add a label. iSCAT has been described previously for detection of purified single proteins (Cole et al. ACS Photonics (2017), 4(2), 211-216 and W02018 / 011591), and for the detection of lipoproteins and determination of concentrations of a molecule in solution (WO2019 / 110977), herein incorporated by reference. Mass photometry in particular has been described in Young et al., Science (April 2018) 360(6387), 423-427 and Li et al., Nucleic Acids Research (August 2020), https: / / doi.org / 10.1093 / nar / gkaa632, herein incorporated by reference.

[0145] Interferometric scattering mass spectrometry, herein denoted mass photometry, is a means for detecting and measuring the mass of single objects and the complexes they form in solution. Mass photometry is, therefore, an iSCAT method. MP detects single molecules by their light scattering as they bind non-specifically or specifically to a surface.

[0146] An iSCAT microscope suitable for use with the present invention is described in pages 41 to 46 and Figure 3 of WO2022 / 058759 A1. This disclosure is incorporated herein by reference.

[0147] The principle behind mass photometry is that a single object (e.g. a molecule) which is in contact with ( / .e. binds non-specifically or specifically to) a measurement surface (e.g. a coverslip) produces a small but measurable light scattering when exposed to a beam of light. Each binding event leads to a change in refractive index at the surface / solution interface, which effectively alters the local light scattering and can be detected with high accuracy by taking advantage of optimized interference between scattered and reflected light. In particular, when a sample comprising said objects is present on a surface and said surface is illuminated by light, some of that light is reflected by the surface and some is scattered by any object in contact with said surface. Interference between the light scattered by said object and the light reflected by the measurement surface is measured ( / .e. detected) by the mass photometer as interferometric contrast. Said interferometric contrast is directly proportional to the object’s mass, whereby the greater the mass of the object, the greater the interferometric contrast, making it possible to weigh single molecules with light. Thus, each event in which an object comes into contact with a mass photometric measurement surface ( / .e. illuminated surface) and causes interference which is detected by mass photometry as interferometric contrast results in an interferometric contrast signal. In other words, an interferometric contrast signal results from detection of light which is scattered, emitted or reflected from an object when in contact with the illuminated surface.

[0148] An interferometric contrast signal may be represented as a point spread function, PSF, i.e. the inverse Fourier transform of the optical transfer function of light that is scattered, emitted or reflected from said object and measured using said mass photometer. In such a representation, it is the amplitude of said PSF which is directly proportional to the object’s mass, from which it follows that the greater the mass of the object, the greater the amplitude of the PSF. By calibration with molecules of known mass, the magnitude of the interferometric contrast signal (or, where said interferometric contrast signal may be represented by a PSF, the amplitude of the PSF) can be converted into a molecular mass for objects (e.g. proteins or polypeptides) with ~2% mass accuracy and up to 20 kDa mass resolution.

[0149] Mass photometry is unique in its capability for accurate mass measurement of single molecules in solution, in their native state (as defined herein) and without the need for labels (e.g. tags).

[0150] In the context of the present invention mass photometry may be used in a time-resolved manner. This means a series of measurements are made at known time points / delays and the data can be used to determine the kinetics of an evolving system. The time between each measurement may be selected from between 1 ms and 100 s. Suitably the time between each measurement is 1 second to 30 seconds.

[0151] A mass photometry measurement may comprise an averaging of data collected over a window of time from 1 ms to 100 s in duration. Suitably the window of time is 1 second to 30 seconds in duration.

[0152] Equilibrium kinetics are readily investigated by numerous methods, including mass photometry, however the kinetics of a system which is not in equilibrium, for example immediately after two objects have been mixed, requires a time-resolved technique. Furthermore, for systems which equilibrate rapidly it is important to capture kinetic information as early as possible in the equilibration of the system, preferably from the start of the process (t=0). The present invention solves this problem by contacting the first and second objects on a modified surface and using mass photometry to detect reversible binding of the first object, the second object and / or any entities derived therefrom to the modified surface. Because the method is configured in this way mass photometry measurements can begin immediately following the contacting of the first and second objects, thus gaining kinetic insight into the initial stages of the interaction between the objects.

[0153] A further complicating factor with making kinetics measurements is that the technique used to monitor the reaction may affect the results. This can be true when mass photometry is used because objects often bind irreversibly to the traditional surfaces used for mass photometry measurements. This binding to the surface effectively removes the object from solution and thus the concentration of the object in solution (which heavily effects the observed kinetics) changes over time. The present invention solves this problem using a modified surface to which the objects and entities bind reversibly and are thus not sequestered on the surface.

[0154] The Modified Surface

[0155] The modified surface may be a surface which is modified such that the release rate of the first object, the second object and / or any entities derived therefrom to the modified surface is at least 4 times greater than the binding rate of the first object, the second object and / or any entities derived therefrom from the modified surface. Alternatively, or in addition, the modified surface may be a surface which is modified such that the quantity of any object and / or entity bound to the modified surface at any given time amounts to less than 20% of the total number of that object and / or entity present in the solution.

[0156] The modified surface may be a surface which is modified such that the residency time of any one object and / or entity on the modified surface is less than 30 seconds. The modified surface may be a surface which is modified such that the residency time of any one object and / or entity on the modified surface is less than 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 seconds.

[0157] Any one or any combination of the above functional definitions of the modified surface results in a surface which does not significantly impact the concentration of any object or entity in solution and thus permits more accurate kinetic measurements.

[0158] The modified surface may comprise, or be formed from, glass, glass derivatives, diamond, sapphire, cubic zirconia or a niobate.

[0159] Suitably, the modified surface may comprise a surface, as defined herein, wherein the chemical structure of the surface has been modified. For example, the surface may be functionalised with a chemical moiety or the surface may include a coating including a chemical moiety, wherein the chemical moiety reduces and / or mitigates the irreversible binding of said objects and entities derived therefrom to the surface. Suitable chemical moieties which may be used to functionalise the surface and / or included in a surface coating T1 include a chemical moiety selected from: an organosilyl, a fluorinated organosilyl, polyalkylenyl, polymeric silyl, poly(oxyalkylene)aminosilyl, poly(aminoalkylene)silyl, (polyalkylenyl ether)alkylenyl, (polyvinylpyrrolidone)aminosilyl, poly(ethylene glycol), poly(ethylene oxide), zwitterionic silyl, hydroxylic silyl moiety, poly(acrylamide), poly(L-lysine)- graft-poly(ethylene)glycol polymer, sulfobetaine, poly(sulfobetaine), carboxybetaine, poly(carboxybetaine), poly(oxazoline), poly(2-methyl-2-oxazoline), poly(2-ethyl-2-oxazoline), poly(2-isopropenyl-2-oxazoline), poly(glycerol), hyperbranched poly(glycerol), phosphorylcholine, poly(phosphorylcholine), or poly(2-methacryloyloxyethyl phosphorylcholine)

[0160] In an embodiment, the modified surface is a surface of glass or crystal which has been functionalised with a chemical moiety and / or includes a coating comprising a chemical moiety, and comprises:

[0161] (I) a surface of a silicate glass or silica crystal, respectively, wherein at least one of the oxygen atoms (formally each such oxygen atom is an oxygen anion) on the surface of said silicate glass or silica crystal which is only bonded to one silicon atom has been: functionalised with a fluorinated organosilyl, polyalkylenyl, polymeric silyl, poly(oxyalkylene)aminosilyl, poly(oxyalkylene)silyl, poly(amidoalkylene)silyl, (polyalkylenyl ether)alkylenyl, (polyvinylpyrrolidone)aminosilyl, zwitterionic silyl or hydroxylic silyl moiety; or deprotonated to form an anion which interacts with poly(L-lysine)-graft- poly(ethylene)glycol polymer; or

[0162] (II) a surface of diamond crystal, sapphire crystal, cubic zirconia crystal or a niobate crystal, wherein the surface of said crystal has been coated with at least one layer, wherein at least one layer is a silicate, wherein at least one of the oxygen atoms (formally, each such oxygen atom is an oxygen anion) on the surface of said silicate which is only bonded to one silicon atom has been: functionalised with a fluorinated organosilyl, polyalkylenyl, polymeric silyl, poly(oxyalkylene)aminosilyl, poly(oxyalkylene)silyl poly(amidoalkylene)silyl, (polyalkylenyl ether)alkylenyl, (polyvinylpyrrolidone)aminosilyl, zwitterionic silyl or hydroxylic silyl moiety; or deprotonated to form an anion which interacts with poly(L-lysine)-graft- poly(ethylene)glycol polymer.

[0163] In other words, said chemical moiety which prevents the object or objects from interacting covalently or electrostatically with the surface of said glass or said crystal for the aforementioned time ranges is a fluorinated organosilyl, polyalkylenyl, polymeric silyl, poly(oxyalkylene)aminosilyl, poly(oxyalkylene)silyl, poly(amidoalkylene)silyl, (polyalkylenyl ether)alkylenyl, (polyvinylpyrrolidone)aminosilyl, zwitterionic silyl, hydroxylic silyl moiety or poly(L-lysine)-graft-poly(ethylene)glycol polymer, respectively, irrespective of whether said surface is a surface of a silicate glass or silica crystal or a surface of a diamond, sapphire, cubic zirconia or niobate crystal which has been coated with a silicate layer.

[0164] An organosilyl moiety may be an alkyl silyl, an alkoxysilyl or comprise a combination of alkyl and alkoxy groups.

[0165] A fluorinated organosilyl moiety may be a (fluoroalkyl)dialkoxysilyl or a (fluoroalkyl)(dialkyl)silyl moiety. Suitably, said (fluoroalkyl)dialkoxysilyl moiety is a (fluoroalkyl)dimethoxysilyl moiety or (fluoroalkyl)diethoxysilyl moiety while said (fluoroalkyl)(dialkyl)silyl moiety is (fluoroalkyl)(dimethyl)silyl moiety or a (fluoroalkyl)(diethyl)silyl moiety. Suitably, said (fluoroalkyl)dialkoxysilyl moiety may be a moiety selected from the group consisting of (tridecafluoro-1 , 1 ,2,2-tetrahydrooctyl)triethoxysilyl, (nonafluoro- 1 , 1 ,2,2- tetrahydrohexyl)triethoxysilyl, (heptadecafluoro-1 ,1 ,2,2-tetrahydrodecyl)triethoxysilyl, (tridecafluoro-1 , 1 ,2,2-tetrahydrooctyl)trimethoxysilyl, (nonafl uoro- 1 , 1 ,2,2- tetrahydrohexyl)trimethoxysilyl and (heptadecafluoro- 1 ,1 , 2, 2-tetrahydrodecyl)trimethoxysilyl moieties, while said (fluoroalkyl)(dialkyl)silyl moiety may be a moiety selected from the group consisting of (tridecafluoro-1 , 1 , 2, 2-tetrahydrooctyl)triethylsilyl, (nonafluoro- 1 , 1 ,2,2- tetrahydrohexyl)triethylsilyl, (heptadecafluoro-1 ,1 ,2,2-tetrahydrodecyl)triethylsilyl,

[0166] (tridecafluoro-1 , 1 ,2,2-tetrahydrooctyl)trimethylsilyl, (nonafluoro-1 , 1 ,2,2- tetrahydrohexyl)trimethylsilyl and (heptadecafluoro-1 , 1 ,2,2-tetrahydrodecyl)trimethylsilyl moieties.

[0167] A polyalkylenyl moiety may be a polyalkylenyl moiety or a substituted form thereof, such as a poly(ethylenyl) or poly(propylenyl) moiety [e.g. a moiety comprising an end portion of a poly(acrylate), poly(methacrylate), poly(acrylamide) or poly(methacrylamide) chain, wherein said portion comprises at least 1 C-C bond which was formed during polymerisation], such as a poly(hydroxy-functionalised ethylenyl) or poly(hydroxy-functionalised propylenyl) moiety [e.g. a moiety comprising an end portion of a poly(hydroxy-functionalised acrylate), poly(hydroxy-functionalised methacrylate), poly(hydroxy-functionalised acrylamide) or poly(hydroxy-functionalised methacrylamide) chain, wherein said portion comprises at least 1 C-C bond which was formed during polymerisation]. Suitably, said polyalkylenyl moiety may be a poly(hydroxyalkylenylcarboxyl-ethylenyl) moiety, poly(hydroxyalkylenylcarboxyl- propylenyl) moiety, poly(hydroxyalkylenylcarboxamidyl-ethylenyl) moiety or poly(hydroxyalkylenylcarboxamidyl-propylenyl) moiety { / .e.. a moiety comprising an end portion of a poly(hydroxyalkylenyl acrylate) [e.g. poly(hydroxyethylenyl acrylate) or poly(hydroxypropylenyl acrylate)], poly(hydroxyalkylenyl methacrylate) [e.g. poly(hydroxyethylenyl methacrylate) or poly(hydroxypropylenyl methacrylate)], poly(hydroxyalkylenyl acrylamide) [e.g. poly(hydroxyethylenyl acrylamide) or poly(hydroxypropylenyl acrylamide)] or poly(hydroxyalkylenyl methacrylamide) [e.g. poly(hydroxyethylenyl methacrylamide) or poly(hydroxypropylenyl methacrylamide)] chain, respectively, wherein said portion comprises at least 1 C-C bond which was formed during polymerisation}, suitably a moiety such as that obtained from polymerisation of 2- hydroxyethyl-methacrylate (HEMA) [ / .e. a moiety comprising an end portion of a poly(hydroxyethylenyl methacrylate) chain, wherein said portion comprises at least 1 C-C bond which was formed during polymerisation] or 3-hydroxypropyl-methacrylate (HPMA) [ / .e. a moiety comprising an end portion of a poly(hydroxypropylenyl methacrylate) chain, wherein said portion comprises at least 1 C-C bond which was formed during polymerisation] or N-(2- hydroxypropyl)methacrylamide {i.e. a moiety comprising an end portion of a poly[N-(2- hydroxypropylenyl) methacrylamide] chain, wherein said portion comprises at least 1 C-C bond which was formed during polymerisation} onto an oxygen atom on the surface of said silicate (e.g. via surface-initiated polymerisation). Herein, the term "alkylenyl" refers to a divalent analog of a linear or branched alkyl group, preferably ethylenyl (-CH2CH2-) or propylenyl [-CH2CH2(CHs)-] radicals.

[0168] A polymeric silyl moiety may be obtained from a poly(acrylamide) chain (PAcrAM) to which poly(2-methyl-2-oxazoline) (PMOXA) or poly(ethylene glycol) (PEG) blocks, amine moieties and silyl moieties have been grafted [i.e. PAcrAM-g-(PMOXA, amine, silyl) or PAcrAM-g-(PEG, amine, silyl), respectively], wherein said poly(2-methyl-2-oxazoline) or poly(ethylene glycol) blocks impart non-fouling properties and said amine moieties help adhesion to the glass surface, and each of said silyl moieties functionalise at least one of the oxygen atoms on the surface of said silicate glass or silica crystal which is only bonded to one silicon atom. Alternatively, a polymeric silyl moiety may be a moiety with the formula R-(R2Si)x- where each R is an alkyl moiety independently selected from the group of Ci-Ce alkyl moieties, and X is a whole number greater than 0. Suitably, each R is selected from the group consisting of methyl, ethyl and propyl moieties, and wherein X is a whole number between 3 and 20, suitably each R is selected from the group consisting of methyl and ethyl moieties, and wherein X is a whole number between 4 and 10.

[0169] A poly(oxyalkylene)aminosilyl may be a moiety with the formula HO-[CH2CH2O]Y- [CH2]Y’C(O)ONRa-(Rb)2Si- where each Rais H or a Ci-Ce alkyl moiety, each Rbis a Ci-Ce alkyl or a Ci-Ce alkoxy moiety, Y is a whole number greater than 0 and Y’ is a whole number selected from 1 to 3. Suitably, each Rais selected from the group consisting of H, methyl, ethyl and propyl moieties, each Rbis selected from the group consisting of methyl, methoxy, ethyl, ethoxy, propyl and propoxy moieties, Y is a whole number between 10 and 10000, and Y’ is a whole number selected from 1 or 2. Suitably, a poly(oxyalkylene)aminosilyl may be obtained by functionalising the surface of said silicate glass or silica crystal with 3- aminopropyltrimethoxy silane and reacting the resulting amine groups with poly(ethylene glycol) which has been modified with / V-hydroxysuccinimide.

[0170] A poly(aminoalkylene)silyl may be a moiety such as a polyalkylene imine silyl moiety, e.g. polyethylene imine silyl moiety, or a polyaziridine silyl moiety.

[0171] A poly(oxyalkylene)silyl may be a moiety with the formula HO-[CH2CH2O]v”-(Rb)2Si- where each Rb’ is a Ci-Ce alkyl or a Ci-Ce alkoxy moiety and Y” is a whole number greater than 0. Suitably, each Rb’ is selected from the group consisting of methyl, methoxy, ethyl, ethoxy, propyl and propoxy moieties, and Y” is a whole number between 10 and 10000. Suitably, a poly(oxyalkylene)silyl is obtained by functionalising PEG with a (triethoxy)silyl moiety [to form PEG-(triethoxy)silane which may be reacted with the surface of said glass or crystal to functionalise it with a PEG-(diethoxy)silyl moiety],

[0172] A poly(amidoalkylene)silyl [ / .e. a moiety comprising an end portion of a poly(oxazoline) chain attached to a silylene moiety, wherein said portion comprises at least 1 N-C bond which was formed during polymerisation] may be a moiety with the formula H-(NRdCH2CH2)z-(Rb)2Si- or where each Rdis a C1-C7 carbonyl ( / .e. acyl) moiety, each Rbis a Ci-Ce alkyl or a Ci-Ce alkoxy moiety, and Z is a whole number greater than 0. Suitably, each Rdis selected from the group consisting of formyl, acetyl, propionyl or benzoyl moieties, each Rbis selected from the group consisting of methyl, methoxy, ethyl, ethoxy, propyl and propoxy moieties, and Z is a whole number between 10 and 10000. Suitably, a poly(aminoalkylene)silyl may be obtained by functionalising poly(2-methyl-2-oxazoline) or poly(2-ethyl-2-oxazoline) with a triethoxysilyl moiety [to form poly(2-methyl-2-oxazoline)-(triethoxy)silane or poly(2-ethyl-2-oxazoline)- (triethoxy)silane, which may be reacted with the surface of said glass or crystal to functionalise it with a poly(2-methyl-2-oxazoline)-(diethoxy)silyl moiety or poly(2-ethyl-2-oxazoline)- (diethoxy)silyl moiety, respectively],

[0173] A (polyalkylenyl ether)alkylenyl may be a moiety with the formula H-[OCH(A)CH(A)]Q- where one A of each [OCH(A)CH(A)] moiety is CH2OH and the other is H, and Q is a whole number greater than 0. Suitably, each [OCH(A)CH(A)] moiety may be a [OCH(CH2OH)CH2] moiety and Q is a whole number between 10 and 10000. Suitably, said (polyalkylenyl ether)alkylenyl may be obtained by ring-opening polymerisation of glycidol onto an oxygen atom of said silicate glass or silica crystal.

[0174] A (polyvinylpyrrolidone)aminosilyl may be a moiety with the formula H-[CH(J)CH(J)]w- [CH2]wC(O)ONRe-(Rf)2Si- where one J of each [CH(J)CH(J)] moiety is a pyrrolidonyl moiety and the other is H, each Reis H or a Ci-Ce alkyl moiety, each Rfis a Ci-Ce alkyl or a Ci-Ce alkoxy moiety, W is a whole number greater than 0 and W’ is a whole number between 0 and 3. Suitably, each -[CH(J)CH(J)]- moiety may be a -[CH2CH(pyrrolidonyl)]- moiety, each Reis selected from the group consisting of H, methyl, ethyl and propyl moieties, each Rfis selected from the group consisting of methyl, methoxy, ethyl, ethoxy, propyl and propoxy moieties, W is a whole number between 10 and 10000, and W’ is a whole number selected from 1 or 2. Suitably, said (polyvinylpyrrolidone)aminosilyl may be obtained by functionalising the surface of said silicate glass or silica crystal with 3-aminopropyltrimethoxy silane and reacting the resulting amine groups with polyvinylpyrrolidone which has been modified with / V- hydroxysuccinimide. A zwitterionic silyl may be a moiety with the formula (T)-(T’)-(R9)2Si- where one of T and T’ is a positively charged moiety and the other is a negatively charged moiety, and each R9is a Ci-Ce alkyl moiety or a Ci-Ce alkoxy moiety. Suitably, T’ is a - [(CRh2)vNR'2]+- moiety and T is a -[(CRh2)vSO3]_- moiety, wherein the N of T’ is bonded to the terminal carbon of the T moiety, wherein v and v’ are each independently selected from the set of whole numbers greater than 0, each Rhis H or a Ci-Ce alkyl moiety, each Rh’ is H or a Ci-Ce alkyl moiety, each R' is H or a Ci-Ce alkyl moiety, and each R9is a C1-C3 alkyl moiety or a C1-C3 alkoxy moiety. Suitably, a zwitterionic silyl moiety is SO3(CH2)2N(CH3)2(CH2)3(CH3O)2Si- ( / .e. 3-{[dimethyl(3- trimethoxysilyl)propyl]ammonio}propane-1 -sulfonate (SBSi), where T is a -[(C^JsSOs]" moiety, T’ is a -[(CH2)3N(CH3)2]+- moiety and R9is a CH3O- moiety). The aforementioned zwitterionic moieties are characterised by their equal positive and negative charge, giving them a net neutral charge overall under the conditions at which mass photometry is carried out (e.g. at physiological pH). Thus, said zwitterionic moieties can be described as biomimetic, as they act in a similar fashion to the hydrophilic phosphorylcholine moiety which is present in the phospholipids found in cell membranes. Once the surface of said glass or crystal has been functionalised ( / .e. coated) with said zwitterionic moiety, it may exhibit an anti-fouling property due to the strong hydration capacity of the zwitterionic moiety, which is achieved through electrostatically-induced hydration via hydrogen bonding. Hence, said zwitterionic silyl moieties (and coatings) may deliver anti-fouling properties to the surface described in the present invention.

[0175] A hydroxylic silyl moiety may be a moiety with the formula (E)-(Rm)2Si-, (E’)-(Rm)2Si- , or - (Rm)2Si-(E”)-(Rm)2Si- where each Rmis independently selected from a Ci-Ce alkyl or a Ci-Ce alkoxy moiety, E is a HO-[C(Rn)2]r- moiety or a HO-[C(Rn)2]f-NR°-[C(Rn)2]f- moiety, E’ is a {HO-[C(Rn)2]r}2-CRn-[C(Rn)2]f- moiety or a {HO-[C(Rn)2]r}2-N-[C(Rn)2]f- moiety, E” is a - [C(Rn)2]f-C{[C(Rn)2]fOH}Rn-[C(Rn)2]f- moiety or a -[C(Rn)2]f-N{[C(Rn)2]fOHHC(Rn)2]f- moiety, each Rnis independently selected from H or a Ci-Ce alkyl and each f is independently selected from the set of whole numbers greater than 0. Suitably, each Rmis independently selected from a C1-C3 alkyl or a C1-C3 alkoxy moiety, E is a HO-[C(Rn)2]f- moiety or a HO- [C(Rn)2]f-NR°-[C(Rn)2]f- moiety, E’ is a {HO-[C(Rn)2]f}2-CRn-[C(Rn)2]f- moiety or a {HO- [C(Rn)2]f}2-N-[C(Rn)2]f- moiety, E” is a -[C(Rn)2]^C{[C(Rn)2]fOH}Rn-[C(Rn)2]f- moiety or a - [C(Rn)2]f-N{[C(Rn)2]fOH}-[C(Rn)2]f- moiety, each Rnis independently selected from H or a C1- C3 alkyl and each f is a whole number independently selected from the group consisting of 1 , 2 and 3. Suitably, a hydroxylic silyl moiety is selected from the group consisting of HOCH2(CH3O)2Si-, HO(CH2)2N(CH3)(CH2)3(CH3O)2Si-, [HO(CH2)2N]2(CH2)3(CH3O)2Si- and -Si(CH3O)2(CH2)3N[(CH2)2OH](CH2)3(CH3O)2Si-.A poly(L-lysine)-graft-poly(ethylene)glycol polymer may be a random graft copolymer with a poly(L-lysine) backbone with poly(ethylene glycol) side chains. The poly(L-lysine) backbone interacts electrostatically with the substrate ( / .e. by deprotonation of the surface of said silicate glass or silica crystal to form an anion which interacts with a cation formed by protonation of said poly(L-lysine) backbone) while the poly(ethylene glycol) side chains extend from the surface and form a densely-packed polymer brush. Said poly(L-lysine)-graft-poly(ethylene)glycol polymer thus typically provides a coating which is non-fouling and provides a low level of non-specific binding of proteins and other macromolecules.

[0176] A poly(ethylene glycol), poly(ethylene oxide), poly(acrylamide), sulfobetaine, poly(sulfobetaine), carboxybetaine, poly(carboxybetaine), poly(oxazoline), poly(2-methyl-2- oxazoline), poly(2-ethyl-2-oxazoline), poly(2-isopropenyl-2-oxazoline), poly(glycerol), hyperbranched poly(glycerol), phosphorylcholine, poly(phosphorylcholine), or poly(methacryloyloxyethyl phosphorylcholine) moiety may also be used to functionalise and / or coat the surface.

[0177] Alternatively, the modified surface may be a surface which has been modified by a process which changes the physical structure of the surface. For example, the process may be selected from one or more of: a cleaning process, a polishing process, an ion exchange process, a basic washing process, an acidic washing process, a washing process comprising use of Piranha solution, a washing process comprising use of a peroxide solution, a sonication process, a plasma treatment process, a corona treatment process, a flame treatment process, a blasting process, an ablation process, or an etching process.

[0178] The modified surface may have any suitable geometry. For example, the modified surface may comprise a flat plate, such as the surface of a sample holder, coverslip, slide, cell, flow cell, flow chamber, microfluidic cell, microfluidic chamber, well, plate, channel or container. The modified surface may be part of a larger geometry or device. In another embodiment of the present invention, the surface is a surface of a sample holder, coverslip, cell or flow cell. In this context “cell” refers to a container for a solution in which the solution can be measured rather than the biological meaning of cell. A flow cell is a cell with a measurement area which is designed such that solutions can be continuously flowed through or across the measurement area.

[0179] The modified surface may form 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 chamber (e.g. a chamber having dimensions of all 3 axes (including the longest axis) of no greater than 10 mm).

[0180] The modified surface may comprise one or more detection areas. A detection area may be any suitable size and shape. Suitably, a detection area is the area which can be analysed by a light scattering microscope at the same time, or in a single observation event. The size and shape of a detection area may be guided by the size and shape of the area illuminated during light scattering, and therefore by the type of microscope used. The modified surface may comprise more than one detection area. Where more than one detection areas are provided on a surface, they may be adjacent, or may overlap. Where more than one detection areas are provided, they may be spaced apart from one another. The modified surface may comprise 1 , 2, 3, 4, 5, 10, 20, 50, 70, 100, 500 or 1000 or more detection areas.

[0181] Suitably, the modified surface is planar or substantially planar. The modified surface may be curved or include some curvature, for example a concave depression or convex structure on a substantially planar surface. The modified surface may not be the surface of a nanoparticle, since small spherical objects would cause light scattering in their own right and prevent an accurate determination of mass of the release event. A nanoparticle or ultrafine particle is usually defined as a particle of matter that is between 1 and 100 nanometres in diameter (preferably wherein all nanoparticles of said matter have dimensions in all 3 axes (including the longest axis) of between 1 and 100 nm, suitably wherein the longest and shortest axes of each nanoparticle differ by a factor of no more than 3), optionally between 1 and 60 nm in diameter, optionally less than 50 nm in diameter, as measured by ISO 19749:2021. Thus, small spherical particles with a diameter of 60 pm or below, optionally 50 pm or below, optionally 40 pm or below, are optionally not used as a surface according to the present invention. A substantially planar modified surface may be preferred.

[0182] Suitably a detection area of the modified surface allows transmission of ultraviolet light (which may be defined herein as having wavelengths in the range from 10 nm to 380 nm); visible light (which may be defined herein as having wavelengths in the range from 380 nm to 740 nm); and / or infrared light (which may be defined herein as having wavelengths in the range from 740 nm to 300 pm). Preferably, a detection area allows transmission of light in the visible light spectrum. Suitably, a detection area is substantially optically transparent (i.e. transmitting at least 85 % of ultraviolet, visible and / or infrared light).

[0183] In the method of the invention the first solution may be contacted with the modified surface before being contacted with the second solution. Optionally the method may further comprise making a mass photometry measurement of the first solution before the first solution is contacted with the second solution. This allows for a “background” or “baseline” measurement to be obtained to determine the behaviour of the first object before contacting the second object. This also enables the user to ensure the mass photometry readings for the first object are stable before contacting with the second object. This means that any changes to the mass photometry measurements after contacting with the second object can be confidently ascribed to the effect of the second object.

[0184] Alternatively, the second solution may be contacted with the modified surface before the first solution such that the first solution is contacted with the second solution and the modified surface simultaneously. This may be of use where the first object has an inherent instability and must be measured quickly. It also allows for a “background” or “baseline” measurement of the second object to be taken.

[0185] In the method of the invention the detection may be carried out immediately after the first solution and second solution are contacted. This allows for the initial kinetics of the interaction of the first and second objects to be determined. Immediately after means there is no substantial delay between the contacting of the first and second solutions and the start of the measurement. There may be a short delay of a few seconds to ensure the first and second solutions are properly mixed. The measurement may instead begin before the first and second solutions are contacted and continue through this process and after.

[0186] The method of the invention may further comprise contacting the first, second or third solution with a further solution comprising a further object. The further object may be selected independently from any of the lists from which the first object is selected. The further object may affect the interaction kinetics of the first and second objects. For example, the further object may be a buffering reagent which, after contact of the further solution with the third solution, results in an altered pH. This may impact the interactions kinetics of the first and second objects which can then be detected using mass photometry as described above. The further object may interact with the first object, second object or entities derived therefrom to form higher order associations.

[0187] There is further provided a kit of parts including a modified surface suitable for use in mass photometry to measure binding interactions between a first object and a second object in solution, wherein: (i) the modified surface comprises, or is formed from, glass, glass derivatives, diamond, sapphire, cubic zirconia or a niobate; and,

[0188] (ii) the modified surface is functionalised with a chemical moiety or comprises a surface coating including a chemical moiety which reduces or mitigates irreversible binding of first said object, second said object and an entity derived from interaction of first and second said objects thereto, and wherein the chemical moiety is selected from: an organosilyl, a fluorinated organosilyl, polyalkylenyl, polymeric silyl, poly(oxyalkylene)aminosilyl, poly(aminoalkylene)silyl, (polyalkylenyl etherjalkylenyl, (polyvinylpyrrolidone)aminosilyl, poly(ethylene glycol), poly(ethylene oxide), zwitterionic silyl, hydroxylic silyl moiety, poly(acrylamide), poly(L-lysine)-graft-poly(ethylene)glycol polymer, sulfobetaine, poly(sulfobetaine), carboxybetaine, poly(carboxybetaine), poly(oxazoline), poly(2-methyl-2- oxazoline), poly(2-ethyl-2-oxazoline), poly(2-isopropenyl-2-oxazoline), poly(glycerol), hyperbranched poly(glycerol), phosphorylcholine, poly(phosphorylcholine), or poly(2-methacryloyloxyethyl phosphorylcholine).

[0189] Further embodiments of the disclosure are as follows:

[0190] Embodiment 1 . A method for measuring binding interactions between a first object and a second object using mass photometry, the method comprising:

[0191] (i) providing a first solution comprising a first object;

[0192] (ii) providing a second solution comprising a second object;

[0193] (iii) contacting the first solution with the second solution on a modified surface to form a mixture of the first and second solutions on the modified surface, said modified surface comprising a surface which is modified to permit the reversible binding thereto of the first object, the second object or an entity derived from the interaction of said first and second objects, or combinations thereof; and,

[0194] (iv) detecting the reversible binding of the first object, the second object and / or an entity derived from the interaction of said first and second objects to the modified surface using mass photometry; and, wherein at least one of said first object, said second object or an entity derived from the interaction of said first and second objects is detectable by mass photometry.

[0195] Embodiment 2. The method of Embodiment 1 , wherein the method comprises:

[0196] (i) providing a first aqueous solution comprising a biomolecule, as defined herein, wherein the biomolecule is detectable using mass photometry;

[0197] (ii) providing a second aqueous solution comprising a second object, wherein the second object is a known or putative reaction partner of the biomolecule; (iii) contacting the first solution with the second solution on a modified surface, as defined herein, to form a mixture of the first and second solutions on the modified surface, said modified surface comprising a surface which is modified to permit the reversible binding thereto of the biomolecule, the second said object or an entity derived from the interaction of the biomolecule and the second said object, or combinations thereof;

[0198] (iv) detecting the reversible binding of the biomolecule, the second object and / or an entity derived from the interaction of the biomolecule and the second said object to the modified surface using mass photometry; and, optionally,

[0199] (v) determining a kinetic parameter of binding interaction, as defined herein, between said first and second biomolecules.

[0200] Embodiment 3. The method of Embodiment 1 or 2, wherein the first object has a mass (i.e. molecular mass) of greater than 10 kDa and the first object is detectable by mass photometry.

[0201] Embodiment 4. The method of any one of Embodiments 1 to 3, wherein the second object has a mass (i.e. molecular mass) of greater than 10 kDa and the second object is detectable by mass photometry.

[0202] Embodiment 5. The method of any one of Embodiments 1 to 3, wherein the second object has a mass of from 100 Da to less than 10 kDa, such as from 200 to 2 kDa, such as from 250 to 2 kDa, and the second object is not detectable by mass photometry.

[0203] Embodiment 6. The method of any one of Embodiments 1 to 5, wherein step (iv) is performed immediately after contacting the first solution with the second solution on the modified surface in step (iii), thereby measuring the binding interactions between the first and second objects at the zero-time point of interaction.

[0204] Embodiment 7. The method of any one of Embodiments 1 to 6, wherein step (iv) is performed repeatedly in a time resolved manner.

[0205] Embodiment 8. The method of any one of Embodiments 1 to 7, wherein step (iv) is performed to measure dynamic binding interactions between said first and second objects.

[0206] Embodiment 9. The method of any one of Embodiments 1 to 8, further including step (v) of determining a kinetic parameter of binding interaction between said first and second objects. Embodiment 10. The method of Embodiment 9, wherein the kinetic parameter of binding interaction comprises a rate of association (ka), a rate of dissociation (kd), or equilibrium dissociation constant (KD), or a combination thereof, between said first and second objects or an entity derived from the interaction of said first and second objects.

[0207] Embodiment 11. The method of any one of Embodiments 1 to 10, wherein step (iii) comprises: a first step where the first solution only is added to the modified surface, and a second subsequent step where the second solution is added to the first solution on the modified surface to form said mixture of first and second solutions on the modified surface.

[0208] Embodiment 12. The method of any one of Embodiments 1 to 11 , further including the step of performing a mass photometry measurement of the first solution on the modified surface before the second subsequent step of adding the second solution to the first solution.

[0209] Embodiment 13. The method of any one of Embodiments 1 to 12, wherein step (iii) comprises a single step of simultaneously adding both the first and second solutions to the modified surface to form said mixture of first and second solutions on the modified surface.

[0210] Embodiment 14. The method of any one of Embodiments 1 to 13, wherein the method further comprises contacting the first solution, the second solution, or the mixture of the first and second solutions with a third solution comprising a third object on the modified surface and detecting the reversible binding of the first object, the second object, the third object and / or an entity derived from an interaction of said first, second, and / or third objects to the modified surface using mass photometry.

[0211] Embodiment 15. The method of any one of Embodiments 1 to 14, wherein the first object is a biomolecule.

[0212] Embodiment 16. The method of any one of Embodiments 1 to 15, wherein the first object is selected from a polypeptide, a protein or a fragment thereof, a lipoprotein, a glycoprotein, a protein complex, an antibody or an antibody fragment thereof, an enzyme, a nucleic acid, a protein-nucleic acid complex, a virus or portion thereof, a virus-like particle, a viral vector such, an exosome, a cell, a nanoparticle, a quantum dot, a liposome, a vesicle, a micelle, a lipid, a carbohydrate, a polysaccharide.

[0213] Embodiment 17. The method of any one of Embodiments 1 to 16, wherein the second object is a known or putative interaction partner of the first object. Embodiment 18. The method of any one of Embodiments 1 to 17, wherein the second object is selected from a polypeptide, a protein or a fragment thereof, a lipoprotein, a glycoprotein, a protein complex, an antibody or an antibody fragment thereof, an enzyme, a nucleic acid, a protein-nucleic acid complex, a virus or portion thereof, a virus-like particle, a viral vector such, an exosome, a cell, a nanoparticle, a quantum dot, a liposome, a vesicle, a micelle, a lipid, a carbohydrate, a polysaccharide, a drug, drug candidate, drug-fragment.

[0214] Embodiment 19. The method of any one of Embodiments 1 to 18, wherein both the first solution and the second solution is an aqueous solution.

[0215] Embodiment 20. The method of any one of Embodiments 1 to 19, wherein the modified surface is a surface comprising, or formed from, a glass, diamond, sapphire, cubic zirconia or a niobate.

[0216] Embodiment 21. The method of any one of Embodiments 1 to 20, wherein the modified surface is a surface which has been modified by a process which changes the physical structure of the surface, wherein the process is selected from one or more of: a cleaning process, a polishing process, an ion exchange process, a basic washing process, an acidic washing process, a washing process comprising use of Piranha solution, a washing process comprising use of a peroxide solution, a sonication process, a plasma treatment process, a corona treatment process, a flame treatment process, a blasting process, an ablation process, or an etching process.

[0217] Embodiment 22. The method of any one of Embodiments 1 to 21 , wherein the modified surface comprises a surface which has been functionalised with a chemical moiety or comprises a surface include a coating including a chemical moiety, wherein the chemical moiety reduces and / or inhibits irreversible binding of said first object, said second object and / or an entity formed from interaction between said first and second objects to said surface.

[0218] Embodiment 23. The method of any one of Embodiments 1 to 22, wherein the chemical moiety is selected from one or more of: an organosilyl, a fluorinated organosilyl, polyalkylenyl, polymeric silyl, poly(oxyalkylene)aminosilyl, poly(aminoalkylene)silyl, (polyalkylenyl etherjalkylenyl, (polyvinylpyrrolidone)aminosilyl, poly(ethylene glycol), poly(ethylene oxide), zwitterionic silyl, hydroxylic silyl moiety, poly(acrylamide), poly(L-lysine)-graft- poly(ethylene)glycol polymer, sulfobetaine, poly(sulfobetaine), carboxybetaine, poly(carboxybetaine), poly(oxazoline), poly(2-methyl-2-oxazoline), poly(2-ethyl-2-oxazoline), poly(2-isopropenyl-2-oxazoline), poly(glycerol), hyperbranched poly(glycerol), phosphorylcholine, poly(phosphorylcholine), or poly(2-methacryloyloxyethyl phosphorylcholine).

[0219] Embodiment 24. The method of Embodiment 22 or 23, wherein the chemical moiety comprises a silyl / silicon containing moiety selected from organosilyl moiety, such as a fluorinated organo silyl, a polymeric silyl moiety, poly(oxyalkylene)aminosilyl moiety, poly(aminoalkylene)silyl moiety, (polyvinylpyrrolidone)aminosilyl moiety, zwitterionic silyl moiety, hydroxyl silyl moiety.

[0220] Embodiment 25. The method of Embodiment 17, wherein the chemical moiety is selected from one or more of:

[0221] / V, / V-bis(2-hydroxyethyl)-3-aminopropyltriethoxysilyl

[0222] (tridecafluoro-1 , 1 ,2,2-tetrahydrooctyl)triethoxysilyl,

[0223] (nonafluoro-1 , 1 ,2,2-tetrahydrohexyl)triethoxysilyl,

[0224] (heptadecafluoro-1 ,1 ,2,2-tetrahydrodecyl)triethoxysilyl,

[0225] (tridecafluoro-1 , 1 ,2,2-tetrahydrooctyl)trimethoxysilyl,

[0226] (nonafluoro-1 , 1 ,2,2-tetrahydrohexyl)trimethoxysilyl,

[0227] (heptadecafluoro-1 ,1 ,2,2-tetrahydrodecyl)trimethoxysilyl,

[0228] (tridecafluoro-1 , 1 , 2 , 2-tetrahyd roocty l)triethy Isi ly I ,

[0229] (nonafluoro-1 , 1 , 2 , 2-tetrahydrohexy l)triethy Isi ly I ,

[0230] (heptadecafluoro-1 ,1 ,2,2-tetrahydrodecyl)triethylsilyl,

[0231] (tridecafluoro-1 , 1 ,2,2-tetrahydrooctyl)trimethylsilyl,

[0232] (nonafluoro-1 , 1 ,2,2-tetrahydrohexyl)trimethylsilyl,

[0233] (heptadecafluoro-1 ,1 ,2,2-tetrahydrodecyl)trimethylsilyl,

[0234] 3-{[dimethyl(3-trimethoxysilyl)propyl]ammonio}propane-1-sulfonate, poly(ethylene glycol), HOCH2(CH3O)2Si-,

[0235] HO(CH2)2N(CH3)(CH2)3(CH3O)2Si-, [HO(CH2)2N]2(CH2)3(CH3O)2Si-, or -Si(CH3O)2(CH2)3N[(CH2)2OH](CH2)3(CH3O)2Si-.

[0236] Embodiment 26. The method of any one of Embodiments 1 to 25, wherein the modified surface comprises a surface which includes, suitably is formed from, a glass, such as borosilicate glass, which has been functionalised with a chemical moiety or includes a coating comprising a chemical moiety, wherein the chemical moiety comprises a poly(oxyalkylene)aminosilyl moiety. Embodiment 27. The method of Embodiment 26, wherein the modified surface comprises a surface which includes, suitably is formed from, a borosilicate glass which has been functionalised with a chemical moiety or includes a coating comprising a chemical moiety, wherein the chemical moiety comprises / V, / V-bis(2-hydroxyethyl)-3- aminopropyltriethoxysilane.

[0237] Embodiment 28. The method of any one of Embodiments 1 to 27, wherein the modified surface is in the form of a plate, or the modified surface forms a part of a flow cell.

[0238] Embodiment 29. The method of any one of Embodiments 1 to 28, wherein the modified surface comprises a surface which is modified such that the release rate, as measured by mass photometry, of the first object, the second object and / or an entity derived from interaction of said first and second objects from the modified surface is at least 4 times greater than the respective binding rate, as measured by mass photometry, of the first object, the second object and / or an entity derived from said first and / or second objects to the modified surface.

[0239] Embodiment 30. The method of any one of Embodiments 1 to 29, wherein the modified surface comprises a surface which is modified such that the residency time, as determined by mass photometry, of the first object, the second object and / or an entity derived from interaction of said first and second objects on the modified surface is less than 30 seconds.

[0240] Embodiment 31. The method of any one of Embodiments 1 to 30, wherein the first object is a protein and the second object is a protein.

[0241] Embodiment 32. The method of any one of Embodiments 1 to 30, wherein the first object is a protein and the second object is a nucleic acid, polysaccharide, PEG-based polymer, pullulan-based nanogel or polymer, glycopolymer, conjugated polymer, polymer having either positive or negative charge, polyampholyte, zwitterionic polymer, vesicle, virus, virus-like particle, particulate polymer conjugate, polymeric nanoparticle, liposome, micelle, dendrimer, carbon nanotube or gold nanoparticle.

[0242] Embodiment 33. The method of any one of Embodiments 1 to 30, wherein the first object is a nucleic acid and the second object is a virus or virus-like particle.

[0243] Embodiment 34. The use of a modified surface, as defined in any one of the preceding claims, to measure zero-time point binding interactions between a first object, as defined in any one of the preceding claims, and a second object, as defined in any one of the preceding claims, in solution using mass photometry. Embodiment 35. A kit of parts including a modified surface suitable for use in mass photometry to measure binding interactions between a first object and a second object in solution, wherein:

[0244] (i) the modified surface comprises, or is formed from, glass, glass derivatives, diamond, sapphire, cubic zirconia or a niobate; and, the modified surface is functionalised with a chemical moiety or comprises a surface coating including a chemical moiety which reduces or mitigates irreversible binding of first said object, second said object and an entity derived from interaction of first and second said objects thereto, and wherein the chemical moiety is selected from: an organosilyl, a fluorinated organosilyl, polyalkylenyl, polymeric silyl, poly(oxyalkylene)aminosilyl, poly(aminoalkylene)silyl, (polyalkylenyl etherjalkylenyl, (polyvinylpyrrolidone)aminosilyl, poly(ethylene glycol), poly(ethylene oxide), zwitterionic silyl, hydroxylic silyl moiety, poly(acrylamide), poly(L-lysine)-graft-poly(ethylene)glycol polymer, sulfobetaine, poly(sulfobetaine), carboxybetaine, poly(carboxybetaine), poly(oxazoline), poly(2-methyl- 2-oxazoline), poly(2-ethyl-2-oxazoline), poly(2-isopropenyl-2-oxazoline), poly(glycerol), hyperbranched poly(glycerol), phosphorylcholine, poly(phosphorylcholine), or poly(2- methacryloyloxyethyl phosphorylcholine).

[0245] The following non-limiting Examples are provided to illustrate the disclosure.

[0246] Examples

[0247] 1. Surface preparation:

[0248] A modified surface was prepared by functionalising Schott D263® M borosilicate glass coverslips with / V, / V-bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane, 62% in ethanol (commercial name SI B1140.0, Gelest supplier). This was achieved by first washing coverslips with 200 mL of 50:50 solution isopropanol (IPA): milliQ water and sonicating for 5 minutes. The waste was discarded and replaced with fresh milliQ, after which the coverslips were sonicated for a further 5 minutes. The glass was subsequently dried with compressed air and plasma treated in air for 2 minutes (P = 500 W, air flow = 250 seem, air pressure = 3 bar). A reaction mixture was prepared with 245 mL toluene and 5 mL SIB114. After the plasma cleaning was complete, the slides were placed on a metal rack and submerged in the reaction mixture for 2 hours with gentle mixing. The slides were then rinsed in 200 mL toluene and allowed to air dry in a fumehood over 10 minutes. The coverslips were then cured in an oven at 100 °C for 2 hours. Thereafter, the slides were washed in 200 mL 50:50 IPA: milliQ water and sonicated for 5 minutes. The waste was discarded, and the slides washed in fresh milliQ water with sonication for 5 minutes. The slides were then rinsed with milliQ and dried with compressed air. The surface was inspected by eye to ensure that it was free of spots.

[0249] The resulting coverslip, thus modified, was adhered to an Ibidi GmbH flow cell, such that the modified surface was able to contact liquid flowing into said cell.

[0250] 2. Kinetics of protein A and human IgG association and dissociation of the resulting human IgG-protein A complex

[0251] Measurements were calibrated using a sample of beta-amylase. The calibration measurement was carried out on a coverslip with an identical surface coating to that described above but without the flow cell attached ( / .e. measurement was carried out in a silicon gasket). For the measurements in the flow cell, an aqueous solution of protein A (pA, =first object, 10 nM, 20pL) was flowed into the flow cell and a recording of the detection was started. While the recording was in progress a sample of human IgG (hlgG, =second object, 10 nM, 20pL) was subsequently flowed therein. The assembly of complexes was monitored for 10 minutes.

[0252] Data was initially analysed using DMP and then moved into a custom Jupyter Notebook. Figure 1 shows that formation of a hlgG-pA complex (hlgG-pA, third object) is almost immediate and species of larger mass take longer to form. At longer times, the proportion of hlgG-protein A complex decreases more than hlgG does, suggesting it is more important in forming higher order oligomers (complexes).

[0253] Association of protein A (pA) and human IgG (hlgG), as well as dissociation of the resulting human IgG-protein A complex (hlgG-pA) may be described by the following equation (I):

[0254] (I) pA + hlgG hlgG-pA

[0255] Further association of protein A (pA) and the human IgG-protein A complex (hlgG-pA) into higher order complexes that can be seen in the resulting mass histogram can be described by the following equations(ll-IV):

[0256] (II) hlgG-pA + hlgG (hlgG)2-pA

[0257] (III) pA + (hlgG)2-pA (hlgG)2-pA2

[0258] (IV) (hlgG)2.pA + hlgG-pA (hlgG)3.pA2

[0259] Note that using this methodology it is possible to propose alternative schemes for the formation of complexes and test which schemes show the greatest agreement with the experimental data. Once association had been allowed to take place for approximately 10 minutes, Dulbecco’s phosphate buffer was added to allow dissociation to take place. An initial dilution (10 pL) resulted in relatively little change in the proportion of species, as shown in Figure 2, but after a second dilution (10 pL extra to a total dilution volume of 20 pL) a large reduction in the amount of species was observed and lots of protein A was released, as shown in Figure 3, suggesting dilution resulted in dissociation of higher order oligomers (complexes). This was confirmed when repeating the experiment at large field of view to detect light from more interferometric contrast events, resulting in less statistical variation for the same temporal resolution.

[0260] Since the initial concentration of protein A was known, it was possible to calculate the concentration of protein A at multiple intervals after hlgG was flowed into the flow cell, as well as the concentration of hlgG, the hlgG-protein A complex and the hlgG-(protein A)2 complex at multiple intervals.

[0261] For equations (l-l V) the forward and backward rates are governed by the association rate constant (a,x) and the dissociation rate constant (d, x) where x refers to the equation number for each step. For each species we can then write down the following differential equations, each describing the rate of change in concentration of a given object:

[0262] + ka.,iv[(hIgG)3 — pA2]

[0263] Said coupled sets of differential equations were then fitted to the respective calculated concentrations of said objects at each interval using the Levenberg Marquardt least-squares method (Figure 4). In the fitting procedure the initial period where only the first object, protein A, is present and where no detection takes place due to the addition of the second object, human IgG, is explicitly modelled. Once fitted, the best fit values for the rate constants were then found to be: ka I= 3.12 x 106± 2.26 x 105moZ-1cZm3s-1kd I= 0.0051 ± 5.8 x 10-4s-1kaJ1= 3.81 X 106± 1.97 x 106mo1dm3s-1kdJ1= 0.042 ± 0.024 s-1ka,m=1-52 x 106+ 4.21 x 105mol-1dm3s-1kd,m = 6.48 X 10-4± 0.0011 s-1ka,iv = 1.31 x 106+ 4.20 x 105moZ-1dm3s-1kd IV= 0.0045 ± 0.0022 s-1.

[0264] Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0265] “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other.

[0266] For example “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[0267] It will further be appreciated by those skilled in the art that although the invention has been described by way of example with reference to several embodiments, it is not limited to the disclosed embodiments and that alternative embodiments could be constructed without departing from the scope of the invention as defined in the appended claims.

Claims

CLAIMS1. A method for measuring binding interactions between a first object and a second object using mass photometry, the method comprising:(i) providing a first solution comprising a first object;(ii) providing a second solution comprising a second object;(iii) contacting the first solution with the second solution on a modified surface to form a mixture of the first and second solutions on the modified surface, said modified surface comprising a surface which is modified to permit the reversible binding thereto of the first object, the second object or an entity derived from the interaction of said first and second objects, or combinations thereof; and,(iv) detecting the reversible binding of the first object, the second object and / or an entity derived from the interaction of said first and second objects to the modified surface using mass photometry; and, wherein at least one of said first object, said second object or an entity derived from the interaction of said first and second objects is detectable by mass photometry.

2. A method as claimed in claim 1 , wherein the first object has a mass (i.e. molecular mass) of greater than 10 kDa and the first object is detectable by mass photometry.

3. A method as claimed in claim 1 or 2, wherein the second object has a mass (i.e. molecular mass) of greater than 10 kDa and the second object is detectable by mass photometry.

4. A method as claimed in claim 1 or 2, wherein the second object has a mass of from 100 Da to less than 10 kDa, such as from 200 to 2 kDa, such as from 250 to 2 kDa, and the second object is not detectable by mass photometry.

5. The method as claimed in any one of the preceding claims, wherein step (iv) is performed immediately after contacting the first solution with the second solution on the modified surface in step (iii), thereby measuring the binding interactions between the first and second objects at the zero-time point of interaction.

6. The method as claimed in any one of the preceding claims, wherein step (iv) is performed repeatedly in a time resolved manner.

7. The method as claimed in any one of the preceding claims, wherein step (iv) is performed to measure dynamic binding interactions between said first and second objects.

8. The method as claimed in any one of the preceding claims, further including step (v) of determining a kinetic parameter of binding interaction between said first and second objects.

9. The method as claimed in claim 8, wherein the kinetic parameter of binding interaction comprises a rate of association (ka), a rate of dissociation (kd), or equilibrium dissociation constant (KD), or a combination thereof, between said first and second objects or an entity derived from the interaction of said first and second objects.

10. The method as claimed in any one the preceding claims, wherein step (iii) comprises: a first step where the first solution only is added to the modified surface, and a second subsequent step where the second solution is added to the first solution on the modified surface to form said mixture of first and second solutions on the modified surface.

11. The method as claimed in claim 10, further including the step of performing a mass photometry measurement of the first solution on the modified surface before the second subsequent step of adding the second solution to the first solution.

12. The method as claimed in any one of claims 1 to 9, wherein step (iii) comprises a single step of simultaneously adding both the first and second solutions to the modified surface to form said mixture of first and second solutions on the modified surface.

13. The method as claimed in any one of the previous claims, wherein the method further comprises contacting the first solution, the second solution, or the mixture of the first and second solutions with a third solution comprising a third object on the modified surface and detecting the reversible binding of the first object, the second object, the third object and / or an entity derived from an interaction of said first, second, and / or third objects to the modified surface using mass photometry.

14. The method as claimed in any one of the preceding claims, wherein the first object is a biomolecule.

15. The method as claimed in any one of the preceding claims, wherein the first object is selected from a polypeptide, a protein or a fragment thereof, a lipoprotein, a glycoprotein, a protein complex, an antibody or an antibody fragment thereof, an enzyme, a nucleic acid, aprotein-nucleic acid complex, a virus or portion thereof, a virus-like particle, a viral vector such, an exosome, a cell, a nanoparticle, a quantum dot, a liposome, a vesicle, a micelle, a lipid, a carbohydrate, a polysaccharide.

16. The method as claimed in any one of the preceding claims, wherein the second object is a known or putative interaction partner of the first object.

17. The method as claimed in any one of the preceding claims, wherein the second object is selected from a polypeptide, a protein or a fragment thereof, a lipoprotein, a glycoprotein, a protein complex, an antibody or an antibody fragment thereof, an enzyme, a nucleic acid, a protein-nucleic acid complex, a virus or portion thereof, a virus-like particle, a viral vector such, an exosome, a cell, a nanoparticle, a quantum dot, a liposome, a vesicle, a micelle, a lipid, a carbohydrate, a polysaccharide, drug, drug candidate.

18. The method as claimed in any one of the preceding claims, wherein both the first solution and the second solution is an aqueous solution.

19. The method as claimed in any one of the preceding claims, wherein the modified surface is a surface comprising, or formed from, a glass, diamond, sapphire, cubic zirconia or a niobate.

20. The method as claimed in any one of the preceding claims, wherein the modified surface is a surface which has been modified by a process which changes the physical structure of the surface, wherein the process is selected from one or more of: a cleaning process, a polishing process, an ion exchange process, a basic washing process, an acidic washing process, a washing process comprising use of Piranha solution, a washing process comprising use of a peroxide solution, a sonication process, a plasma treatment process, a corona treatment process, a flame treatment process, a blasting process, an ablation process, or an etching process.

21. The method as claimed in any one of the preceding claims, wherein the modified surface comprises a surface which has been functionalised with a chemical moiety or comprises a surface include a coating including a chemical moiety, wherein the chemical moiety reduces and / or inhibits irreversible binding of said first object, said second object and / or an entity formed from interaction between said first and second objects to said surface.

22. The method as claimed in claim 21, wherein the chemical moiety is selected from one or more of: an organosilyl, a fluorinated organosilyl, polyalkylenyl, polymeric silyl, poly(oxyalkylene)aminosilyl, poly(aminoalkylene)silyl, (polyalkylenyl ether)alkylenyl, (polyvinylpyrrolidone)aminosilyl, poly(ethylene glycol), poly(ethylene oxide), zwitterionic silyl, hydroxylic silyl moiety, poly(acrylamide), poly(L-lysine)-graft-poly(ethylene)glycol polymer, sulfobetaine, poly(sulfobetaine), carboxybetaine, poly(carboxybetaine), poly(oxazoline), poly(2-methyl-2-oxazoline), poly(2-ethyl-2-oxazoline), poly(2-isopropenyl-2-oxazoline), poly(glycerol), hyperbranched poly(glycerol), phosphorylcholine, poly(phosphorylcholine), or poly(2-methacryloyloxyethyl phosphorylcholine).

23. The method as claimed in claim 22, wherein the chemical moiety comprises a silyl / silicon containing moiety selected from organosilyl moiety, such as a fluorinated organo silyl, a polymeric silyl moiety, poly(oxyalkylene)aminosilyl moiety, poly(aminoalkylene)silyl moiety, (polyvinylpyrrolidone)aminosilyl moiety, zwitterionic silyl moiety, hydroxyl silyl moiety.

24. The method as claimed in claim 21, wherein the chemical moiety of the coating is selected from one or more of: / V, / V-bis(2-hydroxyethyl)-3-aminopropyltriethoxysilyl(tridecafluoro-1 , 1 ,2,2-tetrahydrooctyl)triethoxysilyl, (nonafluoro-1 , 1 ,2,2-tetrahydrohexyl)triethoxysilyl, (heptadecafluoro-1,1,2,2-tetrahydrodecyl)triethoxysilyl, (tridecafluoro-1 , 1 ,2,2-tetrahydrooctyl)trimethoxysilyl, (nonafluoro-1 , 1 ,2,2-tetrahydrohexyl)trimethoxysilyl, (heptadecafluoro-1,1,2,2-tetrahydrodecyl)trimethoxysilyl, (tridecafluoro-1 , 1 , 2 , 2-tetrahyd roocty l)triethy Isi ly I , (nonafluoro-1 , 1 , 2 , 2-tetrahydrohexy l)triethy Isi ly I , (heptadecafluoro-1,1,2,2-tetrahydrodecyl)triethylsilyl, (tridecafluoro-1 , 1 ,2,2-tetrahydrooctyl)trimethylsilyl, (nonafluoro-1 , 1 ,2,2-tetrahydrohexyl)trimethylsilyl, (heptadecafluoro-1,1,2,2-tetrahydrodecyl)trimethylsilyl, 3-{[dimethyl(3-trimethoxysilyl)propyl]ammonio}propane-1-sulfonate, poly(ethylene glycol), HOCH2(CH3O)2Si-,HO(CH2)2N(CH3)(CH2)3(CH3O)2Si-, [HO(CH2)2N]2(CH2)3(CH3O)2Si-, or -Si(CH3O)2(CH2)3N[(CH2)2OH](CH2)3(CH3O)2Si-.

25. The method as claimed in any one of the preceding claims, wherein the modified surface comprises a surface which includes, suitably is formed from, a glass, such as borosilicate glass, which has been functionalised with a chemical moiety or includes a coating comprising a chemical moiety, wherein the chemical moiety comprises a poly(oxyalkylene)aminosilyl moiety.

26. The method as claimed in any one of the preceding claims, wherein the modified surface is in the form of a plate, or the modified surface forms a part of a flow cell.

27. The method as claimed in any one of the preceding claims, wherein the modified surface comprises a surface which is modified such that the release rate, as measured by mass photometry, of the first object, the second object and / or an entity derived from interaction of said first and second objects from the modified surface is at least 4 times greater than the respective binding rate, as measured by mass photometry, of the first object, the second object and / or an entity derived from said first and / or second objects to the modified surface.

28. The method as claimed in any one of the preceding claims, wherein the modified surface comprises a surface which is modified such that the residency time, as determined by mass photometry, of the first object, the second object and / or an entity derived from interaction of said first and second objects on the modified surface is less than 30 seconds.

29. The use of a modified surface, as defined in any one of the preceding claims, to measure zero-time point binding interactions between a first object, as defined in any one of the preceding claims, and a second object, as defined in any one of the preceding claims, in solution using mass photometry.

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