Methods and systems for detecting antibody self-association

Abstract

The present disclosure provides methods and systems for detecting antibody protein product self-association that include inducing co-agglutination of nanoparticles and a solid support to capture nanoparticles, each coated with a ligand specific for an immunoglobulin, and utilizing a fluidic device to detect the co-agglutination and antibody protein product self-association.

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 302,341, filed January 24, 2022, and U.S. Provisional Patent Application No. 63 / 306,842, filed February 4, 2022, both of which are incorporated by reference in their entireties herein.

[0002] The present disclosure provides methods and systems for detecting protein self-association that involve inducing co-agglutination of nanoparticles and a solid support to capture nanoparticles coated with immunoglobulin or receptor-specific ligands, respectively, and utilizing a fluidic device to detect the co-agglutination and antibody protein product self-association. [Background technology]

[0003] During the development of therapeutic antibodies, much time and resources are invested in identifying potentially problematic physicochemical properties of the antibody to reduce the risk of costly late-stage failure in the clinic. Early evaluation of antibody properties, especially before purification, is an efficient and resource-saving approach to guide the molecular optimization process.

[0004] Fluidic systems can be used for the manipulation and analysis of samples containing proteins such as antibodies. Fluidic optics provide high-throughput single-cell screening capabilities based on nanofluidic and optoelectronic positioning technology. This technology is based on light-induced electrical motion that produces specified forces in both solid and fluidic structures (Jorgolli et al., Biotechnol Bioeng 2019, 116(9), 2393-2411). For example, commercially available fluidic devices such as the integrated technology of the Berkeley Lights (BLI) Beacon® Optofluidic System (Emeryville, CA) are flexible and have a wide range of applications applicable to commercial large molecule drug development, including antibody discovery, clonal selection, gene editing, phenotype-genotype correlation, and cell line development. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Jorgolli et al.,Biotechnol Bioeng 2019,116(9),2393-2411 Summary of the Invention [Means for solving the problem]

[0006] There is a need to develop "on-a-chip" assays that predict molecule performance in shake flasks and bioreactors. Disclosed herein are methods for detecting and characterizing proteins, such as antibody protein products, such as human or humanized antibodies, that tend to self-associate. The methods can be performed in fluidic devices, such as the chip of the BLI Beacon Optofluidic system, utilizing solid supports, such as micrometer-sized beads, and nanoparticles, both coated with ligands specific for antibody protein products, such as anti-human IgG (huIgG) antibodies. Methods to detect antibodies that tend to self-associate relate to solubility, aggregation, and viscosity, attributes that are important for feasibility assessment.

[0007] Methods and systems for detecting antibody protein product association and aggregation are described. It is contemplated that co-agglutination of nanoparticles, such as capture beads, and solid supports results in detectable changes in optical signals. Examples of suitable optical signals include light emission (such as fluorescent emission), optical patterns, and light scattering (such as dynamic light scattering). First, antibody protein product aggregates can be incubated with nanoparticles that bind to the antibody protein products, and then with capture beads that further bind to the antibody protein products, thereby inducing co-agglutination of the nanoparticles and capture beads. Co-agglutination results in a change in absorption, such that the aggregates can be detected, for example, by detecting a change in absorption or light emission. Furthermore, capture beads that induce aggregation advantageously allow visualization of the aggregates without the need to measure a change in absorption or light emission. This process can be performed in a fluidic device, and aggregation can be detected in situ.

[0008] In some embodiments, nanometer-scale anti-Fc coated nanoparticles are mixed with a sample of antibody protein product. If the antibody protein product is self-associated, the nanoparticles form clusters. Then, micrometer-scale anti-Fc capture beads are added. If the antibody protein product is self-associated, the capture beads co-agglutinate with the nanoparticle clusters. The presence of co-agglutination structures can be detected as a change in optical pattern or as a change in absorption or light emission. This assay can be performed in situ in a fluidic device, such as in a BLI pen. This assay can be used to select clones that have a low risk of producing antibodies that are at risk of self-association.

[0009] For example, in the disclosed methods, cells expressing antibody protein products are seeded into individual pens or channels on a fluidic device. In some embodiments, the individual pens are placed on a microfluidic chip containing thousands of pens in a Beacon® system through a light-induced electric field that places each cell in an empty pen. The antibody protein products secreted by the cells in each pen are then analyzed for their properties using reagents brought on the chip, and images are obtained through bright field or filter cubes with a charge-coupled device (CCD) camera. The method may further include quantification of the light emission of the capture beads, which allows the identification of cells expressing antibody protein products with a high risk of self-association, attributes related to solubility, aggregation and viscosity.

[0010] The present disclosure provides a method and system for detecting antibody protein product association, which comprises: a) contacting a sample containing an antibody protein product with nanoparticles coated with a ligand for the antibody protein product, which contacting occurs in a fluidic device under conditions that allow the antibody protein product to self-associate, thereby forming clusters of nanoparticles; b) contacting the clusters of nanoparticles with a solid support coated with a ligand for the antibody protein product of (a); and c) detecting a change in an optical signal of the nanoparticles and / or the solid support, the change in the optical signal being indicative of antibody protein product association. Examples of optical signals include a change in light emission (such as a change in fluorescence emission), a change in light scattering (e.g., dynamic light scattering), and a change in an optical pattern. For example, a change in absorption can be observed indirectly by measuring light emission. Aggregation of metal nanoparticles, such as gold nanoparticles, can cause a change in absorption, and thus metal nanoparticles can be useful for detecting changes in light emission. In some embodiments, the optical signal is detected using an emission filter. In some embodiments, the optical signal is detected using a fluorescent scan, e.g., a change in the optical signal is detected using a fluorescent scan, and the solid support has an average of 6.5 to 10 μm, e.g., about 6.5 μm.

[0011] For aggregation of nanoparticles of metal and non-metallic materials, changes in light scattering, such as dynamic light scattering, can be observed, for example, metal (e.g., gold) as well as non-metal (e.g., polymers such as polystyrene) nanoparticles can be useful for detection of changes in light scattering (e.g., dynamic light scattering). Changes in optical patterns can be observed for aggregation of nanoparticles of any material. Microscopy can be used to detect changes in optical patterns. It is contemplated that machine learning can be useful in high-throughput identification of changes in optical patterns on fluidic devices, for example, including hundreds or thousands of pens. Machine learning algorithms can be trained with images of aggregated and / or non-aggregated nanoparticles and solid supports, which can be used to identify optical patterns indicative of aggregation.

[0012] In some embodiments, the disclosed methods and systems are used for the identification of cells expressing antibody protein products, such as monoclonal antibodies or fragments thereof, that have a high risk of self-association. In further embodiments, the disclosed methods and systems are used to select clones that are less likely to express antibody protein products, such as monoclonal antibodies or fragments thereof, that have a high risk of self-association. In some embodiments, the sample comprises or consists of conditioned medium.

[0013] In any of the disclosed methods, the antibody protein product comprises or consists of a large peptide (e.g., a peptide containing at least 100 amino acid residues), an antibody, an antibody fragment, an antibody fusion peptide, or an antigen-binding fragment thereof. In an exemplary embodiment, the antibody protein product is a human or humanized monoclonal antibody.

[0014] In some embodiments, the disclosed methods and systems are carried out using nanoparticles that include or consist of metals or polymers. Examples of metals for nanoparticles include gold, silver, and palladium. Examples of polymers for nanoparticles include polystyrene, poly(lactide-co-glycolide) (PLGA), polyethylene oxide (PEO), polyethylene glycol (PEG), and polyvinyl alcohol (PVA). Additionally, nanoparticles can include fused silica, quartz, chitosan, dextran, alginate, gadolinium, or carbon. It is contemplated that metal nanoparticles can be used to detect changes in absorption and / or light emission, and that nanoparticles of any material described herein can be used to detect changes in light scattering (such as dynamic light scattering) or optical patterns. Changes in the aggregation of metals can cause changes in absorption, which can be detected as changes in light emission. Changes in optical patterns can be detected by microscopy. Since some metal nanoparticles, such as silver and palladium, can be dispersed in solution, electron microscopy can be used to detect changes in optical patterns, such as metal nanoparticles. Without being limited by theory, it is contemplated that silver is negatively charged and cannot be passively coated with protein ligands such as antibodies, and therefore gold nanoparticles may have an advantage over silver nanoparticles for coating the nanoparticles with ligands.

[0015] In some embodiments, the disclosed methods and systems are carried out with nanoparticles having an average diameter ranging from about 1 nm to about 100 nm, or about 10 nm to about 50 nm, or about 20 nm to about 60 nm, or about 30 nm to about 70 nm, or about 40 nm to about 80 nm, or about 50 nm to about 100 nm, or about 10 nm to about 40 nm, or about 10 to about 30 nm, or about 20 nm to about 50 nm, or about 30 nm to about 50 nm. In representative embodiments, the disclosed methods are carried out with nanoparticles having an average diameter ranging from about 10 nm, or about 20 nm, or about 30 nm, or about 40 nm, or about 50 nm, or about 60 nm, or about 70 nm, or about 80 nm, or about 90 nm, or about 100 nm.

[0016] As used herein, a "ligand" for an antibody protein product refers to a substance that binds to the antibody protein product, such as an antibody that binds to the antibody protein product, or a binding partner to the antibody protein product. Examples of ligands for purposes herein include an antigen to the antibody protein product, an anti-idiotypic antibody, an anti-Fc antibody, Protein A, or Protein G. In representative embodiments, the solid support (e.g., beads) comprises an anti-FC protein, Protein A, or Protein G, or the solid support (e.g., beads) comprises Protein A or Protein G. In some embodiments, the disclosed methods are performed with nanoparticles coated with a ligand for an antibody, where the ligand is an antibody or fragment thereof that specifically binds to human Fc protein, or an antibody or fragment thereof that binds to human immunoglobulin, such as anti-human IgG, anti-human IgM, anti-human IgA, or anti-human IgD, or anti-human IgE. In other embodiments, the nanoparticles are coated with a ligand for an antibody, where the ligand is Protein A or Protein G, or a combination thereof. The term "coated" refers to attaching or covalently coupling the ligand to the nanoparticle, including, for example, passive absorption.

[0017] In various embodiments, the disclosed methods and systems are carried out using a solid support that is a bead or microsphere, a membrane, a nanofiber, a nanotube, a resin, or agarose. For example, the solid support can be polymer-based, such as polystyrene beads, poly(lactide-co-glycolide) (PLGA) beads, polyethylene oxide (PEO) beads, polyethylene glycol (PEG) beads, polyvinyl alcohol (PVA) beads, or metal-based, such as gold beads. Furthermore, the solid support can be chitosan, dextran, alginate, gadolinium-based, carbon-based, silica-based, or iron-based. When the solid support is a resin, the resin can be a polymeric resin, such as cellulose, polystyrene, agarose, polyacrylamide, or agarose. In some methods, the beads can be magnetic beads.

[0018] For example, the solid support may comprise or consist of beads having an average diameter of at least about 1 μm, 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 6.5 μm, 7 μm, 8 μm, 9 μm, 10 μm, 20 μm, 30 μm, 40 μm, or 50 μm (including ranges between any two of the recited values), such as about 1-5 μm, about 1-10 μm, about 1-20 μm, about 1-40 μm, about 5-10 μm, about 5-20 μm, about 5-40 μm, about 6.5-10 μm, about 10-20 μm, or about 10-40 μm. Without being limited by theory, it is contemplated that solid supports having a larger diameter than the nanoparticles may have a higher antibody protein product binding capacity than each nanoparticle, facilitating coaggregation reactions. By way of example, the average diameter of the solid support may be at least 2x, 5x, 10x, 50x, 100x, 500x or 1000x that of the nanoparticles, such as 2x-100x, 2x-500x, 2x-1000x, 10x-100x, 10x-500x, 10x-1000x, 100x-500x or 100x-1000x that of the nanoparticles. The diameter may be determined as the average diameter for a population of solid supports or nanoparticles.

[0019] In any of the disclosed methods, the solid support is coated with an antibody or fragment thereof that specifically binds to an antibody protein product, such as anti-human IgG, anti-human IgM, anti-human IgA, or anti-human IgD or anti-human IgE. In other embodiments, the solid support is coated with a ligand for the antibody protein product, the ligand being Protein A or Protein G, or a combination thereof. The term "coated" refers to attaching or covalently coupling a ligand to the nanoparticle.

[0020] In some embodiments, the solid support comprises a unique barcode. Examples of suitable barcodes include a peptide or nucleic acid with a unique sequence or molecular weight, a pigment or combination of pigments, a glycan and / or carbohydrate or combination thereof, or a fluorophore or combination of fluorophores.

[0021] In any of the disclosed methods, the sample is any fluid or formulation containing an antibody, antibody protein product, or fragment thereof. In various embodiments, the sample is a fluid containing an antibody, antibody protein product, or fragment thereof that is to be processed, measured, or analyzed for stability and / or structural integrity or other properties. In some embodiments, the sample comprises or consists of conditioned medium or any fluid from which the antibody, antibody protein product, or fragment thereof is purified or isolated. In some embodiments, the sample comprises cells expressing the antibody, antibody protein product, or fragment thereof.

[0022] In any of the disclosed methods, the sample comprises an antibody protein product, such as an antibody, an antibody protein product, a bispecific T cell engager (BiTE®) molecule, an antibody fragment, an antibody fusion peptide, or an antigen-binding fragment or peptide thereof. In related embodiments, the antibody is a polyclonal antibody or a monoclonal antibody. As used herein, the term "antibody protein product" refers to an antibody, as well as any one of several antibody surrogates that are based on the structure of an antibody, but in various instances are not found in nature. "Antibody" is a subgenus of antibody protein products. It refers to any isotype of immunoglobulin that has specific binding to a target antigen, including, for example, monoclonal antibodies. The antibody may be of any suitable host species, such as chimeric, humanized, fully human, fully mouse, fully rabbit, or fully llama. An antibody generally comprises two full-length heavy chains and two full-length light chains. For example, human antibodies can be of any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE. In some embodiments, the molecular weight of the antibody protein product is in the range of at least about 12 kDa to 10 MDa, e.g., at least about 12 kDa to 5 MDa, 12 kDa to 1 MDa, 12 kDa to 750 kDa, at least about 12 kDa to 250 kDa, or at least about 12 kDa to 150 kDa. In certain embodiments, the antibody protein product has a valency (n) ranging from monomer (n=1) to dimer (n=2), trimer (n=3), to tetramer (n=4), unless of higher valency. Antibody protein products are in some embodiments based on the complete antibody structure and / or mimic antibody fragments that retain full antigen-binding capacity, such as scFv, Fab and VHH / VH (discussed below). The smallest antigen-binding antibody fragment that retains a complete antigen-binding site is the Fv fragment, consisting entirely of the variable (V) region. Soluble flexible amino acid peptide linkers are used to link the V region to scFv (single chain fragment variable) fragments to stabilize the molecule, or constant (C) domains are added to the V region to generate Fab fragments (fragment, antigen-binding).Both scFv and Fab fragments can be easily produced in host cells, e.g., prokaryotic host cells. Other antibody protein products include disulfide bond stabilized scFv (ds-scFv), single chain Fab (scFab) and dimeric and multimeric antibody formats, such as diabodies, triabodies and tetrabodies or minibodies (miniAbs), including different types that include scFv linked to oligomerization domains. The smallest fragments are the VHH / VH of camelized heavy chain Abs and single domain Abs (sdAbs), including the UniDab® construct-containing molecules and the UniAb® constructs (TeneoBio). The building blocks most frequently used to create new antibody formats are single chain variable (V) domain antibody fragments (scFv), which contain V domains (VH and VL domains) from heavy and light chains linked by a peptide linker of about 15 amino acid residues. Peptibodies or peptide-Fc fusions are yet another antibody protein product. The structure of peptibodies comprises a bioactive peptide grafted onto the Fc domain. Peptibodies are well described in the art. See, for example, Shimamoto et al., mAbs 4(5):586-591 (2012). Other antibody protein products include single chain antibodies (SCAs); diabodies; triabodies; tetrabodies; bispecific or trispecific antibodies, etc. Bispecific antibodies can be categorized into five major classes: BsIgG, adducted IgG, BsAb fragments, bispecific fusion proteins, and BsAb conjugates. See, for example, Spiess et al., Molecular Immunology 67(2)Part A:97-106 (2015). In an exemplary embodiment, the antibody protein product comprises or consists of a Bispecific T Cell Engager (BiTE®) molecule, which is an artificial bispecific monoclonal antibody. BiTE® molecules are fusion proteins that contain two scFvs of different antibodies, one that binds CD3 and the other that binds to a target antigen. BiTE® molecules are known in the art.See, e.g., Huehls et al., Immuno Cell Biol 93(3):290-296(2015); Rossi et al., MAbs 6(2):381-91(2014); Ross et al., PLoS One 12(8):e0183390.

[0023] In any of the disclosed methods, the method may be performed using any fluidic system, fluidic element, or fluidic device known in the art, such as a fluidic optical element. A fluidic element (or fluidic device) is an element that includes one or more separate circuits configured to hold a fluid, each circuit being composed of fluidically interconnected circuit elements. The circuit elements, including but not limited to regions, flow paths, channels, chambers, and / or pens, and at least one port, are configured to allow fluid to flow into and / or out of the fluidic element. The fluidic circuit may be configured to have a first end that is fluidly coupled to a first port (e.g., an inlet) in the fluidic element, and a second end that is fluidly coupled to a second port (e.g., an outlet) in the fluidic element or coupled to a second fluidic element or a second region, flow path, channel, chamber, or pen in the fluidic element. The fluidic element may be a microfluidic element, although other scales, such as nanoscale, may also be suitable. For example, the fluidic device can be a microfluidic chip, a microfluidic channel, a microfluidic cell, a nanofluidic chip, a nanofluidic channel, a nanofluidic cell, or an isolation pen. In some embodiments, the fluidic system includes a multiwell plate, such as a 96 or 384 well plate. The multiwell plate can be in fluid communication with the circuit.

[0024] For microfluidic devices, the circuit includes a fluidic region, which may include a microfluidic channel and at least one chamber, and holds a volume of fluid of less than about 1 mL, such as less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, or 2 μL. In certain embodiments, the circuit holds about 1-2, 1-3, 1-4, 1-5, 2-5, 2-8, 2-10, 2-12, 2-15, 2-20, 5-20, 5-30, 5-40, 5-50, 10-50, 10-75, 10-100, 20-100, 20-150, 20-200, 50-200, 50-250, or 50-300 μL. The circuit can be configured to have a first end that is fluidly coupled to a first port (e.g., an inlet) in the microfluidic device and a second end that is fluidly coupled to a second port (e.g., an outlet) in the microfluidic device.

[0025] As used herein, a "nanofluidic device" or "nanofluidic device" is a type of fluidic device having a fluidic circuit containing at least one circuit element configured to hold a volume of fluid of less than about 1 μL, e.g., less than about 750, 500, 250, 200, 150, 100, 75, 50, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 nL or less. A nanofluidic device can include a plurality of circuit elements (e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000, 10,000 or more). In certain embodiments, one or more (e.g., all) of the at least one circuit element are configured to hold a volume of fluid of about 100 pL to 1 nL, 100 pL to 2 nL, 100 pL to 5 nL, 250 pL to 2 nL, 250 pL to 5 nL, 250 pL to 10 nL, 500 pL to 5 nL, 500 pL to 10 nL, 500 pL to 15 nL, 750 pL to 10 nL, 750 pL to 15 nL, 750 pL to 20 nL, 1 to 10 nL, 1 to 15 nL, 1 to 20 nL, 1 to 25 nL, or 1 to 50 nL. In other embodiments, one or more (e.g., all) of the at least one circuit element are configured to hold a volume of fluid of about 20nL-200nL, 100-200nL, 100-300nL, 100-400nL, 100-500nL, 200-300nL, 200-400nL, 200-500nL, 200-600nL, 200-700nL, 250-400nL, 250-500nL, 250-600nL, or 250-750nL.

[0026] "Fluid channel" or "flow channel", as used herein, refers to a flow region of a fluidic device whose length is significantly longer than both its horizontal and vertical dimensions. For example, a flow channel can be at least 5 times longer, e.g., at least 10 times longer, at least 25 times longer, at least 100 times longer, at least 200 times longer, at least 500 times longer, at least 1,000 times longer, at least 5,000 times longer or more than the length of either the horizontal or vertical distance. In some embodiments, the length of the flow channel ranges from about 50,000 microns to about 500,000 microns, including all values ​​therebetween. In some embodiments, the horizontal distance ranges from about 100 microns to about 1000 microns (e.g., about 150 to about 500 microns) and the vertical distance ranges from about 25 microns to about 200 microns, e.g., about 40 to about 150 microns. It is noted that flow channels can have a variety of different spatial configurations in a fluidic device and are therefore not limited to being perfectly linear elements. For example, a flow channel may include one or more sections having any of the following configurations: curved, bent, spiraling, sloping, descending, forking (e.g., multiple different flow paths), and any combination thereof. Additionally, a flow channel may expand and contract to have different cross-sectional areas along its path to provide a desired flow path therein.

[0027] In some embodiments, a system is described. The system may include a fluidic device. The system may further include nanoparticles coated with a ligand for the antibody protein product. The system may further include a solid support coated with a ligand for the antibody protein product. For some systems, the average diameter of the solid support is greater than the average diameter of the nanoparticles. The system may further include an optical detector configured to detect a change in an optical signal of the solid support, such as a change in absorption or light emission. [Brief description of the drawings]

[0028] [Figure 1]FIG. 1 is a schematic illustrating a capture bead and nanoparticle co-agglutination assay. [Diagram 2] FIG. 2 provides brightfield OEP and filtered TRed images showing that when anti-huFc coated gold nanoparticles were mixed with anti-huIgG coated capture beads in the absence of added antibody, there was no detectable coaggregation pattern or detectable luminescence signal. [Diagram 3] Figure 3 provides brightfield OEP and filtered TRed images after mixing 6.5 μM anti-huFc coated gold nanoparticles and anti-huIgG coated capture beads with mAb1 (negative control). No bead coagglutination or luminescence signal was detected in the presence of 1 μM mAb1. [Figure 4] Figure 4 provides brightfield OEP and filtered TRed images after mixing anti-huFc coated gold nanoparticles and 6.5 μM anti-huIgG coated capture beads with mAb2 (positive control). Islands of coaggregation of anti-huIgG capture beads and anti-huFc gold nanoparticles were detected in the presence of 1 μM mAb2 along with an increase in luminescence signal. [Diagram 5] Figure 5 is a bright field OEP image showing that anti-huFc coated gold nanoparticles (left panel) produced more visible small dots compared to uncoated gold nanoparticles (right panel) after 1 hour incubation with a positive control antibody, mAb2, in the absence of capture beads. [Figure 6] FIG. 6 shows brightfield OEP and filtered TRed images showing that no coaggregation pattern or luminescence signal was detected when anti-huFc coated gold nanoparticles were mixed with 3 μM anti-huIgG coated capture beads in the presence of CHOK1 growth medium but no added antibody. [Figure 7]Figure 7 provides brightfield OEP and filtered TRed images after mixing mAb1 in CHO-K1 medium (negative control) with anti-huFc coated gold nanoparticles and 3 μM anti-huIgG coated capture beads. No bead coagglutination or luminescence signal was detected in the presence of 1 μM mAb1. [Figure 8] Figure 8 provides brightfield OEP and filtered TRed images after mixing mAb2 (positive control) in CHO-K1 medium with anti-huFc coated gold nanoparticles and 3 μM anti-huIgG coated capture beads. Islands of coaggregation of anti-huIgG capture beads and anti-huFc gold nanoparticles were detected with strong luminescence signal in the presence of 1 μM mAb2. [Figure 9] FIG. 9 provides brightfield OEP and filtered TRed images taken from a Beacon® pen containing a live culture of CHO-K1 cells expressing mAb mixed with 40 nm gold nanoparticles coated with anti-huFc and capture beads coated with 3 μM anti-huIgG prior to imaging. [Figure 10A] Figures 10A-10C show the increase in peak fluorescence emission detected for mAb2 (positive control) and anti-huFc gold nanoparticle mixtures in the presence of capture beads compared to mAb1 (negative control). Figure 10A shows that 10 μm capture beads produced a separation of peak fluorescence emission between mAb2 and mAb1. Figure 10B shows that 6.5 μm capture beads produced the greatest separation of peak fluorescence emission between mAb2 and mAb1. [Figure 10B] Figures 10A-10C show the increase in peak fluorescence emission detected for mAb2 (positive control) and anti-huFc gold nanoparticle mixtures in the presence of capture beads compared to mAb1 (negative control). Figure 10A shows that 10 μm capture beads produced a separation of peak fluorescence emission between mAb2 and mAb1. Figure 10B shows that 6.5 μm capture beads produced the greatest separation of peak fluorescence emission between mAb2 and mAb1. [Figure 10C]Figures 10A-10C show the increase in peak fluorescence emission detected for mAb2 (positive control) and anti-huFc gold nanoparticle mixtures in the presence of capture beads compared to mAb1 (negative control). Figure 10A shows that 10 μm capture beads produced a separation of peak fluorescence emission between mAb2 and mAb1. Figure 10B shows that 6.5 μm capture beads produced the greatest separation of peak fluorescence emission between mAb2 and mAb1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The disclosed method utilizes nanoparticles coated with a ligand for an antibody protein product, such as an anti-human immunoglobulin, e.g., an anti-huIgG antibody, in an affinity-capture self-assembly nanoparticle spectroscopy measurement for the detection of low concentrations of antibody protein product self-association. Antibody protein products that tend to self-associate will cause the nanoparticles to cluster, resulting in a change in optical signal, e.g., a change in absorption or light emission. Since there is no optical signal measurement in some fluidic systems, another form of signal detection may be used. A solid support also coated with a ligand for the antibody protein product, such as polystyrene beads coated with anti-human immunoglobulin (e.g., anti-huIgG), may be used to capture clusters of nanoparticles and amplify the signal through co-agglutination of the nanoparticles on the surface of the solid support. The amplified signal may be detected through an emission filter on a fluidic optical device such as the Beacon® system. In some methods, the change in the pattern of the solid support may be detected by microscopy. The detection of the change in the pattern may further include image processing. The image processing may be automated and may include machine learning.

[0030] Since cells in each pen of the chip on the Beacon® system continuously express antibodies, in the exemplary disclosed method, the secreted antibodies are captured by the introduced nanoparticles diffusing in the solution. The presence of self-associating antibodies causes the particles to form clusters, and the addition of micrometer-sized capture solid supports (e.g., capture beads) to each pen causes the nanoparticle clusters to co-aggregate on the solid support. The co-aggregation reaction on the solid support results in a change in the solid support pattern and light emission, which is captured by the CCD camera. Thus, quantification of the emission signal intensity allows the identification of cells expressing antibodies with a high risk of self-association. Also, changing the filter cube excitation and emission wavelengths is believed to improve the signal-to-noise ratio for detection at the optimal wavelength.

[0031] In some embodiments, the solid support is a capture bead with a large antibody binding capacity of 1e5 molecules / bead, while there are only about 170 antibody molecules per nanoparticle, so the inclusion of the capture solid support allows detection of antibodies at low concentrations. If the particle-bound antibodies have a strong tendency to self-associate, particle aggregation and changes in absorption (and light emission) will result. The small size of nanoparticles allows them to be densely packed, which is an advantage for detection. Co-agglutination reactions are used for image-based detection with changes in bead patterns and absorption or light emission measurements for quantification.

[0032] The fluidics device allows for the growth and expansion of single cells in a chamber or isolation pen, followed by clonal selection of cells that produce the antibody protein product to be detected. Clonal selection allows for the selection of clones for large scale protein production and purification during drug discovery and biopharmaceutical manufacturing, such as antibody production. The disclosed method also allows for the continuous analysis of cells as they are expanded, and assays can be repeated on the same growing cells.

[0033] A colony of biological cells is "clonal" if all living cells in the regrowable colony are daughter cells derived from a single progenitor cell. In certain embodiments, all daughter cells in a clonal colony are derived from a single progenitor cell by no more than 10 divisions. In other embodiments, all daughter cells in a clonal colony are derived from a single progenitor cell by no more than 14 divisions. In other embodiments, all daughter cells in a clonal colony are derived from a single progenitor cell by no more than 17 divisions. In other embodiments, all daughter cells in a clonal colony are derived from a single progenitor cell by no more than 20 divisions. The term "clonal cells" refers to cells of the same clonal colony.

[0034] As used herein, a "colony" of biological cells refers to two or more cells (e.g., about 2 to about 20, about 4 to about 40, about 6 to about 60, about 8 to about 80, about 10 to about 100, about 20 to about 200, about 40 to about 400, about 60 to about 600, about 80 to about 800, about 100 to about 1000, or more than 1000 cells).

[0035] As used herein, the term "(a) maintaining cells" refers to providing an environment that includes both liquid and gaseous components and, optionally, surfaces that provide the conditions necessary for cells to be maintained to survive and / or expand.

[0036] As used herein, the term "expand," when referring to cells, refers to an increase in cell number.

[0037] In some embodiments, the sample comprises or consists of conditioned medium or any liquid from which antibodies, antibody protein products or fragments thereof may be purified or isolated. In various embodiments, the sample so subjected to the methods disclosed herein comprises or consists of large peptides, antibodies, antibody fragments, antibody fusion peptides or antigen-binding fragments thereof. In related embodiments, the antibody is a polyclonal or monoclonal antibody.

[0038] Fluidic Devices Fluidic devices refer to devices that use small amounts of fluid to perform various types of analyses. Fluidic devices include one or more separate circuits configured to hold fluid, with each circuit being made up of fluidically interconnected circuit elements. The circuit elements, including but not limited to regions, flow paths, channels, chambers, and / or pens and at least one port, are configured to allow fluid to flow into and / or out of the fluidic device. These devices use chips, cells, channels, or isolated pens that contain the fluids for the analyses.

[0039] Fluidic devices, such as microfluidic devices, generally have one or more channels with at least one dimension less than 1 mm. Common fluids used in fluidic devices include whole blood samples, bacterial cell suspensions, protein or antibody solutions, and various buffers. Fluidic devices can be used to obtain a variety of measurements including molecular diffusion coefficients, fluid viscosity, pH, chemical binding coefficients, and enzyme reaction kinetics. Other applications for fluidic devices include capillary electrophoresis, isoelectric focusing, immunoassays, flow cytometry, sample injection of proteins for analysis via mass spectrometry, PCR amplification, DNA analysis, cell manipulation, cell separation, cell patterning, and chemical gradient formation. Many of these applications have utility for clinical diagnostics.

[0040] Advantages to using fluidic devices include that the volume of fluid in these channels is very small, usually a few nanoliters, and only small amounts of reagents and analytes are used. Furthermore, when analyzing protein-producing cells, a relatively small number of cells (or even a single cell) can produce sufficient amounts and concentrations of protein for analysis, reducing or avoiding incubation times for colony expansion growth. The fabrication techniques used to build microfluidic devices are relatively inexpensive and highly amenable to both highly sophisticated multiplexed devices and also mass production. Fluidic technology allows the manufacture of highly integrated devices to perform several different functions on the same substrate chip.

[0041] Any fluidic device may be used (or adapted for use) in the disclosed methods, including commercially available devices. The fluidic device may be configured for use in a fluidic optical system and may use light to manipulate materials in the fluidic device, such as cells. As an exemplary microfluidic device, a chip containing a Berkley Lights (BLI) pen is described herein. For example, the BLI pen may be analyzed in a Beacon® fluidic optical system, a Lightning™ Optofluidic System, or a Culture Station System (BLI. Emeryville, CA). Another exemplary fluidic optical system is the Cyto-Mine® System (Sphere Fluidics, Great Abington, Cambridge, UK). EXAMPLES

[0042] Example 1 General Method The nanoparticles and capture beads used in the experiments described herein are available from commercial sources as listed in Table 1. Purified antibodies were used as controls: an antibody known to exhibit high self-association and viscosity scores (designated as mAb2) was used as a positive control; an antibody known to exhibit low self-association and viscosity scores (designated as mAb1) was used as a negative control. The characteristics of the control antibodies are listed in Table 2.

[0043] The control antibodies to be tested were stored at 4°C prior to testing and were not stressed prior to or during testing. The control antibodies were premixed with gold nanoparticles, followed by the addition of capture beads to the mixture. The mixture was then loaded onto the Beacon® system. PBS pH 7.4 buffer and CHO-K1 growth medium were used for background detection. Images were acquired on the Beacon® system under bright field (OEP) and filter cubes at excitation 562 / 40 nm and emission 624 / 40 nm (TRed 620 nm).

[0044] [Table 1]

[0045] [Table 2]

[0046] FIG. 1 summarizes the experiments carried out in the disclosed method and in the following examples to detect antibody association and aggregation. Gold nanoparticles (40 nm) coated with polyclonal goat anti-human Fc fragments were used to bind the mAb of interest in a first incubation step (shown as "premix"). For associative mAbs, the gold nanoparticles cluster in the premix. Then, micrometer-scale anti-Fc solid supports (e.g., "capture beads") that further bind the mAb of interest are added to the premix, which leads to co-aggregation of the gold nanoparticles and the capture beads. The presence of co-aggregation structures caused a change in the light absorption and a change in the optical pattern of the mixture. Thus, quantification of the absorption change or image-based detection of the optical pattern change allowed the identification of cells expressing antibodies with a high risk of self-association, a hallmark of solubility, aggregation and viscosity. In contrast, non-associative antibodies do not cause clustering of the gold nanoparticles or co-aggregation of the capture beads, resulting in no shift in the light absorption or optical pattern.

[0047] Example 2 Detection of antibody self-association using gold nanoparticles and micrometer beads A combination of antibody-coated gold nanoparticles and antibody-coated solid supports, i.e., anti-IgG-coated capture beads, was used to detect antibody self-association in an optical fluidic device. PBS buffer (10 μl) or 1 μM purified antibodies (10 μl mAb1 or mAb2) were mixed with anti-huFc_coated 40 nm gold nanoparticles (10 μl) in a tube at room temperature for 15 min (indicated as "premix"). 120 μl of 6.5 μm anti-hu-IgG (heavy and light chain) coated beads (indicated as capture beads) were concentrated by centrifugation, and then the capture beads were resuspended in 10 μl of premix. The mixture was then loaded into a channel of the chip 3500 on the Beacon® system. Images were recorded through bright field (OEP) and TRed filters. The assay was also performed by mixing the premix in the absence of capture beads, which was also loaded into the channel of the chip 3500 on the Beacon® system for imaging.

[0048] A premix containing PBS buffer (without antibody addition) was incubated with the capture beads as described above. As shown in Figure 2, there was no coaggregation pattern of anti-huFc_gold nanoparticles mixed with the capture beads when detected using bright field OEP. Furthermore, no luminescence signal was detected through the filter TRed.

[0049] A premix containing 1 μM mAb1 (negative control) was incubated with the capture beads as described above. As shown in Figure 3, there was no coaggregation pattern of anti-FC gold nanoparticles mixed with the capture beads when detected using bright-field OEP. Furthermore, no luminescence signal was detected through the filter TRed.

[0050] A premix containing 1 μM mAb2 (positive control) was incubated with the capture beads as described above. However, Figure 4 shows the detection of a clear coaggregation reaction pattern of gold nanoparticles with the capture beads, revealing an increase in the luminescence signal through the filter TRed in the presence of the positive control antibody (mAb2). This set of results shows that the approach of adding capture beads to a mixture of antibodies and gold nanoparticles was able to distinguish purified antibodies with a high risk of self-association from those with a low risk of self-association on the Beacon® system. As shown in the bright field images in Figure 5, a premix containing 1 μM mAb2 and 40 nm anti-huFc-coated gold nanoparticles (left panel) without capture beads gave rise to more obvious small dots compared to unconjugated 20 nm gold nanoparticles (right panel). This experiment reveals that the changes in bright field OEP indicate that anti-hu-IgG-coated nanoparticles specifically bind mAb2 with strong affinity, while unconjugated gold nanoparticles rely on the slow passive adsorption of the antibody onto the positively charged gold. This experiment demonstrates that gold nanoparticles are capable of forming clusters in the presence of mAb2.

[0051] Example 3 Detection of antibody self-association using gold nanoparticles and micrometer beads in the presence of cell culture medium To determine whether capture bead and gold nanoparticle coaggregation assays and imaging can be performed in the presence of culture medium, a similar set of experiments to those described in the preceding examples was performed using the same purified control antibody and 3 μm anti-human Fc coated capture beads in the presence of fresh CHO-K1 culture medium. Here, 1 μM purified antibody (5 μl) in CHO-K1 growth medium (5 μl) or CHO-K1 growth medium (5 μl) alone was mixed with 40 nm anti-huFc_gold nanoparticles (5 μl) in a tube (denoted as "premix") for 15 min at room temperature. 120 μl of 6.5 μm anti-hu-IgG (heavy and light chain) coated beads (denoted as capture beads) were concentrated by centrifugation and then the capture beads were resuspended in 10 μl of premix. The mixture was loaded into the channel of the chip 3500 on the Beacon® system. Images were recorded through bright field (OEP) and filter TRed.

[0052] As shown in Figures 6 and 7, no coaggregation or luminescence signal was observed in the presence of fresh CHO-K1 culture medium alone or in the presence of the negative control antibody mAb1 in the culture medium, indicating that there was no nonspecific binding interference by the culture medium. A premix containing 1 μM mAb2 (positive control) in culture medium was incubated with the capture beads as described above. As shown in Figure 8, there was a clear coaggregation pattern of gold nanoparticles mixed with 3 μm anti-huFc coated beads, and an increase in luminescence signal was detected through the filter TRed. These results further suggest that the capture bead and gold nanoparticle coaggregation method can be used to evaluate the tendency of antibody self-association.

[0053] Example 4 Detection of antibody self-association in conditioned medium in an optical fluid device An experiment was performed to test the coaggregation assay using gold nanoparticles and capture beads coated in a Beacon® pen containing live CHO-K1 cells expressing monoclonal antibodies. Cells were cultured on the fluidic chip for 48 hours in growth medium. A premix of 40 nm anti-huFc_gold nanoparticles and 3 μm anti-huFc coated beads was then loaded into the fluidic pen and incubated for 5 hours before imaging. After 5 hours of loading, images were captured through bright field and cube filter TRed 620 nm. Changes in the bead optical pattern and luminescence signal were observed in some pens, as shown in Figure 9. This experiment demonstrates that the coaggregation assay can be performed in conditioned medium of live cells growing in a fluidic optical device.

[0054] Example 5 Detection of antibody self-association using fluorescence scanning An experiment was performed to determine whether fluorescence scanning could be used to detect antibody aggregates when detected using the methods described in the above examples. To detect antibody self-association using fluorescence scanning, a combination of antibody-coated gold nanoparticles and antibody-coated solid support, i.e., anti-IgG-coated capture beads, was used. Equal volumes of 1 μM purified antibodies (mAb1-negative control or mAb2-positive control) were mixed with 40 nm gold nanoparticles coated with anti-huFc (shown as "premix") in a tube at room temperature for 15 minutes. 100 μl of 6.5 μm anti-hu-IgG (heavy and light chain) coated beads (shown as capture beads) were concentrated by centrifugation, and then the capture beads were resuspended in 10 μl of premix. An aliquot of the premix was added to an equal volume of 5% capture beads of sizes 10 μm, 6.5 μm, and 3 μm, respectively.

[0055] After 15 min incubation at room temperature, fluorescence scans were performed on a black plate on a Tecan SAFIRE II at an excitation wavelength of 520 nm. As shown in Figure 10A-C, an increase in peak fluorescence emission was detected for the positive control mAb2 compared to the negative control mAb1. At an excitation wavelength of 520 nm, solid supports with average diameters of 6.5-10 μm showed a larger separation than solid supports with an average diameter of 3 μm. Capture beads with a size of 6.5 μm produced the largest separation of peak fluorescence emission between mAb1 and mAb2 (see Figure 10B).

[0056] This data demonstrates that fluorescence scanning can be used to detect aggregation. In particular, this experiment demonstrates that peak fluorescence emission can be used to distinguish between antibodies with different risks of self-association.

Claims

1. 1. A method for detecting antibody protein product association, comprising: a) contacting a sample containing an antibody protein product with nanoparticles coated with a ligand for the antibody protein product, said contacting occurring in a fluidic device under conditions that allow the antibody protein product to self-associate, thereby forming clusters of nanoparticles; b) contacting said clusters of nanoparticles with a solid support coated with a ligand for said antibody protein product of (a); c) detecting a change in the optical signal of the nanoparticles and / or the solid support, wherein the change in optical signal indicates antibody protein product association; A method comprising:

2. The method of claim 1 , wherein the sample comprises conditioned medium.

3. 2. The method of claim 1, wherein the antibody protein product comprises or consists of a large peptide, an antibody, an antibody fragment, an antibody fusion peptide, or an antigen-binding fragment thereof.

4. The method of claim 1 , wherein the nanoparticles have an average diameter in the size range of about 10 nm to about 50 nm.

5. 10. The method of claim 1, wherein the nanoparticles have an average diameter of about 20 nm or about 30 nm.

6. 2. The method of claim 1, wherein the nanoparticles are coated with an antibody or fragment thereof that specifically binds to human Fc protein, protein A, or protein G, or a combination thereof.

7. The method of claim 1 , wherein the solid support is a bead, a resin, or agarose.

8. The method of claim 1 , wherein the nanoparticles comprise or consist of a metal or a polymer.

9. 9. The method of claim 8, wherein the nanoparticles comprise or consist of gold.

10. 2. The method of claim 1, wherein the solid support is coated with an antibody or fragment thereof that specifically binds to human Fc protein, protein A, or protein G, or a combination thereof.

11. 10. The method of claim 1, wherein the solid support is a bead having an average diameter at least 2x, 5x, 10x, 50x, 100x, 500x, or 1000x the average diameter of the nanoparticles.

12. 10. The method of claim 1, wherein the solid support is a bead having an average diameter ranging in size from about 1 μm to about 10 μm or from about 6.5 μm to about 10 μm.

13. 8. The method of claim 7, wherein the solid support is a bead having an average diameter of about 3 μm or about 6.5 μm.

14. The method of claim 1 , wherein the fluidic device comprises or consists of a microfluidic chip or an isolation pen.

15. The method of claim 1 , wherein a single clone of cells is seeded into the fluidic device.

16. The method of claim 1 , wherein the optical signal comprises light emission, an optical pattern, or light scattering.

17. The method of claim 16 , wherein the optical radiation comprises fluorescent radiation.

18. The method of claim 1 , wherein the change in the optical signal is detected using an emission filter.

19. The method of claim 1 , wherein the change in the optical signal is detected using a fluorescence scan.

20. 20. The method of claim 19, wherein the solid support has an average size of 6.5 to 10 μm.

21. a fluidic element; nanoparticles coated with a ligand for the antibody protein product; a solid support coated with a ligand for said antibody protein product; A system comprising: Optionally, the system, wherein the average diameter of the solid support is greater than the average diameter of the nanoparticles.

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