Tools for characterizing antibodies

Abstract

The present disclosure provides tools (e.g., uses, nanoparticles, solutions, methods, kits, and systems) for screening target proteins for self-association properties (viscosity and opalescence) in ultra-dilute solutions, providing a means to screen large numbers of target proteins at concentrations orders of magnitude lower than the end-use formulation.

Description

[Technical field]

[0001] The present disclosure provides tools (eg, uses, nanoparticles, solutions, methods, kits, and systems) useful for detecting the self-association properties of proteins (eg, therapeutic antibodies) using ultradilute solution measurements. [Background technology]

[0002] Monoclonal antibodies (mAbs) are among the most successful medical therapeutics and are used to treat a variety of diseases. Despite this success, mAbs are prone to development issues that pose significant manufacturing, stability, and delivery challenges. Specifically, the development of mAbs intended for subcutaneous administration can be hindered by the need for highly concentrated formulations, which are often limited by poor solution behavior in the form of high viscosity, opalescence, phase separation, and aggregation. While intravenous administration of mAbs in hospital settings is routine, subcutaneous administration is less invasive, more assured of patient compliance, and easily adaptable in settings with limited medical infrastructure. Rapid selection of viable therapeutic mAb candidates is always desirable, but this urgency is even more acute when fighting infectious disease pandemics. When issues with developability arise, resource-intensive mitigation strategies must be implemented, which inevitably cause delays and do not guarantee success. Therefore, it would be much more efficient to early select mAbs with favorable solution properties, placing a strong emphasis on identifying variants with drug-like properties at the early stages of discovery. Summary of the Invention [Means for solving the problem]

[0003] Disclosed herein in a first aspect is the use of a positively charged polymer to stabilize nanoparticles that include a capture agent on their surface.

[0004] Disclosed herein, in a second aspect, is a nanoparticle comprising a capture agent and a positively charged polymer on a surface thereof.

[0005] Disclosed herein in a third aspect is a solution comprising a plurality of nanoparticles of the second aspect.

[0006] Disclosed herein in a fourth aspect is a kit that includes a nanoparticle and a positively charged polymer.

[0007] Disclosed herein in a fifth aspect is a method for determining the self-association tendency of a target protein, the method comprising: (i) capturing the target protein in a solution as defined in the third aspect; (ii) determining the color of the solution. Including, A change in colour of this solution compared to a control solution as defined in the third aspect not containing the target protein indicates the tendency of the target protein to self-associate. This is the method.

[0008] Disclosed herein, in a sixth aspect, is a method for screening a target protein at dilute concentrations for self-association properties, the method comprising: adsorbing a capture agent and a positively charged polymer onto a surface (e.g., a nanoparticle (e.g., a metal nanoparticle)) to form a capture agent conjugate; incubating the capture agent conjugate with the target protein in a solution (e.g., a buffer solution) to form a target protein conjugate; measuring the absorbance of the target protein conjugate at multiple wavelengths in the range of 450 nm to about 750 nm; and identifying the plasmon wavelength as the wavelength at which the absorbance by the target protein conjugate is greatest.

[0009] Disclosed herein, in a seventh aspect, is a system or kit that includes one or more of each of a capture agent, a positively charged polymer, a capture surface (e.g., a metal nanoparticle), a solution (e.g., a buffer), a target protein; at least one calibration protein; and a spectrophotometer.

[0010] Other aspects and embodiments of the present disclosure will become apparent in view of the following detailed description and accompanying drawings. [Brief description of the drawings]

[0011] [Figure 1] Schematic of an exemplary research and development process for monoclonal antibodies. An antibody discovery campaign begins with the identification and validation of thousands of candidate antibodies that recognize a target biomolecule. The large pool is condensed to the order of hundreds during the optimization phase, and candidates undergo affinity maturation and humanization. During these early stages, only minute amounts of material are available (ng-μg range), making evaluation of developability quality attributes such as viscosity, opalescence, and aggregation infeasible. Because of this, it is not uncommon for molecules with poor developability attributes to progress to clinical development, significantly increasing the amount of time and resources spent on generating a viable drug product. Evaluation of key developability attributes during lead optimization using ultra-low consumption, high throughput assays such as the CS-SINS approach described herein will facilitate the advancement of antibody candidates with reduced risk of complications during manufacturing and formulation in later stages of development, expediting timelines and reducing associated resource consumption. [Figure 2-1]Figures 2A-2D show the evaluation of antibody-gold conjugate stabilization using polylysine. Immunogold conjugates from a standard AC-SINS run (Figure 2A, left) were evaluated for stability under typical formulation and process conditions (10 mM acetate and histidine buffer, pH 4.04-6.5). By dynamic light scattering measurements, we observed rapid aggregation of the conjugates beginning at pH 5.0 and continuing throughout the relevant conditions up to pH 6.5 (Figure 2B, left). A simultaneous shift in the plasmon wavelength of the aggregated conjugates was also observed, measured by absorbance spectroscopy (Figure 2C, left). We measured the zeta potential across the pH range and observed a consistent decrease in potential as the pH was increased from 4.0, crossing zero near pH 5.75 and turning negative thereafter (Figure 2D, left). To achieve stability of the immunogold conjugate in the formulation space, a small amount (3% by weight) of polylysine, a highly charged biopolymer, was incorporated (Figure 2, center). Stabilization of the particles was observed up to pH 6.0 using both DLS measurements of particle size (Figure 2B, center) and optical measurements of plasmon wavelength (Figure 2C, center). The zeta potential of the conjugate remained positive over the assayed pH range (Figure 2D, center). However, the absolute value of the potential near pH 6.0 was close to zero, which is associated with aggregation in similar experiments with unstable conjugates. As demonstrated by the highly stabilized 15% polylysine condition (Figure 2A, right), stabilization increased as polylysine was increased within the range of 3% to 15%. The conjugate was stable over the relevant pH range as shown by DLS size measurements (Figure 2B, right) and plasmon wavelength (Figure 2C, right). The zeta potential of the highly stabilized particles was positive over the measured range (Figure 2D, right). FIG. 2E is an exemplary graph of absorbance as a function of wavelength used to determine the plasmon wavelength of a nanoparticle. [Figure 2-2] Continued from Figure 2-1. [Figure 2-3] Continued from Figure 2-2. [Figure 3-1]Figure 3A-H shows the application of charge-stabilized immunogold conjugates for the assessment of weak protein-protein interactions. A diverse panel of 56 monoclonal antibodies was utilized to evaluate the utility of charge-stabilized immunogold conjugates in identifying antibody-self-interactions. The panel represented an accurate reproduction of the clinical antibody situation, as evidenced by the similarity of biophysical properties with a dataset of 500 molecules extracted from the Therapeutic Antibody Database (TABS) (Figure 3A-3D). The robustness of the calibration approach (described in Methods) was demonstrated by comparing the results of experiments performed with various concentrations of polylysine (3% vs. 10%). An excellent correlation was observed between the two conditions, demonstrating that the assay exhibits resistance to effects from small changes in polylysine concentration that may occur due to variability in reagent preparation (Figure 3E). We compared CS-SINS scores to direct measures of important developability properties (see Kingsbury, JS et al. Sci Adv 6, eabb0372, doi:10.1126 / sciadv.abb0372 (2020), incorporated herein by reference in its entirety), which experimentally established that mAbs with viscosity >30 cP or opalescence >12 NTU indicate problems in manufacturing and formulation during clinical development. Applying these benchmarks, we found that using a CS-SINS threshold of 0.35 indicated good behavior (viscosity <30 cP, and opalescence <12 NTU), viscosity (>30 cP), or opalescence (>12 NTU), or problematic solution behavior in >85% of the mAb panel (Figures 3F-3H). [Figure 3-2] Continued from Figure 3-1. [Figure 3-3] Continued from Figure 3-2. [Diagram 3-4] Continued from Figure 3-3. [Figure 3-5] Continued from Figure 3-4. [Diagram 3-6] Continued from Figure 3-5. [Figure 4] We present the methodology for the CS-SINS calibration process. CS-SINS measurements are calibrated and evaluated using two tests. Only CS-SINS measurements that pass both tests should be accepted. [Figure 5-1]5A-C show an exemplary calibration of CS-SINS measurements. Examples of experiments where the calibration process passed (FIG. 5A) and failed (FIG. 5B-5C) due to gentle mixing of goat anti-human Fc antibody with gold particles (FIG. 5B) or addition of 50% of the target mAb concentration (FIG. 5C). To pass test number 1, the antibody-gold conjugate exhibits a plasmon wavelength below 534 nm (human polyclonal antibody) and 533 nm (NIST mAb). To pass test number 2, the conjugate exhibits linear fit parameters of the calibration panel to a reference panel of mAbs that are within 10% of the theoretical values ​​(1 for slope, 0 for intercept, and 1 for R2). The CS-SINS score is calculated as described herein. [Figure 5-2] Continued from Figure 5-1. [Figure 6A] The calibrated CS-SINS results show a strong correlation for conjugates prepared using various polylysine concentrations. A robust calibration protocol was developed to normalize results from experiments with various amounts of polylysine. Plasmon shift measurements for a panel of mAbs using polylysine-stabilized goat anti-human Fc antibody conjugates at polylysine / IgG mass fractions of 0.03 (97% IgG) and 0.10 (90% IgG) (Figure 6A). Figure 6B is a graph of the calibration between CS-SINS scores for a panel of mAbs measured at two polylysine / IgG mass fractions for the plasmon measurements reported in Figure 6A, using the calibration detailed in Figures 4 and 5. [Figure 6B]The calibrated CS-SINS results show a strong correlation for conjugates prepared using various polylysine concentrations. A robust calibration protocol was developed to normalize results from experiments with various amounts of polylysine. Plasmon shift measurements for a panel of mAbs using polylysine-stabilized goat anti-human Fc antibody conjugates at polylysine / IgG mass fractions of 0.03 (97% IgG) and 0.10 (90% IgG) (Figure 6A). Figure 6B is a graph of the calibration between CS-SINS scores for a panel of mAbs measured at two polylysine / IgG mass fractions for the plasmon measurements reported in Figure 6A, using the calibration detailed in Figures 4 and 5. [Figure 7-1] Figures 7A-C show that CS-SINS measurements are strongly correlated with diffusion interaction parameters in IgG subclass-specific behavior. CS-SINS scores (0.01 mg / mL) correlate well with kD measurements (1-10 mg / mL) (Figure 7A, top). CS-SINS scores are even better correlated with kD measurements for the IgG1 / IgG2 antibody subset (Figure 7A, middle) and less well correlated for the IgG4 antibody subset (Figure 7A, bottom). Solution behavior in the context of subclass reveals that the proposed 0.35 threshold for CS-SINS measurements effectively identifies all of the poorly behaving molecules in the IgG1 / IgG2 subclass grouping (Figure 7B). Solution behavior for IgG4 is more difficult to predict, as most tend to be highly opalescent but do not always react in the CS-SINS assay (Figure 7C). [Figure 7-2] Continued from Figure 7-1. [Figure 8-1]Figures 8A-C are graphs of the evaluation of the use of polylysine-stabilized gold conjugates to measure mAb self-association in histidine formulations (pH 6). Gold particles were first coated with goat anti-human Fc IgG and polylysine (>70 kDa) (Figure 8A), goat non-specific IgG and polylysine (Figure 8B), or polylysine alone (Figure 8C), and the conjugates were then incubated with a panel of mAbs with various levels of self-association. To evaluate the dependence of the plasmon shift on the amount of mAb adsorbed, the conjugates (after the first-stage conjugation step) were incubated with a fixed concentration of human antibody (0.01 mg / mL) consisting of human polyclonal antibody alone (0% mAb), human mAb alone (100% mAb), or a combination of these. The plasmon shift reported is relative to human polyclonal antibody. In Figures 8A and 8B, the mass ratio of polylysine to IgG was 0.03, and the concentrations of polylysine and IgG used in the conjugation were 0.012 and 0.388 mg / mL, respectively, and in Figure 8C, the concentration of polylysine used in the conjugation was 0.4 mg / mL. [Figure 8-2] Continued from Figure 8-1. [Figure 9] 9A-9C are graphs showing the effect of polylysine size on CS-SINS measurements of plasmon shift in histidine formulations (pH 6). Conjugates were prepared by co-adsorbing goat anti-human Fc antibodies with polylysine polymers of various sizes, and the conjugates were used to measure the plasmon shift of a panel of human mAbs. FIG. 9A shows the correlation between the plasmon shift measured using 30-70 kDa polylysine (0.03 polylysine / IgG mass ratio) and ≥70 kDa polylysine (0.03 polylysine / IgG mass ratio). FIG. 9B shows the correlation between the plasmon shift measured using 15-30 kDa polylysine polymers (0.10 polylysine / IgG mass ratio) and ≥70 kDa polylysine polymers (0.03 polylysine / IgG mass ratio). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present disclosure provides methods to predict problematic properties (eg, self-association) of protein formulations using small amounts of protein in ultra-dilute solution.

[0013] Antibodies with low levels of self-association have a low risk of exhibiting unfavorable solution behavior during manufacturing, formulation, and delivery at high concentrations. Although dilute solution interactions represent a valid approach for predicting antibody solution behavior, it is difficult to employ appropriate screening methods for large numbers of antibodies during early discovery (Figure 1). Conventional techniques require relatively concentrated protein solutions (>1 mg / mL) to perform such measurements. This single challenge related to antibody concentration has prevented large-scale systematic analysis of antibody self-association during antibody lead optimization (Figure 1). In a manner predictive of high-concentration solution behavior (e.g., viscosity at 150 mg / mL), assaying antibody interactions at much lower concentrations (1–10 μg / mL) would allow selection of well-behaved mAbs from a much larger pool of candidates during early discovery.

[0014] An approach already in use (affinity capture nanoparticle spectroscopy, AC-SINS) involved adsorbing an anti-human capture antibody to gold nanoparticles (20 nm) and using this conjugate to capture a human mAb of interest. By capturing multiple mAbs in close proximity, colloidal interactions were amplified and antibody self-interactions were sensitively detected via measurable changes in the optical properties intrinsic to the gold nanoparticles. However, the main limitation of AC-SINS was its incompatibility with process stream and formulation conditions in the pH 4.5-7 range and low ionic strength commonly utilized during antibody development. Gold nanoparticles coated with capture (anti-human) antibodies are unstable under such conditions and readily aggregate.

[0015] Described herein is the development of a system and method termed Charge-Stabilized Self-Interaction Nanoparticle Spectroscopy (CS-SINS) that overcomes the limitations of AC-SINS by using positively charged polymers to stabilize antibody-gold conjugates. Moreover, CS-SINS could reliably assess the weak self-interactions of mAbs at ultra-dilute concentrations (10 μg / mL) predicting high concentrations and opalescent antibodies that are problematic at concentrations four orders of magnitude higher (150 mg / ml).

[0016] The section headings used in this section and throughout this disclosure are for organizational purposes only and are not intended to be limiting.

[0017] 1.Definition The terms "comprise(s)", "include(s)", "having", "has", "can", "contain(s)", and variations thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not exclude the possibility of additional acts or structures. The singular forms "a", "an", and "the" include plural references unless the context clearly indicates otherwise. The present disclosure also contemplates other embodiments that "comprise", "consist of", and "consist essentially of" the embodiments or elements presented herein, whether or not expressly stated.

[0018] In reciting numerical ranges herein, each intervening number is expressly contemplated with the same degree of precision, for example, in the range 6 to 9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and in the range 6.0 to 7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are expressly contemplated.

[0019] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art. For example, any nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, gene and protein and nucleic acid chemistry and hybridization described herein are known and commonly used in the art. The meaning and scope of the terms should be clear, but in the event of potential ambiguity, the definitions set forth herein take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include the plural, and plural terms shall include the singular.

[0020] The term "antibody", as used herein, refers to a protein that is used inherently by the immune system to identify and neutralize foreign agents such as bacteria and viruses. Typically, an antibody is a protein that contains at least one complementarity determining region (CDR). The CDRs form the "hypervariable region" of the antibody, which is responsible for antigen binding (discussed further below). A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide, and two identical copies of a light (L) chain polypeptide. Each heavy chain contains one N-terminal variable (V H ) region and three C-terminal constant (C H1 , C H2 , and C H3 ) region, and each light chain comprises one N-terminal variable (V L ) region and one C-terminal constant (C L) region. The light chain of an antibody can be assigned to one of two different types, either kappa (κ) or lambda (λ), based on the amino acid sequence of its constant domain. In a typical antibody, each light chain is linked to a heavy chain by a disulfide bond, and the two heavy chains are linked to each other by disulfide bonds. The light chain variable region is aligned with the variable region of the heavy chain, and the light chain constant region is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chains are aligned with each other. The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. V H Area and V L The regions are identical in overall structure, and each region contains four framework (FW or FR) regions. The term "framework region" as used herein refers to a relatively conserved amino acid sequence in the variable region located between the CDRs. There are four framework regions in each variable domain, and they are called FR1, FR2, FR3, and FR4, respectively. The framework regions form a β-sheet that provides the structural framework of the variable region (see, for example, CA Janeway et al. (eds.), Immunobiology, 5th ed., Garland Publishing, New York, NY (2001)). The framework regions are linked by three CDRs. As discussed above, the three CDRs (known as CDR1, CDR2, and CDR3) form the "hypervariable region" of the antibody, which is involved in antigen binding. The CDRs form loops that connect, and in some cases are part of, the β-sheet structure formed by the framework regions. The constant regions of the light and heavy chains are not directly involved in the binding of the antibody to the antigen, but the constant regions can affect the orientation of the variable region. The constant regions also exhibit various effector functions, such as participation in antibody-dependent complement-mediated lysis or antibody-dependent cellular cytotoxicity, through interactions with effector molecules and cells.

[0021] The terms "fragment of an antibody," "antibody fragment," and "antigen-binding fragment" of an antibody are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (see generally Holliger et al., Nat. Biotech., 23(9):1126-1129 (2005)). Any antigen-binding fragment of an antibody described herein is within the scope of the present invention. An antibody fragment desirably contains, for example, one or more CDRs, a variable region (or a portion thereof), a constant region (or a portion thereof), or a combination thereof. Examples of antibody fragments include, but are not limited to, the following: (i) a V L Domain, V H Domain, C L domain, and C H1 (ii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; and (iii) a V fragment of a single arm of an antibody. L Domains and V H (iv) Fab' fragments, which are generated by cleaving the disulfide bridges of the F(ab')2 fragment using mild reducing conditions; (v) disulfide-stabilized Fv fragments (dsFv); and (vi) antibody single variable region domains (V H or V L ) Domain antibodies (dAbs) are polypeptides.

[0022] A "spectrophotometer" is any type of device that measures the absorption and / or transmission of light at various wavelengths. Spectrophotometers are most commonly applied to ultraviolet radiation (185-400 nm), visible radiation (400-700 nm), and infrared radiation (700-15000 nm), but some spectrophotometers may examine broad bands of the electromagnetic spectrum, including x-ray, ultraviolet, visible, infrared, and / or microwave wavelengths. A spectrophotometer has a light source, a spectrograph, a wavelength selector, a sample holder, and a detector. Any spectrophotometer capable of processing solution-based samples may be used (e.g., those with cuvette sample holders, plater readers, and the like).

[0023] A "polypeptide", "protein" or "peptide" is a linked sequence of two or more amino acids linked by peptide bonds. Polypeptides can be natural, synthetic, or modified or combinations of natural and synthetic. Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies. Proteins can be modified by the addition of sugars, lipids, or other moieties not contained in the amino acid chain. The terms "polypeptide", "protein", and "peptide" are used interchangeably herein.

[0024] A positively charged polymer can be any of a variety of compounds that have a net positive charge. Positively charged polymers useful in the present invention include positively charged peptides and proteins (both natural and synthetic), as well as polyamines, carbohydrates, or synthetic polycationic polymers. Positively charged polymers can be linear or branched polymers and can have interconnections between repeat units in addition to backbone linkages. A positively charged polymer composition can have any level of polydispersity, from substantially monodisperse to substantially polydisperse. A substantially monodisperse composition comprises polymer molecules that are substantially all the same chain length. A substantially polydisperse composition comprises polymer molecules of various chain lengths (and therefore molecular weights).

[0025] The concentration of the positively charged polymer can vary, but is directed to a concentration that prevents self-aggregation of the capture agent conjugate in the absence of the target protein. Aggregation of the capture agent conjugate can be measured under various solution conditions (pH, conjugate concentration, temperature, ionic strength) by a number of methods known in the art (e.g., dynamic light scattering as shown herein).

[0026] The concentration of the positively charged polymer may be greater than about 0.1% by weight of the positively charged polymer and the capture agent (i.e., 0.1% positively charged polymer and 99.9% capture agent), greater than about 1% by weight of the positively charged polymer and the capture agent, greater than about 3% by weight of the positively charged polymer and the capture agent, greater than about 5% by weight of the positively charged polymer and the capture agent, greater than about 10% by weight of the positively charged polymer and the capture agent, greater than about 15% by weight of the positively charged polymer and the capture agent, greater than about 20% by weight of the positively charged polymer and the capture agent, greater than about 30% by weight of the positively charged polymer and the capture agent, or greater than about 40% by weight of the positively charged polymer and the capture agent. In some embodiments, the positively charged polymer is added at a concentration of greater than 3% by weight of the positively charged polymer and the capture agent.

[0027] The concentration of the positively charged polymer can be from about 0.1% to about 50% by weight of the positively charged polymer and the capture agent. The concentration of the positively charged polymer is about 0.1% by mass to about 50% by mass, about 1% by mass to about 50% by mass, about 3% by mass to about 50% by mass, about 5% by mass to about 50% by mass, about 10% by mass to about 50% by mass, about 20% by mass to about 50% by mass, about 30% by mass to about 50% by mass, about 0.1% by mass to about 40% by mass, about 1% by mass to about 40% by mass, about 3% by mass to about 40% by mass, about 5% by mass to about 50% by mass, about 10% by mass to about 50% by mass, about 20% by mass to about 50% by mass, about 30% by mass to about 50% by mass, about 0.1% by mass to about 30% by mass, about 1% by mass to about 30% by mass, about 3% by mass to about 30% by mass, about 5% by mass to about 30% by mass, about 10% by mass to about 30% by mass, about 2 ... 0 mass% to about 30 mass%, about 0.1 mass% to about 20 mass%, about 1 mass% to about 20 mass%, about 3 mass% to about 20 mass%, about 5 mass% to about 20 mass%, about 10 mass% ~about 20% by mass, about 0.1% by mass - about 15% by mass, about 1% by mass - about 15% by mass, about 3% by mass - about 15% by mass, about 5% by mass - about 15% by mass, about 10% by mass - about 15% by mass mass%, about 0.1 mass% to about 10 mass%, about 1 mass% to about 10 mass%, about 3 mass% to about 10 mass%, about 5 mass% to about 10% mass, about 0.1 mass% to about 5 mass%, It can be about 1% to about 5% by weight, about 3% to about 5% by weight, about 0.1% to about 3% by weight, about 1% to about 3% by weight, or about 0.1% to about 1% by weight. In some embodiments, the positively charged polymer is added at a concentration greater than 3% by weight of the positively charged polymer and the capture agent, hi some embodiments, the positively charged polymer is added at a concentration of about 3% to about 15% by weight of the positively charged polymer and the capture agent.

[0028] The positively charged polymer may have a wide range of molecular weights. In some embodiments, the positively charged polymer may have a molecular weight of more than about 10 kD, more than about 15 kD, more than about 20 kD, more than about 25 kD, more than about 30 kD, more than about 40 kD, more than about 50 kD, more than about 60 kD, more than about 70 kD, more than about 80 kD, more than about 90 kD, more than about 100 kD, more than about 150 kD, more than about 200 kD, or more. In other embodiments, the positively charged polymer may have a molecular weight of 10-500 kDa, 10-250 kDa, 10-200 kDa, 15-70 kDa, 30-70 kDa, or 15-30 kDa. However, other sizes that prevent self-aggregation of the capture agent conjugate may also be used. Molecular weight is determined by one of skill in the art by methods such as size exclusion chromatography and / or multi-angle laser light scattering techniques.

[0029] In certain embodiments of the invention, the positively charged polymer may be in the class of synthetic polypeptides, also known as polyamino acids. The synthetic polypeptide may be a homopolymer of one of the positively charged (i.e., basic) amino acids (e.g., lysine, arginine, or histidine) or a heteropolymer of two or more positively charged amino acids. In some embodiments, the polycation may be polylysine (e.g., poly-D-lysine, poly-L-lysine, and poly-DL-lysine), polyarginine, and polyhistidine, and may be polylysine in particular. In addition, the polymer may contain one or more positively charged non-standard amino acids (e.g., ornithine, 5-hydroxylysine, and the like). Alternatively, the polypeptide may be functionalized with other groups, such as poly(γ-benzyl-L-glutamic acid). Any combination of amino acids may be polymerized into linear, branched, or cross-linked chains. Such polycationic polypeptides may contain at least 50 amino acid residues, at least 100 amino acid residues, at least 200 amino acid residues, at least 300 amino acid residues, at least 500 amino acid residues, at least 750 amino acids, at least 1000 amino acids, at least 2000 amino acids, at least 3000 amino acids, at least 4000 amino acids or more (e.g., about 50 to about 500 amino acid residues, about 50 to about 1000 amino acid residues, or about 100 to about 1000 amino acid residues). Synthetic polypeptides may be produced by methods known to those of skill in the art (e.g., chemical synthesis or recombinant methods). In alternative embodiments, the positively charged polymer is polylysine of about 70 kDa or more. In alternative embodiments, the positively charged polymer is polylysine added at a concentration of 3 to 15% by weight to volume.

[0030] In some embodiments, the positively charged polymer comprises a synthetic polycationic polymer (such as, but not limited to, polyethyleneimine (PEI), polyamidoamine (PAMAM), and the like).

[0031] The term "stabilization" in relation to nanoparticles refers in particular to the prevention or at least reduction of nanoparticle aggregation. "Agglomeration" refers to the formation of aggregates in a suspension and represents a mechanism that leads to the functional destabilization of colloidal systems. During this process, particles dispersed in a liquid phase stick together and spontaneously form irregular particle aggregates, flocs, or aggregates. This phenomenon is also referred to as coagulation or flocculation, and such suspensions are also called unstable.

[0032] As used herein, the term "nanoparticle" refers to a small particle having a size (e.g., diameter) on the scale of 0.001 μm to 1 μm. Nanoparticles can be in the shape of spheres, rods, chains, stars, flowers, reefs, whiskers, fibers, boxes, and the like. Size refers to the maximum distance from one point of the nanoparticle to another (e.g., diameter in the case of a sphere). Nanoparticles can include any material (e.g., metals, semiconductor materials, magnetic materials, and combinations of materials). Specifically contemplated for this disclosure are metal nanoparticles whenever mentioned herein. Metal nanoparticles (e.g., gold or silver nanoparticles) are inherently suited for surface plasmon resonance-based assays because metal spheres have free electrons on their surface that can interact with the electric field from the incident light, resulting in a strong absorbance spectrum. In some embodiments, the nanoparticles include gold nanoparticles having a gold surface or consisting of gold. The size of the nanoparticles can affect the intensity and wavelength of maximum absorbance. In some embodiments, the nanoparticles have a diameter between 1 nm and 1000 nm (e.g., between 1 nm and 200 nm). In some embodiments, the nanoparticles are between 5 nm and 50 nm in size (e.g., diameter), e.g., about 20 nm. In an exemplary embodiment, the nanoparticles comprise or are 20 nm gold nanoparticles. The total amount of nanoparticles in the assay may affect the signal to noise measurement. In some embodiments, 20 nm gold nanoparticles are at least 7.0×10 particles. 9 In some embodiments, 20 nm gold nanoparticles are used at a concentration of 7.0 x 10 particles / mL. 9 ~1.5×109 particles / mL (e.g., 1.0 × 10 particles 10 ~1.5×10 9 It is used at a concentration of (cells / mL).

[0033] A capture agent is any agent capable of binding to a target protein (specifically, "capturing the target protein" in solution as defined in the third aspect). The nature of the capture agent will therefore depend on the type of target protein. For example, the capture agent may comprise a natural or synthetic binding partner of the target protein (e.g., a protein, a nucleic acid, a carbohydrate, a small molecule, or another binding moiety that is specifically recognized by the target protein). In some embodiments, the capture agent comprises an antibody, or a derivative or fragment thereof capable of binding to the target protein. The capture agent may be directly adsorbed to the nanoparticle, or the nanoparticle may comprise a linker that tethers the capture agent to the nanoparticle. In some embodiments, the capture agent is a protein or a protein ligand. In specific embodiments, the capture agent is (or comprises) an antibody, or a protein that comprises an antigen-binding fragment of an antibody. The antibody may in particular be an Fc-specific antibody (i.e., an antibody that binds to the Fc portion of another antibody), for example an IgG-Fc-specific antibody, in particular an IgG1, IgG2, or IgG4-specific antibody, more specifically an IgG1 or IgG4-Fc-specific antibody. In some embodiments, the antibody is an anti-human antibody. The target protein can be any protein for which one wishes to determine the self-association or self-aggregation properties using a small amount of protein in a dilute solution. In some embodiments, the target protein comprises a therapeutic protein. Therapeutic proteins include both purified and synthetic proteins useful for treating diseases and disorders in a subject. Therapeutic proteins can include, but are not limited to, antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, engineered protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic drugs. In some embodiments, the target protein comprises an antibody or a fragment or derivative thereof. In alternative embodiments, the target protein comprises an antibody, specifically a monoclonal antibody, more specifically a human monoclonal antibody. In one embodiment, the antibody is an IgG antibody, for example an IgG1, IgG2, or IgG4 antibody.In alternative embodiments, the antibody is an IgG1 or IgG2 antibody. The target protein can be at any concentration. In the case of a therapeutic protein, the concentration can be much less than that of the therapeutic formulation. In some embodiments, the target protein is at a concentration of less than 1 mg / mL (e.g., less than 500 μg / mL, less than 100 μg / mL, less than 50 μg / mL, or less than 10 μg / mL). The target protein can be at a concentration of 1-1000 μg / mL, 1-100 μg / mL, 1-50 μg / mL, 1-20 μg / mL, or 1-10 μg / mL. In some embodiments, the target protein can be at a concentration of 1-10 μg / mL. In some embodiments, the target protein is present in a solution (e.g., a buffer) having a pH of 3.5-7 (e.g., 4.5-7), specifically 4-6.5 or 4-6. In some embodiments, the solution (buffer) has a low ionic strength.

[0034] Ionic strength is a measure of the concentration of ions in a solution. The term "low ionic strength solution" refers to a solution such as a buffer with a total buffer concentration of less than about 50 mM, and a high ionic strength buffer is a buffer with a concentration of more than about 100 mM. Buffers are known in the art and can be salts of weak acids and weak bases. Examples are carbonates, bicarbonates, and hydrogen phosphates. In a specific embodiment of the present disclosure, the buffer is a histidine and / or acetate buffer or a buffer with substantially the same ionic strength as a histidine and / or acetate buffer. The histidine and / or acetate buffer can have a concentration of less than 50 mM histidine and / or less than 50 mM acetate, for example, less than 25 mM histidine and / or less than 25 mM acetate, specifically about 10 mM histidine and / or 10 mM acetate. "Substantially the same" means ±50%, ±40%, ±30%, ±20%, ±10%, or ±5%.

[0035] The term "conjugation" or "conjugating" refers to the binding of a molecule to a nanoparticle by chemical, physical, or biological means. Conjugation of a nanoparticle to a molecule is usually meant when referring to a nanoparticle that includes a molecule on its surface. A specific meaning of this term is "adsorption" or "adsorbing", which includes physical adsorption (van der Waals forces), chemical adsorption (covalent bonds), and electrostatic attraction. In alternative embodiments, a specific meaning of this term is physical adsorption.

[0036] The term "self-association" refers to the interaction between homogenous proteins (or antibodies) and the formation of multimers. Such multimers may revert to monomers by manipulations such as dilution. The "tendency" of self-association refers to the ease of self-association, particularly by increasing the concentration of the protein, for example, above 50 mg / ml, 100 mg / ml, or 150 mg / ml (referred to herein as "high" or "higher" concentrations). An example of a parameter indicative of self-association is the diffusion interaction parameter. The diffusion interaction parameter is an index of self-association calculated using the concentration dependence of the diffusion coefficient obtained by experimental methods. If this value is -12.4 g / mL or higher, repulsive forces between molecules dominate. If this value is lower, it is reported to be an attractive (self-association) interaction (Saito et al., Pharm. Res., 2013. Vol. 30, p. 1263). The diffusion coefficient is an index of the ease of diffusion of molecules in solution, and is measured by dynamic light scattering or the like. More generally, protein self-association can result in poor solution behavior, such as high viscosity, opalescence, phase separation, and / or aggregation.

[0037] The term "plasmon wavelength" refers to the wavelength of maximum absorbance.

[0038] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in the practice or testing of this disclosure.All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.The materials, methods, and examples disclosed herein are illustrative only and are not intended to be limiting.

[0039] 2. Use of stabilized nanoparticles In a first aspect, the present disclosure relates to the use of positively charged polymers to stabilize nanoparticles that include a capture agent on their surface.

[0040] The capture agent is usually conjugated to a nanoparticle. In a specific embodiment, the nanoparticle is contained in a solution. Thus, the use can be for stabilizing the nanoparticle (particularly a plurality of them) in a solution (i.e. for stabilizing a nanoparticle suspension). The solution can be as defined in the third aspect below.

[0041] The use typically involves conjugating a positively charged polymer to the nanoparticle before, simultaneously, or after (in a specific embodiment, simultaneously) conjugating the nanoparticle to the capture agent. The concentration of the positively charged polymer (by mass of the positively charged polymer and the capture agent) can be as described above. In some embodiments, the use involves determining the stabilization of the nanoparticle by determining the plasmon wavelength of the nanoparticle in solution to 534 nm or less while capturing a human polyclonal antibody (e.g., human polyclonal antibody ChromPure Human IgG) and / or determining the plasmon wavelength of the nanoparticle in solution to 533 nm or less while capturing a NIST monoclonal antibody (reference material RM8671) mAb.

[0042] 3. Nanoparticles In a second aspect, the present disclosure relates to a nanoparticle comprising a capture agent and a positively charged polymer on a surface thereof.

[0043] In some embodiments, the nanoparticles are metal nanoparticles, particularly gold nanoparticles.

[0044] In some embodiments, the capture agent and / or the positively charged polymer are conjugated to the nanoparticle. The amount of the positively charged polymer (by mass of the positively charged polymer and the capture agent) can be as described above. In a specific embodiment, the nanoparticle is a stabilized nanoparticle, i.e., stabilized, for example, according to the use of the first aspect. Specifically, the plasmon wavelength of the nanoparticle in solution during capture of human polyclonal antibody (e.g., human polyclonal antibody ChromPure Human IgG) is 534 nm or less, and / or the plasmon wavelength of the nanoparticle in solution during capture of NIST monoclonal antibody (reference material RM8671) mAb is 533 nm or less.

[0045] 4. Solution containing nanoparticles In a third aspect, the present disclosure relates to a solution comprising a plurality of nanoparticles of the second aspect, the solution therefore also being referred to as a nanoparticle suspension.

[0046] In some embodiments, the solution is a buffer, particularly a low ionic strength buffer. The pH of the solution / buffer can be 3.5 to 7 (e.g., 4.5 to 7), particularly 4 to 6.5 or 4 to 6. Plural means at least 2, at least 10, at least 100, or at least 1000.

[0047] It must be understood that whenever the present disclosure refers to the use of the nanoparticles of the second aspect, it is also intended to refer instead to the use of the solution of the third aspect.

[0048] 5. Nanoparticle-containing Kits In a fourth aspect, the present disclosure relates to a kit comprising a nanoparticle (particularly a plurality of nanoparticles) and a positively charged polymer.

[0049] The kit may further include a solution such as a buffer, in particular a low ionic strength solution / buffer. The pH of the solution / buffer may be 3.5-7 (e.g., 4.5-7), in particular 4-6.5 or 4-6. In a further embodiment, the kit may also include (i) a capture agent (optionally conjugated to a nanoparticle), and / or (ii) a panel of at least two (e.g., at least 3, 4, 5, or 6) calibration proteins. The kit may additionally include instructions providing the plasmon wavelengths of the calibration proteins of the panel of calibration proteins. The plasmon wavelengths are the known or "historical" plasmon wavelengths of the calibration proteins.

[0050] 6. Methods for determining self-association of a target protein In a fifth aspect, the present disclosure provides a method for determining the self-association propensity of a target protein, comprising: (i) capturing the target protein in a solution as defined in the third aspect; (ii) determining the color of the solution. Including, a change in colour of the solution compared to a control solution as defined in the third aspect not containing the target protein indicates a tendency of the target protein to self-associate; It concerns the method.

[0051] The control solution may be the solution before step (i) and before the target protein is added, or it may be a separate solution (e.g., one that has undergone the same process except for step (i)).

[0052] The concentration of the target protein may be as described above for target protein analysis. The capturing step may include mixing and / or incubating the solution (e.g., as described below for the sixth embodiment).

[0053] The color change typically occurs toward higher absorption wavelengths. In some embodiments, determining the color includes determining light absorption, and a change in light absorption compared to a control solution indicates a tendency for the target protein to self-associate. In a specific embodiment, step (ii) includes determining a plasmon wavelength, and a change in plasmon wavelength compared to a control solution indicates a tendency for the target protein to self-associate. This determination may include measuring absorbance at multiple wavelengths ranging from 450 nm to about 750 nm to determine the plasmon wavelength. The wavelength corresponding to the maximum absorbance (plasmon wavelength) shifts to a larger value as the separation distance between the nanoparticles decreases. Thus, when the target protein self-associates, the absorbance spectrum of the nanoparticles changes and the plasmon wavelength increases. For example, if the nanoparticles are gold nanoparticles, a color change to red (e.g., a red shift in the plasmon wavelength) indicates a tendency for the target protein to self-associate. Typically, the degree of change in color, light absorption, or plasmon wavelength, respectively, indicates the strength of the tendency for the target protein to self-associate. However, in some embodiments, this change may be used as a binary indication of self-association and no self-association based on whether the plasmon wavelength changes due to capture of the target protein.

[0054] The solution is usually the solution defined in the third aspect. The nanoparticles (or solution) in the solution have, in an optional embodiment, a plasmon wavelength during capture of a human polyclonal antibody (e.g., human polyclonal antibody ChromPure Human IgG) of 534 nm or less, and / or a plasmon wavelength during capture of a NIST monoclonal antibody (reference substance RM8671) mAb of 533 nm or less. To this end, the method may comprise, prior to step (i), a step of selecting nanoparticles (or a solution) for capture in step (i), wherein the plasmon wavelength of the nanoparticles in the solution during capture of a human polyclonal antibody (e.g., human polyclonal antibody ChromPure Human IgG) is 534 nm or less, and / or the plasmon wavelength of the nanoparticles in the solution during capture of a NIST monoclonal antibody (reference substance RM8671) mAb is 534 nm or less.

[0055] In some embodiments, the method includes calibrating with a panel of at least two (preferably at least three, four, five, or six) calibration proteins (e.g., antibodies, particularly monoclonal antibodies) that differ in their tendency to self-associate, which tendency is known. (i) measuring the plasmon wavelength of each calibration protein of the panel; (ii) calculating the historical CS-SINS score for each calibration protein (cP) according to the following formula: CS-SINS score = (cP - parameter 1) / (parameter 2 - parameter 1), where parameter 1 is the plasmon wavelength of the calibration protein in the panel with the lowest propensity for self-association and parameter 2 is the plasmon wavelength of the calibration protein in the panel with the highest propensity for self-association; (iii) calculating a new CS-SINS score for each calibration protein according to the following formula: CS-SINS score=(cP-parameter 1) / (parameter 2-parameter 1), where parameter 1 is the plasmon wavelength of the calibration protein in the panel with the lowest tendency to self-associate and parameter 2 is the plasmon wavelength of the calibration protein in the panel with the highest tendency to self-associate, and the following term: ((1-slope) 2 +(intercept) 2 ) fitting the parameters to maximize the linear fit agreement between the past parameters and the new parameters by minimizing (iv) Determining whether the calibration satisfies criteria a) through c) below with respect to the linear fit between the new data and the historical data. a) The slope of the linear fit is between 0.9 and 1.1; b) the intercept of the linear fit is between -0.1 and 0.1; c) R of the linear fit 2 is greater than 0.9; And (v) if all criteria of step (iv) are met, calibrating the CS-SINS score of the target protein using the fit parameters identified in step (iii). Step (ii) may be performed before, after, or simultaneously with step (i). In a specific embodiment, a target protein CS-SINS score of about 0.35 or less indicates that the target protein has a low propensity for self-association (i.e., has self-association properties that are advantageous for formulations and compositions that include high concentrations of the target protein (e.g., 100 mg / mL or more of the target protein).

[0056] 7. Methods for screening target proteins The present disclosure provides a method for screening a dilute concentration of a target protein for self-association properties (e.g., opalescence, and viscoelastic properties). In general, the use of the method of the fifth aspect is provided to screen a target protein for its tendency to self-associate. More specifically, in a sixth aspect, the present disclosure relates to a method that may include adsorbing a capture agent and a positively charged polymer onto a nanoparticle (e.g., a metal nanoparticle) to form a capture agent conjugate; incubating the capture agent conjugate with a target protein in a solution (e.g., a buffer solution) to form a target protein conjugate; measuring the absorbance of the target protein conjugate at a plurality of wavelengths ranging from 450 nm to about 750 nm; and identifying the plasmon wavelength as the wavelength at which the absorbance by the target protein conjugate is maximum.

[0057] The positively charged polymer is co-adsorbed onto a surface (eg, a nanoparticle) along with a capture agent.

[0058] The method may include incubating the capture antibody conjugate with the target protein in a solution (e.g., a buffer solution) to form a target protein conjugate. The incubation may be for any length of time required to allow the target protein to bind to the capture agent to form a target protein conjugate. For example, the incubation may be for at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 8 hours, at least 10 hours, or longer.

[0059] The buffer used for incubation can have any desired properties (pH, ionic strength, etc.), but typically mimics the buffer conditions desired for the final downstream use of the target protein. In some embodiments, the buffer conditions are those that are physiologically acceptable for therapy. In some embodiments, the pH of the buffer, and therefore the pH of the incubation, is between 4.5 and 7. The pH of the buffer can be about 4.5, about 5, about 5.5, about 6, about 6.5, or about 7. In some embodiments, the buffer has low ionic strength.

[0060] The method may include measuring the absorbance of the target protein conjugate at multiple wavelengths ranging from 450 nm to about 750 nm, and identifying the plasmon wavelength as the wavelength at which the absorbance by the target protein conjugate is greatest. p ), i.e., the wavelength corresponding to maximum absorbance, shifts to larger values ​​as the separation distance between nanoparticles decreases. Thus, upon self-association of the target protein, the absorbance spectrum of the nanoparticle changes, which is reflected in the identification of the plasmon wavelength. In some embodiments, this change can be used as a binary indication of self-association and no self-association, based on whether the plasmon wavelength changes upon measuring the absorbance of the target protein conjugate to the capture agent conjugate.

[0061] The sensitivity of the plasmon wavelength to the change in the interparticle distance between the antibody-gold conjugates can be used as a measure of the degree or extent of self-association of the target protein. In some embodiments, the method further comprises calculating a charge-stabilized self-interacting nanoparticle spectroscopy (CS-SINS) score. The SC-SINS score is illustrated in Figure 4 (Test No. 2) and is calculated using Equation (1):

number

[0062] In some embodiments, a CS-SINS score of about 0.35 or less indicates that the target protein has self-association properties that are advantageous for formulations and compositions comprising high concentrations of the target protein (e.g., 100 mg / mL or more of the target protein).

[0063] In some embodiments, the method further includes a calibration method comprising capturing a plurality of calibration proteins with the capture agent conjugate to form a plurality of calibration protein conjugates, measuring the absorbance of each of the plurality of calibration protein conjugates at a plurality of wavelengths in the range of 450 nm to about 750 nm, and identifying the plasmon wavelength.

[0064] The calibration method may further include calculating a CS-SINS score for each of the plurality of calibration protein conjugates; and comparing the CS-SINS score for each of the plurality of calibration protein conjugates to the SC-SINS score of the previous calibration protein conjugate for a linear fit. For example, the CS-SINS for each of the plurality of calibration protein conjugates may be calculated using the following term: ((1-slope) 2 +(intercept) 2 )) to maximize the linear fit between the new data and the past CS-SINS scores. This calibration is Slope of linear fit = 0.9 <x<1.1; Linear fit intercept = -0.1 <x<0.1;および R for linear fit 2 ≧0.9 If , the criterion of a linear fit between the new and historical data is met, and therefore it is a proper calibration.

[0065] The plurality of calibration proteins includes proteins having different self-association properties (e.g., opalescence, and viscoelastic properties). The calibration protein may be a protein of the same species as the target protein. For example, if the target protein is an antibody, the calibration proteins are selected from a variety of antibodies having different self-association properties. In some embodiments, each of the plurality of calibration proteins is independently selected from the group consisting of monoclonal antibodies, polyclonal antibodies, and fragments or derivatives thereof.

[0066] In some embodiments, the calibration proteins include a low self-association control protein and a high self-association control protein, and the plasmon wavelengths of these conjugates are used to calculate CS-SINS, as described elsewhere herein.

[0067] In some embodiments, the plurality of calibration proteins includes NIST Reference Antibody RM 8671 and human polyclonal antibody ChromPure Human IgG (whole molecule). In some embodiments, the calibration identifies the plasmon wavelengths of NIST Reference Antibody RM 8671 and human polyclonal antibody ChromPure Human IgG (whole molecule). A suitable calibration would include a plasmon wavelength of NIST Reference Antibody RM 8671 below 533 nm and a plasmon wavelength of human polyclonal antibody ChromPure Human IgG (whole molecule) below 534 nm.

[0068] 8. System or kit for screening target proteins In a seventh aspect, the present disclosure relates to a system or kit (e.g., reagents, computer software, instruments, etc.) for screening dilute concentrations of target proteins for favorable self-association properties (e.g., opalescence, and viscoelastic properties). In some embodiments, the system includes one or more or each of a capture agent, a positively charged polymer, a surface (e.g., metal nanoparticles), a solution (e.g., buffer), a target protein, at least one calibration protein, and a spectrophotometer. The descriptions provided elsewhere herein above regarding the capture agent, the positively charged polymer, the surface (e.g., metal nanoparticles), the solution (e.g., buffer), and the target protein are also applicable to the system of the present disclosure. The spectrophotometer may include any instrument that measures the absorption and / or transmission of light at various wavelengths of light. The individual member components of the system or kit are physically packaged together or separately.

[0069] The system may also include instructions for using the components of the system. The instructions are related materials or methodologies relating to the system. The materials may include any combination of the following: background information, a list of components and their availability information (such as purchasing information), brief or detailed protocols for using the system, troubleshooting, references, technical support, and any other related documentation. The instructions may be provided with the system or as a separate member component, either in paper form, or in electronic form supplied on a computer readable memory device or downloaded from an Internet website, or as a recorded publication.

[0070] It is understood that the systems and kits of the present disclosure are utilized in conjunction with the methods of the present disclosure. EXAMPLES

[0071] 9. Working Example material and method Immunogold conjugate preparation for AC-SINS in PBS Goat anti-human Fcγ-specific antibody (Jackson ImmunoResearch Laboratories, 109-005-008) was buffer exchanged twice into 20 mM acetic acid, pH 4.3 using Zeba desalting columns (Thermo Fisher Scientific, PI-89882). Concentration was determined using UV absorbance at 280 nm and a mass attenuation coefficient of 1.26 mL / mg×cm. Antibody was diluted to 0.4 mg / mL in 20 mM acetic acid, pH 4.3. 20 nm gold nanoparticles (particles 7.0×10 11 One milliliter of the 100% PBS (100% PBS / mL; Ted Pella Inc., 15705) was pelleted in a 1.5 mL microcentrifuge tube (1615-5500, USA Scientific) at 21130 rcf for 6 minutes. 950 μL of the supernatant was then removed and replaced with 950 μL of milliQ water. The resuspended particles were further diluted by the addition of 500 μL of milliQ water (final concentration of 4.67 × 10 particles). 11 900 μL of gold nanoparticles were added to 100 μL of prepared capture antibody. The mixture was incubated overnight at room temperature. Prior to use, the immunogold conjugate was precipitated at 21130 rcf for 6 minutes. 950 microliters of the supernatant was removed and saved, and the particles were resuspended in the remaining supernatant. The volume was carefully readjusted to 50 μL.

[0072] Preparation of polylysine-stabilized immunogold conjugates for CS-SINS Goat anti-human Fcγ-specific antibody (Jackson ImmunoResearch Laboratories, 109-005-008) was buffer exchanged as described above and diluted to a final concentration of 0.8 mg / mL in 20 mM acetic acid, pH 4.3. Polylysine (Fisher Scientific, ICN19454405), initially dissolved in milliQ water at 5 mg / mL, was diluted to either 0.8 mg / mL (0.03 polylysine / IgG fraction), 2.67 mg / mL (0.10 polylysine / IgG fraction), or 4.0 mg / mL (0.15 polylysine / IgG fraction) in 20 mM acetic acid, pH 4.3. For preparation of 100 μL of conjugate, 48.5 μL of capture IgG was mixed with 1.5 μL of polylysine and stored. 1200 microliters of 20 nm gold nanoparticles (Ted Pella, 15705) were sedimented at 21130 rcf for 6 minutes, after which 1150 μL of supernatant was removed. The nanoparticles were resuspended in the remaining supernatant and the volume was carefully adjusted to 50 μL. The nanoparticles were then added to the IgG / polylysine mixture and mixed rapidly by pipetting up and down 20 times. The conjugate was incubated overnight at room temperature. The preparation described is for 100 μL of conjugate, but can be adjusted to prepare larger volumes if necessary. In experiments using non-capturing antibodies, the goat anti-human IgG was replaced with goat polyclonal IgG (Jackson ImmunoResearch Laboratories, 005-000-003).

[0073] Dynamic Light Scattering and Zeta Potential Measurements of Immunogold Conjugates Dynamic light scattering (DLS) experiments were performed using a Zetasizer Nano ZSP (Malvern Panalytical, Worcestershire, UK). To assess the size of immunogold conjugates in a given buffer, 950 μL of buffer was added to 50 μL of conjugate and immediately transferred to a folded capillary zeta cell (DTS1070, Malvern Panalytical). The cuvette was transferred to a Zetasizer instrument and the size was measured at 25 °C using DLS with 173 ° backscattering measurements. The size measurement was the average of at least 30 10 s measurements. If the sample was monodisperse (immunogold conjugates prepared for standard AC-SINS runs), the z-average diameter calculated using cumulant analysis was reported. If the sample was polydisperse (polylysine-stabilized immunogold conjugates), the main peak of the size distribution determined by non-negative least squares analysis was reported. Immediately after particle size measurement, the zeta potential of the particles was measured using laser Doppler velocimetry on the same instrument. Data were collected and analyzed using Zetasizer 7.11 Software (Malvern Panalytical).

[0074] CS-SINS Assay in Histidine Antibodies of interest were buffer exchanged twice into 10 mM histidine, pH 6.0, using Zeba desalting columns (Thermo Fisher Scientific, PI89882). Concentrations were determined using UV absorbance at 280 nm and the mass attenuation coefficients calculated independently for each monoclonal antibody [1.40 mL / mg×cm was used for human polyclonal antibodies (Jackson ImmunoResearch Laboratories, 009-000-003)]. Antibodies were diluted to 11.1 μg / mL in histidine, pH 6.0, prior to use in CS-SINS. 5 μL of immunogold conjugate was added in triplicate to a clear, flat-bottomed 384-well polystyrene plate (Thermo Fisher Scientific, 12565506) for each mAb and human polyclonal antibody assayed. The immunogold conjugates were then mixed by pipetting up and down 10 times (final antibody concentration 10 μg / mL). The mixture was covered and incubated at room temperature for 4 hours, after which the absorbance was measured at 450-650 nm in 1 nm increments (normal scan speed, 8 readings per well) using a BioTek Synergy Neo plate reader (BioTek, Winooski, VT). The plasmon wavelength was determined by fitting a second-order polynomial to 40 data points around the observed maximum absorbance and setting the first derivative to zero. CS-SINS scores were calculated as described below.

[0075] Calibration of the CS-SINS assay CS-SINS measurements were calibrated and evaluated in the following manner. First, each new batch of gold anti-Fc conjugates was evaluated using two control antibodies, namely a human polyclonal antibody (ChromPure Human IgG, whole molecule, Jackson ImmunoReasearch Laboratories 009-000-003) and NIST mAb RM 8671 (NIST mAb) (see Karageorgos, I. et al., Biologicals 2017, 50, pp. 27-34, which is incorporated herein by reference in its entirety). If the plasmon wavelength of these conjugates adsorbed with human antibodies was above 534 nm (human polyclonal antibody) or 533 nm (NIST mAb), the experiment was terminated and the conjugate was prepared again until it passed this test (test no. 1). Gold anti-Fc conjugates that passed test no. 1 were then evaluated using a panel of six control mAbs (in this test, mAbs A, C, D, E, F, and K) spanning both low and high self-association behavior, and their plasmon wavelengths (in the form of CS-SINS scores) were compared to reference data for the same antibodies. For the reference data, the CS-SINS scores were calculated as (plasmon wavelength of the given mAb - plasmon wavelength of the least self-associating mAb (e.g., mAb A in this test)) / (plasmon wavelength of the most self-associating mAb (e.g., mAb K in this test) - plasmon wavelength of the least self-associating mAb (e.g., mAb A in this test). For measurements of the panel of mAbs in each new experiment (i.e., calibration measurements), the CS-SINS scores were calculated as (plasmon wavelength of the given mAb - parameter 1) / (parameter 2 - parameter 1). These two parameters were then calculated as ((1 - slope) 2 +(intercept) 2 ) are fitted to minimize the sum of 2 If one of the three values ​​of the value is the ideal value (1 for the slope, 0 for the intercept, and R 2If the result was not within 10% of test #1), the experiment was terminated and the conjugate was prepared again until it passed this test (test #2) in addition to test #1. If the measurement passed both tests, a CS-SINS measurement was performed with the full panel of antibodies and the CS-SINS score was calculated using the parameter fit in test #2 (i.e., parameters 1 and 2). Calibration antibodies included: A, tocilizumab; C, cetuximab; D, evolocumab; E, denosumab; F, pembrolizumab; and K, omalizumab.

[0076] Viscosity and Opalescence Measurements Viscosity and opalescence values ​​for the experimental mAb dataset were obtained from Kingsbury et al. (Sci Adv 6, eabb0372, doi:10.1126 / sciadv.abb0372 (2020) which is incorporated by reference in its entirety). EXAMPLES

[0077] Charge stabilization prevents aggregation of antibody-gold conjugates To understand the source of problems experienced in past attempts to use AC-SINS (Figure 2A, left), aggregation of gold-antibody (anti-human IgG) conjugates was measured in dilution buffers (10 mM acetate and / or histidine) over a range of pH values ​​(pH 4.0-6.5; Figure 2B, left). At pH values ​​ranging from 5.0 to 6.5, we observed high plasmon wavelengths of approximately 590 nm, which differ significantly from typical values ​​(530-535 nm) for stable conjugates in PBS (Figure 2E). In the pH range of 5.0-6.5, we observed substantial particle aggregation, as evidenced by the large apparent diameter of the conjugates detected by dynamic light scattering. Consistent with this result, we observed high plasmon wavelengths of approximately 590 nm, which differ significantly from typical values ​​(530-535 nm) for stable conjugates in PBS (Figure 2C, left). The mechanism of particle aggregation was investigated by measuring the zeta potential of the antibody-gold conjugates (Figure 2D, left side) and observed a steady decrease from a maximum of +16 mV at pH 4.0, crossing zero near pH 5.7, and a minimum of -5 mV at pH 6.5. The trend in zeta potential with increasing pH was also consistent with titration of the immobilized capture antibody with intrinsic charged amino acids. These findings suggest that the low net charge of the conjugates near pH 5.6-6 is related to aggregation in the pH 5-7 range.

[0078] During conjugation preparation, a large positively charged polymer (polylysine, ≥70 kDa) was co-adsorbed with the capture antibody. Addition of even a small amount of polylysine (e.g., 3%, Figure 2A, center) was sufficient to prevent aggregation of the conjugates over a wide pH range of 4 to 6 (Figure 2B, center). Increasing polylysine to 15% (Figure 2A, right) further stabilized the conjugates up to pH 6.5 (Figure 2B, right). The plasmon wavelength of the polylysine-stabilized conjugates showed a similar pH-dependent trend as observed for the apparent size of the conjugates (Figure 2C, center, and Figure 2C, right). We also observed a concomitant increase in the zeta potential of the conjugates to positive values ​​(+2 to +6 mV) over this same pH range (Figure 2D, center, and Figure 2D, right). The fact that a low amount of polylysine (3%) resulted in a zeta potential at pH 6 (+2.3 mB) that would be associated with aggregation of the conjugate in the absence of polylysine suggested an additional steric component to the observed stabilization.

[0079] The stabilized conjugates were used to measure mAb self-interactions by CS-SINS in histidine formulations (Figure 8). Seven mAbs were selected that exhibited a wide range of self-association behavior based on light scattering analysis of mAb-colloid interactions in histidine formulations. CS-SINS analysis revealed a small but detectable mAb-dependent plasmon shift (plasmon shift of less than 1 nm compared to more than 30 nm observed for the same antibodies using conventional AC-SINS in PBS). The capture antibody was replaced with a nonspecific antibody, and seven smaller plasmon shifts were observed for the seven mAbs. Conjugates prepared with polylysine alone before the addition of mAb also showed extremely small plasmon shifts that were independent of mAb. The effect of polylysine size (nominal sizes 30-70 kDa and 15-30 kDa) on the plasmon shift was examined, and a similar trend was observed for conjugates prepared with polylysine of various sizes (Figure 9). These results demonstrated that the CS-SINS assay detects mAb-specific colloid interactions that require antibody-mediated immobilization and are weakly dependent on polylysine size.

[0080] A robust calibration method controls the variability of results that can occur when precise experimental protocols are not followed. Two tests were developed for the calibration and evaluation of CS-SINS measurements (Figures 4 and 5). 1) Anti-Fc gold conjugates for two calibration antibodies (human polyclonal antibody and NIST mAb) should result in plasmon wavelengths below 534 nm for the human polyclonal antibody and below 533 nm for the NIST mAb, as shown in Figure 5A for a successful experiment. 2) A panel of six calibration mAbs was used to calibrate the assay and these measurements were compared to reference (historical) measurements obtained in multiple independent assays. Plasmon wavelengths were converted to calibrated CS-SINS scores by fitting two parameters to maximize the linear fit between the calibration data and the reference data, as described in the methods section and Figure 4. If the slope, intercept, and R2 values ​​were within 10% of the ideal values, the assay passed test number 2 and the full panel of antibodies was evaluated. In the case of the successful experiment in Figure 5, the assay passed test number 2, and the evaluation data of a larger panel of antibodies (without calibration antibodies) showed good performance compared to the reference data. The calibration protocol was applied to CS-SINS measurements obtained with 3% or 10% polylysine for a panel of 25 commercially available mAbs (Figure 6A). When analyzed as plasmon wavelength shift, there was a clear difference in the measurements between the two polylysine conditions. However, when the calibration protocol was applied, there was excellent agreement between the two data sets (Figure 6B). To illustrate the value of this calibration process, further experiments were performed to illustrate how experiments with flawed protocols can be easily identified (Figures 5B-5C). EXAMPLES

[0081] mAb self-interaction as a predictor of solution behavior To evaluate the utility of charge-stabilized immunogold conjugates in measuring mAb self-interactions and, consequently, predicting antibody solution behavior, a diverse set of 56 mAbs, including 43 commercially available products, was utilized. Various properties associated with the amino acid sequence (pI, charge, charge asymmetry, and hydrophobicity) were compared for the mAbs in the dataset with 500 unique antibody sequences collected from the broader clinical setting using the Therapeutic Antibody Database (TABS) (Figure 3A-D). Emphasis was placed on the evaluation of sequence-derived metrics of electrostatic and hydrophobic properties, both of which have previously been shown to be drivers of mAb self-interactions. Based on this analysis, it was demonstrated that the mAb set accurately represents key properties of mAbs from the broader clinical antibody context, such as isoelectric point, net charge (pH6), and Fv charge asymmetry parameters (Fv-CSP, pH6). Furthermore, this panel of antibodies also had similar hydrophobic properties as assessed in terms of the Eisenberg Hydrophobicity Index (EHI) to a large panel of clinical-stage antibodies. This finding indicated that the mAb dataset was well suited for evaluating the general applicability of the CS-SINS assay for assessing antibody solution behavior.

[0082] More than 30% of clinical-stage mAbs, and possibly a higher percentage in early preclinical stages, are expected to exhibit poor solution behavior, such as high solution viscosity and opalescence, which are only apparent at higher concentrations (>100 mg / mL). High mAb solution viscosity is particularly problematic during subcutaneous delivery by autoinjector and ultra / diafiltration purification unit operations. High opalescence may indicate a predisposition to phase separation and aggregation. Recently, the diffusion interaction parameter (k DMeasurement of weak colloid-self-interactions by CS-SINS was shown to be most useful in predicting mAbs that exhibit a tendency for high viscosity or opalescence compared to a larger set of molecular descriptors. However, the material requirements of such measurements make them feasible only at the later candidate selection stage of the discovery process (Figure 1) for only a small number (~10) of candidates. On the other hand, the CS-SINS assay, which also measures weak colloid-colloid interactions, is more suitable to be performed during candidate optimization for screening hundreds to thousands of variants (Figure 1).

[0083] CS-SINS was performed in ultra-dilute solutions (10 μg / ml) to determine whether it could detect problematic behaviors manifested at antibody concentrations four orders of magnitude higher (>100 mg / ml). Viscous (>30 cP) antibodies consistently had higher CS-SINS scores compared to low-viscosity well-behaved mAbs, and these mAbs with extreme opalescence profiles (>20 NTU) were readily identified using CS-SINS (Figure 3F). With a threshold of 0.35, CS-SINS identified well-behaved mAbs (<30 cP, <12 NTU) with >85% accuracy (Figures 3G-3H). Setting this CS-SINS score threshold facilitated the identification of all 10 (100%) viscous antibodies and 3 of 7 (43%) opalescent antibodies. In addition, this threshold mischaracterized only 4 (10%) of the remaining 39 well-behaved antibodies. Of the 56 mAbs, 35 of 39 were correctly identified as having good behavior and 13 of 17 were identified as having poor solution properties. D The CS-SINS assay was also robust with respect to reproducibility and was supported by detailed calibration and system suitability criteria (Figures 3, 4, and 5).

[0084] Previous studies using a similar panel of antibodies have determined that the diffusion interaction parameter (k D ) and poor solution properties. Furthermore, as specified herein, the significance of k in predicting poor solution properties (especially those related to opalescence) was D IgG subclass-specific behavior regarding the usefulness of DThe relationship between k and the CS-SINS score measured here was examined (Figure 7). Without separation of mAbs by subclass, k D A moderately strong rank correlation was observed between the CS-SINS score and the CS-SINS score (Spearman's ρ -0.80; Figure 7A, top). However, when dividing mAbs into IgG1 / IgG2 vs. IgG4 subclasses, subclass-specific behavior emerged. There was a significant increase in rank correlation for the IgG1 / IgG2 grouping (Spearman's ρ -0.89; Figure 7A, middle) and a decrease for the IgG4 subclass (Spearman's ρ -0.76; Figure 7A, bottom). By extending this analysis to predicting poor solution behavior, it was observed that using a CS-SINS threshold of 0.35 facilitated the detection of all poorly behaving molecules in the IgG1 / IgG2 subclass grouping (Figure 7B). Applying the same criteria to the IgG4 grouping revealed that all of the opalescent molecules with low CS-SINS scores belonged to this subclass (Figure 7C), suggesting that the mode of IgG4 self-association leading to opalescent behavior may be unique compared to IgG1 / 2 antibodies. More generally, these results collectively demonstrate the ability of CS-SINS to identify antibodies with high levels of viscosity and opalescence, particularly for IgG1 and IgG2 mAbs, using ultradilute (10 μg / mL) solution measurements of self-association. The assay is robust and readily feasible for simultaneous high-throughput screening of large numbers (approximately 100-1000) of mAb candidates. The CS-SINS assay represents a major advancement toward identifying developable antibodies with drug-like properties in early discovery.

[0085] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not intended to be limitations on the scope of the present disclosure, which is defined solely by the appended claims and equivalents thereof.

[0086] Various changes and modifications to the embodiments of the present disclosure will be apparent to those skilled in the art and can be made without departing from the spirit and scope thereof.

Claims

1. 1. Use of a positively charged polymer for stabilizing nanoparticles comprising a capture agent on the surface thereof, wherein the positively charged polymer is a homopolymer of a positively charged amino acid or a heteropolymer of two or more positively charged amino acids.

2. The use according to claim 1 , wherein the nanoparticles are stabilized in a solution.

3. A nanoparticle comprising a capture agent and a positively charged polymer on a surface thereof, the positively charged polymer being a homopolymer of a positively charged amino acid or a heteropolymer of two or more positively charged amino acids.

4. The nanoparticle of claim 3 , wherein the nanoparticle is in a solution.

5. A solution comprising a plurality of nanoparticles according to claim 3 or 4.

6. A kit comprising nanoparticles and a positively charged polymer, the positively charged polymer being a homopolymer of a positively charged amino acid or a heteropolymer of two or more positively charged amino acids.

7. The kit of claim 6, further comprising a solution and / or a capture agent.

8. 1. A method for determining the self-association tendency of a target protein, comprising: (i) capturing a target protein in a solution as defined in claim 5; (ii) determining the color of the solution Including, A method according to claim 5, wherein a change in colour of the solution compared to a control solution not containing the target protein indicates a tendency of the target protein to self-associate.

9. 1. A method for screening a target protein at dilute concentrations for self-association properties comprising: adsorbing a capture agent and a positively charged polymer onto the nanoparticles to form a capture agent conjugate; incubating the capture agent conjugate with the target protein in solution to form a target protein conjugate; Measuring the absorbance of the target protein conjugate at multiple wavelengths ranging from 450 nm to about 750 nm; and Identifying the plasmon wavelength as the wavelength at which absorbance by the target protein conjugate is maximal Including, The method, wherein the positively charged polymer is a homopolymer of a positively charged amino acid or a heteropolymer of two or more positively charged amino acids.

10. 1. A system for screening target proteins at dilute concentrations for self-association properties, comprising: scavenger; a positively charged polymer which is a homopolymer of a positively charged amino acid or a heteropolymer of two or more positively charged amino acids; Nanoparticles; solution; Target protein; at least one calibration protein; and spectrophotometer A system including one or more of the above.

11. The use, nanoparticles, solution, kit, method or system according to any one of claims 1 to 10, wherein the nanoparticles are metal nanoparticles, preferably gold nanoparticles.

12. The use, nanoparticle, kit, method or system according to any one of claims 1 to 11, wherein the positively charged polymer is polylysine.

13. The use, nanoparticle, kit, method or system according to any one of claims 1 to 12, wherein the capture agent is an antibody.

14. The method or system of any one of claims 1 to 13, wherein the target protein is an antibody.

15. The use, nanoparticles, solution, kit, method or system according to any one of claims 1 to 14, wherein the solution has a low ionic strength and / or a pH of 3.5 to 7.

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