Means 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] This disclosure provides useful means (e.g., uses, nanoparticles, solutions, methods, kits, and systems) for detecting the self-assembly properties of proteins (e.g., therapeutic antibodies) using ultradilute solution measurements. [Background technology]

[0002] Monoclonal antibodies (mAbs) are one of the most successful medical therapies and are used to treat a wide range of diseases. Despite this success, mAbs are prone to developmental challenges that present significant challenges in manufacturing, stability, and delivery. Specifically, the development of mAbs intended for subcutaneous administration can be hampered by the need for high-concentration formulations, which are often limited by poor solution behavior in the form of high viscosity, opacity, phase separation, and aggregation. While intravenous administration of mAbs is routine in hospital settings, subcutaneous administration is less invasive, ensures better patient compliance, and is easily adaptable in environments with inadequate medical infrastructure. The rapid selection of developable therapeutic mAb candidates is always desirable, but this urgency is even more pronounced when combating infectious disease pandemics. When developmental feasibility issues arise, resource-intensive mitigation strategies must be employed, which inevitably cause delays and do not guarantee success. Therefore, it is far more efficient to select mAbs with advantageous solution properties early on, while placing considerable emphasis on identifying variants with drug-like properties in the early stages of discovery. [Overview of the initiative] [Means for solving the problem]

[0003] Disclosed herein, in a first aspect, is the use of a positively charged polymer for stabilizing nanoparticles containing a scavenger on a surface.

[0004] Disclosed herein, in a second aspect, are nanoparticles comprising a scavenger and a positively charged polymer on a surface.

[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 comprising nanoparticles 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, (i) A step of capturing a target protein in a solution defined in the third aspect, (ii) Steps to determine the color of this solution Includes, The color change of this solution compared to a control solution defined in a third aspect, which does not contain the target protein, indicates the self-association tendency of the target protein. It is a method.

[0008] Disclosed herein, in a sixth aspect, is a method for screening a target protein at dilute concentrations with respect to its self-assembly properties, comprising: adsorbing a scavenger and a positively charged polymer onto a surface (e.g., nanoparticles (e.g., metal nanoparticles)) to form a scavenger conjugate; incubating the scavenger conjugate with the target protein in a solution (e.g., a buffer) 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 a plasmon wavelength as the wavelength at which the absorbance of the target protein conjugate is maximized.

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

[0010] Other aspects and embodiments of the present disclosure will be apparent upon consideration of the following detailed description and the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] [Figure 1] It is a schematic diagram 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. A large pool is condensed to the order of hundreds during the optimization stage, and candidates undergo affinity maturation and humanization. During these early stages, the available material is in trace amounts (in the range of ng to μg), which makes it unfeasible to evaluate developability quality attributes such as viscosity, opalescence, and aggregation. For this reason, it is not uncommon for molecules with poor developability attributes to progress to clinical development, which greatly increases the amount of time and resources spent on generating viable pharmaceutical products. Evaluation of key developability attributes during lead optimization using an ultra-low-consumption, high-throughput assay 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, accelerate development timelines, and also reduce the consumption of associated resources. [Figure 2-1]Figures 2A–2D show the evaluation of the stabilization of antibody-gold conjugates using polylysine. Immunogold conjugates from a standard AC-SINS run (Figure 2A, left) were evaluated for stability under common formulation and process conditions (10 mM acetate and histidine buffer, pH 4.04–6.5). Dynamic light scattering measurements revealed rapid aggregation of the conjugates starting at pH 5.0 and continuing up to pH 6.5 throughout the relevant conditions (Figure 2B, left). A simultaneous shift in the plasmon wavelength of the aggregated conjugates was also observed and measured by absorbance spectroscopy (Figure 2C, left). Zeta potentials were measured over the pH range and a consistent decrease in potential was observed as pH increased from 4.0, crossing zero around pH 5.75 and then turning negative (Figure 2D, left). To achieve stability of the immunogold conjugate in the formulation space, a small amount (3% by mass) of polylysine, a highly charged biopolymer, was incorporated (Figure 2, center). Particle stabilization 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 data 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 due to unstable conjugates. Stabilization increased as polylysine increased in the range of 3% to 15%, as demonstrated by the highly stabilized 15% polylysine condition (Figure 2A, right). This 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 measurement range (Figure 2D, right). Figure 2E is an illustrative graph of absorbance as a function of wavelength, used to determine the plasmon wavelength of nanoparticles. [Figure 2-2] Continuation of Figure 2-1. [Figure 2-3] Continuation of Figure 2-2. [Figure 3-1]Figures 3A to 3H show the application of charge-stabilized immunogold conjugates for evaluating weak protein-protein interactions. A diverse panel of 56 monoclonal antibodies was used to evaluate the utility of charge-stabilized immunogold conjugates in identifying antibody self-interactions. This panel represents an accurate representation of the clinical antibody context, as evidenced by the similarity of biophysical properties to a dataset of 500 molecules extracted from the Therapeutic Antibody Database (TABS) (Figures 3A to 3D). By comparing the results of experiments performed at different concentrations of polylysine (3% vs 10%), the robustness of the calibration approach (described in the Methods) was demonstrated. An excellent correlation was observed between the two conditions, demonstrating that the assay is resistant to the effects from small changes in polylysine concentration that may occur due to variations in reagent preparation (Figure 3E). The CS-SINS score was compared to direct measurements of key developability characteristics (see Kingsbury, J. S. et al., Sci Adv 6, eabb0372, doi: 10.1126 / sciadv.abb0372 (2020), which is incorporated herein by reference in its entirety), and it has been experimentally established that mAbs with viscosity >30 cP or opalescence >12 NTU indicate problems in manufacturing and formulation during clinical development. Applying these benchmarks, it was 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 to 3H). [Figure 3-2] Continuation of Figure 3-1. [Figure 3-3] Continuation of Figure 3-2. [Figure 3-4] Continuation of Figure 3-3. [Figure 3-5] Continuation of Figure 3-4. [Figure 3-6] Continuation of Figure 3-5. [Figure 4] The methodology of the CS-SINS calibration process is shown. CS-SINS measurements are calibrated and evaluated using two tests. Only CS-SINS measurements that pass both tests should be accepted. [Figure 5-1]Figures 5A–C show exemplary calibration of CS-SINS measurements. Examples of experiments where the calibration process passed (Figure 5A) and failed (Figures 5B–5C) due to loose mixing of goat anti-human Fc antibody with gold particles (Figure 5B) or addition of 50% of the target mAb concentration (Figure 5C). To pass test number 1, the antibody-gold conjugate exhibits plasmon wavelengths less than 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 the reference panel of mAbs that are within 10% of the theoretical values ​​(1 with respect to slope, 0 with respect to intercept, and 1 with respect to R2). The CS-SINS score is calculated as described herein. [Figure 5-2] Continuation of Figure 5-1. [Figure 6A] Calibrated CS-SINS results show strong correlation with respect to conjugates prepared using various polylysine concentrations. A robust calibration protocol was developed to normalize results from experiments with varying 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 with respect to the plasmon measurements reported in Figure 6A, using the calibration detailed in Figures 4 and 5. [Figure 6B]Calibrated CS-SINS results show strong correlation with respect to conjugates prepared using various polylysine concentrations. A robust calibration protocol was developed to normalize results from experiments with varying 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 with respect to the plasmon measurements reported in Figure 6A, using the calibration detailed in Figures 4 and 5. [Figure 7-1] Figures 7A-7C show that CS-SINS measurements are strongly correlated with diffusion interaction parameters in IgG subclass-specific behavior. The CS-SINS score (0.01 mg / mL) is well correlated with kD measurements (1-10 mg / mL) (Figure 7A, top). The CS-SINS score is even more well correlated with kD measurements for subsets of IgG1 / IgG2 antibodies (Figure 7A, center), but less correlated for subsets of IgG4 antibodies (Figure 7A, bottom). Regarding solution behavior in relation to subclasses, the proposed 0.35 threshold for CS-SINS measurements effectively identifies all molecules exhibiting poor behavior in IgG1 / IgG2 subclass grouping (Figure 7B). Solution behavior for IgG4 is more difficult to predict because it mostly tends to show high opacity, but does not always react in the CS-SINS assay (Figure 7C). [Figure 7-2] Continuation of Figure 7-1. [Figure 8-1]Figures 8A–C are graphs evaluating the use of polylysine-stabilized gold conjugates to measure mAb self-assembly in histidine formulations (pH 6). Gold particles were first coated with goat anti-human Fc IgG and polylysine (≧70 kDa) (Figure 8A), goat nonspecific IgG and polylysine (Figure 8B), or polylysine alone (Figure 8C). These conjugates were then incubated with a panel of mAbs exhibiting varying levels of self-assembly. To assess the dependence of plasmon shift on the amount of adsorbed mAbs, conjugates (after the first conjugation step) were incubated with human polyclonal antibodies alone (0% mAb), human mAbs alone (100% mAb), or a combination thereof at a constant concentration of human antibody (0.01 mg / mL). The reported plasmon shift is associated with human polyclonal antibodies. In Figures 8A and 8B, the mass ratio of polylysine to IgG was 0.03, and the concentrations of polylysine and IgG used during conjugation were 0.012 and 0.388 mg / mL, respectively. In Figure 8C, the concentration of polylysine used during conjugation was 0.4 mg / mL. [Figure 8-2] Continuation of Figure 8-1. [Figure 9] This graph shows the effect of polylysine size on CS-SINS measurements of plasmon shift in histidine preparations (pH 6). Conjugates were prepared by co-adsorbing goat anti-human Fc antibodies with polylysine polymers of various sizes, and the plasmon shift of a panel of human mAbs was measured using these conjugates. Figure 9A shows the correlation between plasmon shifts measured using 30-70 kDa polylysine (0.03 polylysine / IgG mass ratio) and ≥70 kDa polylysine (0.03 polylysine / IgG mass ratio). Figure 9B shows the correlation between plasmon shifts measured using 15-30 kDa polylysine polymers (0.10 polylysine / IgG mass ratio) and ≥70 kDa polylysine polymers (0.03 polylysine / IgG mass ratio). [Modes for carrying out the invention]

[0012] This disclosure provides a method for predicting problematic properties (e.g., self-assembly) of a protein formulation using a small amount of protein in an ultradilute solution.

[0013] Antibodies with low levels of self-association are less likely to exhibit undesirable solution behavior during manufacturing, formulation, and delivery at high concentrations. While dilute solution interactions offer an effective approach to predicting antibody solution behavior, employing appropriate screening methods for a large number of antibodies early in discovery is challenging (Figure 1). Conventional techniques require relatively concentrated protein solutions (>1 mg / mL) to perform such measurements. This single challenge related to antibody concentration hinders large-scale, systematic analysis of antibody self-association during antibody lead optimization (Figure 1). Predicting high-concentration solution behavior (e.g., viscosity at 150 mg / mL) by assaying antibody interactions at much lower concentrations (1–10 μg / mL) would allow for the selection of well-behaving mAbs from a much larger pool of candidates early in discovery.

[0014] The previously used approach (affinity capture nanoparticle spectroscopy, AC-SINS) involved adsorbing anti-human capture antibodies onto gold nanoparticles (20 nm) and using this conjugate to capture human mAbs of interest. By capturing multiple mAbs in close proximity, colloidal interactions were amplified, and antibody self-interactions were detected with high sensitivity via measurable changes in the optical properties inherent to the gold nanoparticles. However, the main limitation of AC-SINS was its incompatibility with process flow and formulation conditions at the pH range of 4.5–7 and low ionic strength commonly used during antibody development. Gold nanoparticles coated with capture (anti-human) antibodies are unstable and readily aggregate under such conditions.

[0015] This specification describes the development of a system and method called charge-stabilized self-interacting nanoparticle spectroscopy (CS-SINS), which overcomes the limitations of AC-SINS by stabilizing antibody-gold conjugates using positively charged polymers. Furthermore, CS-SINS was able to reliably evaluate the weak self-interacting of mAbs at ultradilute concentrations (10 μg / mL) that predict high concentrations and opaque antibodies, which 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 limit the scope of the information.

[0017] 1.Definition The terms “comprise(s),” “include(s),” “having,” “has,” “can,” and “contain(s),” and their variations, when used herein, are intended to be open-ended, provisional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “an,” and “the” include plural references unless otherwise explicitly indicated by the context. This disclosure also contemplates other embodiments “including,” “consisting of,” and “essentially consisting of,” the embodiments or elements presented herein, whether expressly or otherwise.

[0018] In the enumeration of numerical ranges described herein, each digit intervening within them is explicitly intended with a similar degree of precision. For example, in the range 6–9, the digits 7 and 8 are intended in addition to 6 and 9, and in the range 6.0–7.0, the digits 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly intended.

[0019] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have meanings generally 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 publicly known and commonly used in the art. The meaning and scope of terms should be clear, but where ambiguity exists, the definitions provided herein shall take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms.

[0020] As used herein, the term “antibody” refers to a protein intrinsically used by the immune system to identify and neutralize foreign substances such as bacteria and viruses. Typically, an antibody is a protein containing at least one complementarity-determining region (CDR). The CDR forms the “hypervariable region” of the antibody, which is involved in antigen binding (further discussed below). A total antibody typically consists of the following four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain has one N-terminal variable (V) H ) region and three C-terminal constant (C H1 , C H2 , and C H3 ) region and each light chain has one N-terminal variable (V L ) region and one C-terminal stationary (C LThe antibody light chain may be assigned to one of two distinct types, 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 the heavy chain by a disulfide bond, and the two heavy chains are linked to each other by disulfide bonds. The variable region of the light chain is aligned with the variable region of the heavy chain, and the constant region of the light chain is aligned with the first constant region of the heavy chain. The remaining constant regions of the heavy chain are aligned with each other. The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. H Region and V L The regions share an identical 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 within the variable region located between the CDRs. Within each variable domain, there are four framework regions, referred to as FR1, FR2, FR3, and FR4, respectively. The framework regions form a β-sheet that provides the structural framework for the variable region (see, e.g., CAJaneway et al., Immunobiology, 5th edition, 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 antibody's "hypervariable region" involved in antigen binding. The CDRs link the β-sheet structure formed by the framework regions, forming loops that may, in some cases, constitute part of it. While the constant regions of the light and heavy chains are not directly involved in antibody binding to the antigen, they can influence the orientation of the variable region. The constant region also exhibits various effector functions, such as involvement in antibody-dependent complement-mediated lysis or antibody-dependent cytotoxicity, through interactions with effector molecules and cells.

[0021] The terms "antibody fragment", "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 of the antigen-binding fragments of an antibody described herein are within the scope of the present invention. The antibody fragment desirably comprises, 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: (i) V L domain, V H domain, C L domain, and C H1 Fab fragment, which is a monovalent fragment consisting of domains, (ii) F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) V of a single arm of an antibody L domain and V H Fv fragment consisting of domains, (iv) Fab' fragment generated by cleaving the disulfide bridge of an F(ab')2 fragment using mild reducing conditions, (v) disulfide-stabilized Fv fragment (dsFv), and (vi) an antibody single variable domain (V H or V L ) domain antibody (dAb), which is a polypeptide.

[0022] A spectrophotometer is any type of instrument that measures the absorption and / or transmission of light at various wavelengths. While spectrophotometers are most commonly applied to ultraviolet (185–400 nm), visible (400–700 nm), and infrared (700–15000 nm) radiation, some spectrophotometers can explore a broad band of the electromagnetic spectrum, including X-ray, ultraviolet, visible, infrared, and / or microwave wavelengths. A spectrophotometer comprises a light source, a spectrometer, a wavelength selector, a sample holder, and a detector. Any spectrophotometer capable of handling solution-based samples (e.g., those equipped with cuvette sample holders, plate readers, and similar devices) can be used.

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

[0024] Positively charged polymers can be any of a variety of compounds having a net positive charge. Useful positively charged polymers in this 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 may have interconnections between repeating units in addition to main chain links. Positively charged polymer compositions can have any level of polydispersity, from substantially monodisperse to substantially polydisperse. A substantially monodisperse composition contains polymer molecules that are substantially all of the same chain length. A substantially polydisperse composition contains polymer molecules of varying chain lengths (and therefore molecular weights).

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

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

[0027] The concentration of the positively charged polymer can be approximately 0.1% to 50% by mass of the positively charged polymer and the scavenger. The concentrations of the positively charged polymer and the scavenger are approximately 0.1% to 50% by mass, approximately 1% to 50% by mass, approximately 3% to 50% by mass, approximately 5% to 50% by mass, approximately 10% to 50% by mass, approximately 20% to 50% by mass, approximately 30% to 50% by mass, approximately 0.1% to 40% by mass, approximately 1% to 40% by mass, approximately 3% to 40% by mass, approximately 5% to 50% by mass, approximately 10% to 50% by mass, approximately 20% to 50% by mass, approximately 30% to 50% by mass, approximately 0.1% to 30% by mass, approximately 1% to 30% by mass, approximately 3% to 30% by mass, approximately 5% to 30% by mass, approximately 10% to 30% by mass, and approximately 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% ~20% by mass, approximately 0.1% by mass – approximately 15% by mass, approximately 1% by mass – approximately 15% by mass, approximately 3% by mass – approximately 15% by mass, approximately 5% by mass – approximately 15% by mass, approximately 10% by mass – approximately 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 mass of the positively charged polymer and the scavenger. In some embodiments, the positively charged polymer is added at a concentration of about 3% to about 15% by mass of the positively charged polymer and the scavenger.

[0028] Positively charged polymers can have a wide range of molecular weights. In some embodiments, positively charged polymers may have molecular weights greater than about 10 kD, greater than about 15 kD, greater than about 20 kD, greater than about 25 kD, greater than about 30 kD, greater than about 40 kD, greater than about 50 kD, greater than about 60 kD, greater than about 70 kD, greater than about 80 kD, greater than about 90 kD, greater than about 100 kD, greater than about 150 kD, greater than about 200 kD, or larger. In other embodiments, positively charged polymers may have molecular weights of 10-500 kDa, 10-250 kDa, 10-200 kDa, 15-70 kDa, 30-70 kDa, or 15-30 kDa. However, other sizes may also be used to prevent self-aggregation of the scavenger conjugate. The molecular weight can be determined by those skilled in the art by methods such as size exclusion chromatography and / or multi-angle laser light scattering techniques.

[0029] In certain embodiments of the present invention, positively charged polymers may fall into a 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 particularly polylysine. In addition, the polymer may contain one or more positively charged nonstandard 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 can be polymerized into linear, branched, or crosslinked 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, or 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 can be produced by methods known to those skilled in the art (e.g., chemical synthesis or recombinant methods). In a selective embodiment, the positively charged polymer is polylysine with a charge of about 70 kDa or more. In a selective embodiment, the positively charged polymer is polylysine added at a concentration of 3 to 15% by weight relative to the volume.

[0030] In some embodiments, the positively charged polymer includes synthetic polycationic polymers (e.g., polyethyleneimine (PEI), polyamidoamine (PAMAM), and the like).

[0031] The term "stabilization" in relation to nanoparticles specifically refers to preventing or at least reducing the aggregation of nanoparticles. "Aggregation" refers to the formation of aggregates in a suspension and represents a mechanism that leads to the functional destabilization of a colloidal system. During this process, particles dispersed in the liquid phase stick together, spontaneously forming irregular particle aggregates, flocs, or condensations. This phenomenon is also called solidification or coagulation, and such suspensions are also called unstable.

[0032] As used herein, the term “nanoparticles” refers to small particles 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, rocks, whiskers, fibers, boxes, and the like. Size refers to the maximum distance from one point to another on a nanoparticle (e.g., the diameter in the case of a sphere). Nanoparticles may include any material (e.g., metals, semiconductor materials, magnetic materials, and combinations of materials). Specifically assumed in this disclosure, whenever referred to herein, are metallic nanoparticles. Metallic nanoparticles (e.g., gold or silver nanoparticles) are inherently suitable for surface plasmon resonance-based assays because metallic spheres have free electrons on their surface that can interact with an electric field from incident light, resulting in strong absorbance spectra. In some embodiments, nanoparticles include gold nanoparticles having a gold surface or being made of gold. The size of the nanoparticles can affect the intensity and wavelength of the maximum absorbance. In some embodiments, the nanoparticles have a diameter of 1 nm to 1000 nm (e.g., 1 nm to 200 nm). In some embodiments, the nanoparticles are 5 nm to 50 nm in size (e.g., diameter), for example, about 20 nm. In exemplary embodiments, the nanoparticles include or consist of 20 nm gold nanoparticles. The total amount of nanoparticles in the assay may affect the signal-to-noise measurement. In some embodiments, the 20 nm gold nanoparticles are at least 7.0 × 10⁶ particles. 9 It is used at a concentration of particles / mL. In some embodiments, 20 nm gold nanoparticles are used in particles of 7.0 × 10⁶ 9 ~1.5×109 particles / mL (for example, 1.0 × 10⁶ particles) 10 ~1.5×10 9 It is used at a concentration of (particles / mL).

[0033] The capture agent is any agent capable of binding to the target protein (specifically, "capturing the target protein" in a solution as defined in the third embodiment). Therefore, the properties of the capture agent will depend on the type of target protein. For example, the capture agent may include a natural or synthetic binding partner for the target protein (e.g., a protein, nucleic acid, carbohydrate, small molecule, or another binding site specifically recognized by the target protein). In some embodiments, the capture agent includes an antibody, or a derivative or fragment thereof capable of binding to the target protein. The capture agent may be directly adsorbed onto nanoparticles, or the nanoparticles may include a linker that anchors the capture agent to these nanoparticles. In some embodiments, the capture agent is a protein or protein ligand. In specific embodiments, the capture agent is (or includes) an antibody, or a protein containing an antigen-binding fragment of an antibody. The antibody may be, in particular, an Fc-specific antibody (i.e., an antibody that binds to the Fc portion of another antibody), for example, an IgG-Fc-specific antibody, specifically 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 whose self-associating or self-aggregating properties are to be determined using a small amount of protein in a dilute solution. In some embodiments, the target protein includes a therapeutic protein. Therapeutic proteins include both purified and synthetic proteins useful for treating diseases and disorders in a subject. Examples of therapeutic proteins include, but are not limited to, antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, modified protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytics. In some embodiments, the target protein includes an antibody or a fragment or derivative thereof. In selective embodiments, the target protein includes an antibody, specifically a monoclonal antibody, and more specifically a human monoclonal antibody. In one embodiment, the antibody is an IgG antibody, for example, an IgG1, IgG2, or IgG4 antibody.In selective embodiments, the antibody is either 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 lower 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 concentrations 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) with 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 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 in which the total buffer concentration is less than about 50 mM, while a high ionic strength buffer is a buffer in which the concentration is greater than about 100 mM. Buffers are known in the art and may be salts of a weak acid and a weak base. Examples include carbonates, bicarbonates, and hydrogen phosphates. In specific embodiments of this disclosure, the buffer is either a histidine and / or acetate buffer or a buffer having substantially the same ionic strength as a histidine and / or acetate buffer. The histidine and / or acetate buffer is obtained having a histidine and / or acetate concentration of less than 50 mM, for example, having a histidine and / or acetate concentration of less than 25 mM, specifically having a histidine and / or acetate concentration of about 10 mM. "Substantially identical" means ±50%, ±40%, ±30%, ±20%, ±10%, or ±5%.

[0035] The term “conjugation” or “conjugating” refers to the binding of molecules to nanoparticles by chemical, physical, or biological means. The conjugation of nanoparticles to molecules is typically used when referring to nanoparticles that contain molecules on their surface. A specific meaning of this term is “adsorption” or “adsorption,” including physiadsorption (van der Waals forces), chemiadsorption (covalent bonding), and electrostatic attraction. In selective embodiments, the specific meaning of this term is physiadsorption.

[0036] The term "self-association" refers to the interaction between homogeneous proteins (or antibodies) and the formation of multimers. Such multimer formation may revert to monomers through operations such as dilution. The "tendency" towards self-association refers specifically to the ease with which self-association occurs when the protein concentration is increased beyond, for example, 50 mg / ml, 100 mg / ml, or 150 mg / ml (referred to herein as "high" or "higher" concentrations). An example of a parameter indicating self-association is the diffusion interaction parameter. The diffusion interaction parameter is an indicator of self-association calculated using the concentration dependence of the diffusion coefficient obtained by experimental methods. When this value is greater than -12.4 g / mL, intermolecular repulsion is dominant. When this value is less than that, attractive (self-association) interaction has been reported (Saito et al., Pharm. Res., 2013, Vol. 30, p. 1263). The diffusion coefficient is an indicator of how easily molecules diffuse in solution and is measured by dynamic light scattering or similar methods. More generally, protein self-association can lead to poor solution behavior, such as high viscosity, opacity, phase separation, and / or aggregation.

[0037] The term "plasmon wavelength" refers to the wavelength at which maximum absorbance occurs.

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

[0039] 2. Use of stabilized nanoparticles In a first aspect, the disclosure relates to the use of a positively charged polymer to stabilize nanoparticles containing a scavenger on a surface.

[0040] The scavenger is typically conjugated to nanoparticles. In specific embodiments, these nanoparticles are contained in a solution. Thus, this use may be for stabilizing nanoparticles (especially a plurality of them) in a solution (i.e., for stabilizing a nanoparticle suspension). This solution may be defined in the third embodiment described below.

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

[0042] 3. Nanoparticles In a second aspect, the disclosure relates to nanoparticles comprising a scavenger and a positively charged polymer on a surface.

[0043] In some embodiments, these nanoparticles are metal nanoparticles, and in particular, gold nanoparticles.

[0044] In some embodiments, the scavenger and / or positively charged polymer are conjugated to the nanoparticles. The amount of positively charged polymer (depending on the mass of the positively charged polymer and the scavenger) may be as described above. In specific embodiments, the nanoparticles are stable nanoparticles, i.e., stabilized according to the use of the first embodiment, for example. Specifically, the plasmon wavelength of the nanoparticles in solution while capturing a human polyclonal antibody (e.g., the human polyclonal antibody ChromPure Human IgG) is 534 nm or less, and / or the plasmon wavelength of the nanoparticles in solution while capturing a NIST monoclonal antibody (reference substance RM8671) mAb is 533 nm or less.

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

[0046] In some embodiments, this solution is a buffer, and in particular a low ionic strength buffer. The pH of this solution / buffer may be 3.5–7 (e.g., 4.5–7), and specifically 4–6.5 or 4–6. The plural means at least 2, at least 10, at least 100, or at least 1000.

[0047] Whenever this disclosure refers to the use of nanoparticles in the second aspect, it should be understood that it is also intended to refer to the use of the solution in the third aspect instead.

[0048] 5. Kits containing nanoparticles In a fourth aspect, the disclosure relates to a kit comprising nanoparticles (particularly multiple nanoparticles) and a positively charged polymer.

[0049] The kit may further include a solution such as a buffer, and in particular a low ionic strength solution / buffer. The pH of this solution / buffer may be 3.5 to 7 (e.g., 4.5 to 7), specifically 4 to 6.5 or 4 to 6. In further embodiments, the kit may also include (i) a scavenger (optionally conjugated to nanoparticles) and / or (ii) a panel of at least two (e.g., at least three, four, five, or six) calibration proteins. The kit may also include instructions for use that provide the plasmon wavelengths of the calibration proteins in the panel of calibration proteins. These plasmon wavelengths are known or "past" plasmon wavelengths of the calibration proteins.

[0050] 6. Method for determining the self-assembly of target proteins In a fifth aspect, the present disclosure relates to a method for determining the self-association tendency of a target protein, (i) A step of capturing a target protein in a solution defined in the third aspect, (ii) Steps to determine the color of this solution Includes, The color change of the solution compared to a control solution defined in a third aspect, which does not contain the target protein, indicates the self-association tendency of the target protein. Regarding the method.

[0051] The control solution may be the solution used before step (i) and before the target protein was added, or it may be a separate solution (for example, one that has undergone the same procedure except for step (i)).

[0052] The concentration of the target protein may be as described above with respect to the target protein analysis. The capture step may include mixing and / or incubating the solution (for example, as described below with respect to the sixth aspect).

[0053] The change in color typically occurs towards higher absorption wavelengths. In some embodiments, determining the color involves determining the light absorption, and the change in light absorption compared to a control solution indicates the self-association tendency of the target protein. In specific embodiments, step (ii) involves determining the plasmon wavelength, and the change in plasmon wavelength compared to a control solution indicates the self-association tendency of the target protein. This determination may involve measuring absorbance at multiple wavelengths in the range of 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 nanoparticles decreases. Therefore, when the target protein self-associates, the absorbance spectrum of the nanoparticles changes, and the plasmon wavelength lengthens. For example, if the nanoparticles are gold nanoparticles, a change in color to red (e.g., a red shift at the plasmon wavelength) indicates the self-association tendency of the target protein. Typically, the degree of change in color, light absorption, or plasmon wavelength indicates the strength of the self-association tendency of the target protein. However, in some embodiments, this change can be used as a binary representation of self-association and non-self-association, based on whether the plasmon wavelength changes due to the capture of the target protein.

[0054] This solution is typically the solution defined in the third embodiment. In a selective embodiment, the nanoparticles (or solution) in the solution have a plasmon wavelength of 534 nm or less for capturing human polyclonal antibodies (e.g., human polyclonal antibody ChromPure Human IgG) and / or a plasmon wavelength of 533 nm or less for capturing NIST monoclonal antibodies (reference substance RM8671) mAb. For this purpose, the method may include a step prior to step (i) of selecting nanoparticles (or solution) for capture in step (i), wherein the plasmon wavelength of the nanoparticles in the solution for capturing human polyclonal antibodies (e.g., human polyclonal antibody ChromPure Human IgG) is 534 nm or less, and / or the plasmon wavelength of the nanoparticles in the solution for capturing NIST monoclonal antibodies (reference substance RM8671) mAb is 534 nm or less.

[0055] In some embodiments, the method includes a step of calibration using a panel of at least two (preferably at least three, four, five, or six) calibration proteins (e.g., antibodies, specifically monoclonal antibodies) with different tendencies to self-associate. These tendencies are known. This calibration is performed as follows: (i) A step of measuring the plasmon wavelength of each calibration protein in this panel, (ii) A step to calculate the past CS-SINS score of each calibration protein (cP) according to the following formula: CS-SINS score = (cP - parameter 1) / (parameter 2 - parameter 1) (wherein parameter 1 is the plasmon wavelength of the calibration protein with the lowest self-association tendency of the panel, and parameter 2 is the plasmon wavelength of the calibration protein with the highest self-association tendency of the panel), (iii) Calculate a new CS-SINS score for each calibration protein according to the following formula: CS-SINS score = (cP - parameter 1) / (parameter 2 - parameter 1) (wherein parameter 1 is the plasmon wavelength of the calibration protein with the lowest self-association tendency of the panel, and parameter 2 is the plasmon wavelength of the calibration protein with the highest self-association tendency of the panel), and the following item: ((1 - slope) 2 +(intercept) 2 By minimizing ), the parameters are fitted to maximize the linear fit agreement between past parameters and new parameters. (iv) Determine whether the calibration satisfies the following criteria a) to c) with respect to the linear fit between new data and 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) Linear fit R 2 However, it is greater than 0.9; Furthermore (v) If all criteria in step (iv) are met, calibrate the CS-SINS score of the target protein using the fit parameters identified in step (iii). This may include. Step (ii) may be performed before, after, or simultaneously with step (i). In specific embodiments, a target protein CS-SINS score of approximately 0.35 or less indicates that the target protein has a low tendency to self-associate (i.e., it has favorable self-association properties for formulations and compositions containing high concentrations of the target protein (e.g., 100 mg / mL or more of the target protein)).

[0056] 7. Methods for screening target proteins This disclosure provides a method for screening dilute concentrations of target proteins with respect to their self-association properties (e.g., opacity and viscoelastic properties). Generally, the use of the method in the fifth embodiment is provided for screening target proteins with respect to their tendency to self-associate. More specifically, in the sixth embodiment, this disclosure relates to a method which may include: adsorbing a scavenger and a positively charged polymer onto nanoparticles (e.g., metal nanoparticles) to form a scavenger conjugate; incubating this scavenger conjugate with a target protein in a solution (e.g., a buffer) to form a target protein conjugate; measuring the absorbance of this target protein conjugate at multiple wavelengths in the range of 450 nm to about 750 nm; and identifying a plasmon wavelength as the wavelength at which the absorbance of this target protein conjugate is maximum.

[0057] Positively charged polymers are co-adsorbed onto a surface (e.g., nanoparticles) together with a scavenging agent.

[0058] This method may involve incubating a capture antibody conjugate and a target protein in a solution (e.g., a buffer) to form a target protein conjugate. This incubation can be for any length of time required to bind the target protein to the capture agent and form the target protein conjugate. For example, this incubation may be 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 may have any desired properties (pH, ionic strength, etc.), but typically mimics buffer conditions desirable for the final downstream use of the target protein. In some embodiments, the buffer conditions are physiologically acceptable in therapeutic use. In some embodiments, the pH of the buffer, and therefore the pH of the incubation, is 4.5–7. The pH of the buffer may be approximately 4.5, approximately 5, approximately 5.5, approximately 6, approximately 6.5, or approximately 7. In some embodiments, the buffer has low ionic strength.

[0060] This method may include measuring the absorbance of the target protein conjugate at multiple wavelengths in the range of 450 nm to approximately 750 nm, and identifying the plasmon wavelength as the wavelength at which the absorbance of the target protein conjugate is maximized. p ), that is, the wavelength corresponding to the maximum absorbance shifts to a larger value as the separation distance between nanoparticles decreases. Therefore, when the target protein self-associates, the absorbance spectrum of the nanoparticles changes, and this is reflected in the identification of the plasmon wavelength. In some embodiments, this change can be used as a binary representation of self-association present and self-association absent, based on whether the plasmon wavelength changes when measuring the absorbance of the target protein conjugate relative to the capture agent conjugate.

[0061] The sensitivity of plasmon wavelengths to changes in interparticle distance between 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 includes calculating a charge-stabilized self-interacting nanoparticle spectroscopy (CS-SINS) score. The SC-SINS score is illustrated in Figure 4 (test number 2) and is given by equation (1):

number

[0062] In some embodiments, a CS-SINS score of approximately 0.35 or less indicates that the target protein has self-associating properties that are advantageous for formulations and compositions containing 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 multiple calibration proteins with a capture agent conjugate to form multiple calibration protein conjugates, measuring the absorbance of each of the multiple calibration protein conjugates at multiple wavelengths in the range of 450 nm to about 750 nm, and identifying plasmon wavelengths.

[0064] The calibration method may further include calculating the CS-SINS score for each of several calibration protein conjugates; and, for linear fitting, comparing the CS-SINS score for each of the several calibration protein conjugates with the SC-SINS score of past calibration protein conjugates. For example, the CS-SINS for each of the several calibration protein conjugates may be compared using the following term: ((1-slope) 2 +(intercept) 2 By minimizing ), the fit is adjusted to maximize the linear fit agreement between the new data and the historical CS-SINS scores. This calibration is performed • Linear fit slope = 0.9 <x<1.1; • Intercept of linear fit = -0.1 <x<0.1;および • Linear fit R 2 ≥0.9 In this case, the criteria for linear fitting between new data and historical data are met, and therefore it is an appropriate calibration.

[0065] Multiple calibration proteins include proteins with various self-assembly properties (e.g., opacity and viscoelasticity). Calibration proteins may be the same type of protein as the target protein. For example, if the target protein is an antibody, the calibration proteins are selected from various antibodies with different self-assembly properties. In some embodiments, each of the multiple calibration proteins is independently selected from the group consisting of monoclonal antibodies, polyclonal antibodies, and their fragments or derivatives.

[0066] In some embodiments, calibration proteins include a low self-associating control protein and a high self-associating control protein, and the CS-SINS is calculated using the plasmon wavelengths of these conjugates as described elsewhere in this specification.

[0067] In some embodiments, the calibration proteins include NIST Reference Antibody RM 8671 and the human polyclonal antibody ChromPure Human IgG (whole molecule). In some embodiments, the calibration identifies the plasmon wavelengths of NIST Reference Antibody RM 8671 and the human polyclonal antibody ChromPure Human IgG (whole molecule). Appropriate calibration would include plasmon wavelengths of NIST Reference Antibody RM 8671 less than 533 nm and plasmon wavelengths of human polyclonal antibody ChromPure Human IgG (whole molecule) less than 534 nm.

[0068] 8. Systems or kits for screening target proteins In a seventh aspect, the disclosure relates to a system or kit (e.g., reagents, computer software, instruments, etc.) for screening a target protein at dilute concentrations with respect to preferred self-association properties (e.g., opacity and viscoelastic properties). In some embodiments, the system comprises one or more, or each of, a scavenger, a positively charged polymer, a surface (e.g., metal nanoparticles), a solution (e.g., a buffer), a target protein, at least one calibration protein, and a spectrophotometer. The descriptions above regarding the scavenger, positively charged polymer, surface (e.g., metal nanoparticles), solution (e.g., a buffer), and target protein provided elsewhere in this specification are also applicable to the system of the 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 packaged physically together or separately.

[0069] This system may also include instructions for using its components. These instructions constitute related documentation or methodologies for this system. Examples of such documentation include any combination of the following: background information, a list of components and their availability information (e.g., purchase information), concise or detailed protocols for using the system, troubleshooting, references, technical support, and any other relevant documentation. The instructions may be provided with the system or as a separate member component, either in paper form, electronically supplied on a computer-readable memory device or downloaded from an internet website, or as a recorded announcement.

[0070] The systems and kits described herein are understood to be used in connection with the methods described herein. [Examples]

[0071] 9. Examples material and method Preparation of immunogold conjugates for AC-SINS in PBS: Goat anti-human Fcγ-specific antibody (Jackson ImmunoResearch Laboratories, 109-005-008) was buffered twice with 20 mM acetic acid (pH 4.3) using a Zeba desalting column (Thermo Fisher Scientific, PI-89882). The concentration was determined using UV absorbance at 280 nm and a mass decay coefficient of 1.26 mL / mg × cm. The antibody was diluted to 0.4 mg / mL with 20 mM acetic acid (pH 4.3). 20 nm gold nanoparticles (particle size 7.0 × 10⁶) 11 (particles / mL; Ted Pella Inc., 15705) 1 ml was allowed to settle in a 1.5 mL microcentrifuge tube (1615-5500, USA Scientific) at 21130 rcf for 6 minutes. Next, 950 μL of the supernatant was removed and replaced with 950 μL of milliQ water. The resuspended particles were further diluted by adding 500 μL of milliQ water (final concentration of particles: 4.67 × 10⁶). 11 (particles / mL). 900 μL of gold nanoparticles were added to 100 μL of the prepared capture antibody. This mixture was incubated overnight at room temperature. Before use, the immunogold conjugate was allowed to precipitate at 21130 rcf for 6 minutes. 950 microliters of the supernatant was removed and stored, 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 buffered as described above and diluted to a final concentration of 0.8 mg / mL with 20 mM acetic acid (pH 4.3). Polylysine (Fisher Scientific, ICN19454405), initially dissolved at 5 mg / mL in milliQ water, was diluted with 20 mM acetic acid (pH 4.3) 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). For the preparation of 100 μL of conjugate, 48.5 μL of captured IgG was mixed with 1.5 μL of polylysine and stored. 1200 microliters of 20 nm gold nanoparticles (Ted Pella, 15705) were allowed to settle at 21130 rcf for 6 minutes, after which 1150 μL of the supernatant was removed. The nanoparticles were resuspended in the remaining supernatant, and the volume was carefully adjusted to 50 μL. These nanoparticles were then added to the IgG / polylysine mixture and rapidly mixed by pipetting up and down 20 times. This conjugate was incubated overnight at room temperature. The preparation described relates to 100 μL of conjugate, but can be adjusted to prepare larger volumes as needed. In experiments using non-capture antibodies, goat anti-human IgG was replaced with goat polyclonal IgG (Jackson ImmunoResearch Laboratories, 005-000-003).

[0073] Dynamic Light Scattering and Zeta Potential Measurement of Immunogold Conjugates Dynamic light scattering (DLS) experiments were performed using a Zetasizer Nano ZSP (Malvern Panalytical, Worcestershire, UK). To evaluate 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 saturate cell (DTS1070, Malvern Panalytical). The cuvette was transferred to the Zetasizer instrument and the size was measured at 25°C using DLS with 173° backscatter measurements. The size measurements were the average of at least 30 10-second measurements. If the sample was monodisperse (immunogold conjugate prepared for a standard AC-SINS run), the z-mean diameter calculated using cumulant analysis was reported. If the sample was polydisperse (polylysine-stabilized immunogold conjugate), 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 with the same instrument. The data was collected and analyzed using Zetasizer 7.11 Software (Malvern Panalytical).

[0074] CS-SINS assay in histidine: The target antibody was buffered twice with 10 mM histidine (pH 6.0) using a Zeba desalting column (Thermo Fisher Scientific, PI89882). The concentration was determined using UV absorbance at 280 nm and a mass decay coefficient independently calculated for each monoclonal antibody [for human polyclonal antibody (Jackson ImmunoResearch Laboratories, 009-000-003), 1.40 mL / mg × cm was used]. The antibodies were diluted to 11.1 μg / mL in histidine (pH 6.0) before use in CS-SINS. 5 μL of immunogold conjugate was added three times to a clear, flat-bottomed 384-well polystyrene plate (Thermo Fisher Scientific, 12565506) for each mAb and human polyclonal antibody that was assayed. Next, 45 μL of each antibody was added to the immunogold conjugate and mixed by pipetting up and down 10 times (final antibody concentration 10 μg / mL). This mixture was covered and incubated at room temperature for 4 hours, after which absorbance was measured at 450–650 nm in 1 nm increments (normal scanning speed, 8 readings per well) using a BioTek Synergy Neo plate reader (BioTek, Winooski, VT). The plasmon wavelength was determined by fitting a quadratic polynomial to 40 data points near the observed maximum absorbance and setting the first derivative to 0. The CS-SINS score was calculated as described below.

[0075] Calibration of CS-SINS Assay The CS-SINS measurements were calibrated and evaluated using the following method. First, each new batch of gold anti-Fc conjugates was evaluated using two control antibodies (i.e., human polyclonal antibody (ChromPure Human IgG, whole molecule, Jackson ImmunoResearch 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 with adsorbed human antibodies exceeded 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 number 1). Next, the gold anti-Fc conjugates that passed Test No. 1 were evaluated using a panel of six control mAbs (mAb A, C, D, E, F, and K in this test) exhibiting 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 antibody. With respect to the reference data, the CS-SINS score was calculated as (plasmon wavelength of a given mAb - plasmon wavelength of the lowest self-associating mAb (e.g., mAb A in this test)) / (plasmon wavelength of the highest self-associating mAb (e.g., mAb K in this test) - plasmon wavelength of the lowest self-associating mAb (e.g., mAb A in this test)). For each new experiment, the CS-SINS score for the mAb panel measurements (i.e., calibrated measurements) was calculated as (plasmon wavelength of a given mAb - parameter 1) / (parameter 2 - parameter 1). These two parameters were then divided into ((1 - slope)). 2 +(intercept) 2 The system is fitted to minimize the sum of the terms of the slope and intercept, and these terms are obtained from linear regression between the calibration and the reference CS-SINS score. The slope, intercept, or R of this linear fit is then used. 2 One of the three values ​​of the value is the ideal value (1 for slope, 0 for intercept, and R). 2If the result was not within 10% of 1), the experiment was terminated, and the conjugate was re-prepared until it passed both test number 1 and this test (test number 2). If the measurement passed both tests, CS-SINS measurement was performed on the full panel of antibodies, and the CS-SINS score was calculated using the parameter fit from test number 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 Opaqueness Measurement: Viscosity and opaqueness 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 herein by reference in its entirety. [Examples]

[0077] Charge stabilization prevents aggregation of the antibody-gold conjugate. To understand the cause of problems experienced in previous attempts using AC-SINS (Figure 2A, left), aggregation of gold-antibody (anti-human IgG) conjugates was measured in dilution buffer (10 mM acetic acid and / or histidine) over a pH range (pH 4.0–6.5; Figure 2B, left). At pH values ​​in the 5.0–6.5 range, high plasmon wavelengths of approximately 590 nm were observed, which are significantly different from the typical values ​​(530–535 nm) for stable conjugates in PBS (Figure 2E). Substantial particle aggregation was observed in the pH 5.0–6.5 range, as is evident from the large apparent diameter of the conjugates detected by dynamic light scattering. Consistent with this result, high plasmon wavelengths of approximately 590 nm were observed, which are significantly different from the typical values ​​(530–535 nm) for stable conjugates in PBS (Figure 2C, left). By measuring the zeta potential of the antibody-gold conjugate, we investigated the mechanism of particle aggregation (Figure 2D, left). We observed a steady decrease from a maximum of +16mV at pH 4.0, crossing zero around pH 5.7, and reaching a minimum of -5mV at pH 6.5. The trend of zeta potential with increasing pH was consistent with the titration of the immobilized capture antibody-specific charged amino acids. These findings suggest that the low net charge of the conjugate around 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. Even small amounts of polylysine (e.g., 3%, Figure 2A, center) were sufficient to prevent conjugate aggregation over a wide pH range of 4–6 (Figure 2B, center). Increasing the polylysine content to 15% (Figure 2A, right) further stabilized the conjugate down to pH 6.5 (Figure 2B, right). The plasmon wavelength of the polylysine-stabilized conjugate showed a pH-dependent trend similar to that observed with the apparent size of the conjugate (Figure 2C, center, and Figure 2C, right). A simultaneous increase in the zeta potential of the conjugate up to positive values ​​(+2 to +6 mV) was also observed 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 (+2.3 mB) at pH 6, which is likely related to the aggregation of the conjugate in the absence of polylysine, suggests further steric elements for the observed stabilization.

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

[0080] Robust calibration methods control the variability in results that can occur if 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 yield plasmon wavelengths of less than 534 nm for the human polyclonal antibody and less than 533 nm for the NIST mAb, as shown in Figure 5A for successful experiments. 2) The assay was calibrated using a panel of six calibration mAbs, and these measurements were compared to reference (past) measurements obtained from 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. The assay passed test number 2 if the slope, intercept, and R2 values ​​were within 10% of the ideal values, evaluating the complete panel of antibodies. In the successful experiment shown in Figure 5, the assay passed test number 2, and evaluation data for a larger panel of antibodies (excluding the calibrated antibody) 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 measurements between the two polylysine conditions. However, when the calibration protocol was applied, excellent agreement was observed between the two datasets (Figure 6B). To illustrate the value of this calibration process, further experiments were conducted to demonstrate how easily experiments with flawed protocols can be identified (Figures 5B-5C). [Examples]

[0081] mAb self-interaction as a prediction of solution behavior To evaluate the usefulness of charge-stabilized immunogold conjugates in measuring mAb self-interactions and predicting the resulting antibody solution behavior, a diverse set of 56 mAbs, including 43 commercially available products, was utilized. Various amino acid sequence-related properties (pI, charge, charge asymmetry, and hydrophobicity) were compared to 500 unique antibody sequences collected from a broader clinical setting for the mAbs in the dataset, using the Therapeutic Antibody Database (TABS) (Figures 3A-D). Emphasis was placed on evaluating sequence-derived metrics of electrostatic and hydrophobic properties, both of which have already been shown to be driving forces of mAb self-interactions. Based on this analysis, it was demonstrated that the mAb set accurately represented key mAb properties from a broader clinical antibody context, such as isoelectric point, net charge (pH6), and Fv charge asymmetry parameter (Fv-CSP, pH6). Furthermore, this antibody panel also exhibited similar hydrophobic properties, when evaluated against the Eisenberg Hydrophobicity Index (EHI), to a larger panel of clinical-stage antibodies. This finding demonstrates that mAb datasets are well-suited for evaluating the general applicability of CS-SINS assays for assessing antibody solution behavior.

[0082] More than 30% of mAbs in the clinical stage, and possibly even higher percentages in the early preclinical stage, are expected to exhibit poor solution behavior, such as high solution viscosity and opacity (these only appear at higher concentrations (>100 mg / mL)). High mAb solution viscosity is particularly problematic during subcutaneous delivery by autoinjector and during ultra / diafiltration purification unit operations. High opacity may indicate a predisposition to phase separation and aggregation. In recent years, the diffusion interaction parameter (k DMeasuring weak colloidal self-interactions using CS-SINS assays has been shown to be most effective in predicting mAbs that exhibit a high viscosity or milky light tendency compared to a large set of molecular descriptors. However, the material requirements of such measurements make them only feasible in the candidate selection stage after a discovery process of only a small number (about 10) of candidates (Figure 1). On the other hand, CS-SINS assays that also measure weak colloidal interactions are suitable for execution during candidate optimization screening of hundreds to thousands of variants (Figure 1).

[0083] CS-SINS was performed in an ultradilute solution (10 μg / ml) to determine whether it could detect the problematic behavior that appears at antibody concentrations four orders of magnitude higher (>100 mg / ml). Viscous antibodies (>30 cP) consistently scored higher in CS-SINS compared to low-viscosity, well-behaving mAbs, and these mAbs with an extreme opacity profile (>20 NTU) were readily identified using CS-SINS (Figure 3F). With a threshold of 0.35, CS-SINS identified well-behaving mAbs (<30 cP, <12 NTU) with >85% accuracy (Figures 3G-3H). Setting this CS-SINS score threshold facilitated the identification of all 10 viscous antibodies (100%) and 3 out of 7 opacity antibodies (43%). In addition, this threshold mischaracterized only 4 out of the remaining 39 well-behaving antibodies (10%). Of the 56 types of mAbs, 35 out of 39 were identified as exhibiting good behavior, and 13 out of 17 were identified as having poor solution properties. D The classification was equivalent to that obtained by [another method]. The CS-SINS assay was also robust in terms of reproducibility, supported by detailed calibration and system suitability criteria (Figures 3, 4, and 5).

[0084] From previous studies using a similar panel of antibodies, the diffusion interaction parameter (k D A strong relationship was revealed between k and poor solution properties. Furthermore, as specified herein, k in the prediction of poor solution properties (particularly those relating to opacity) D IgG subclass-specific behavior regarding the usefulness of k. DThe relationship between this and the CS-SINS score measured in this specification was investigated (Figure 7). If mAb separation by subclass is not performed, k D A moderately strong rank correlation was observed between the CS-SINS score and the mAb (Spearman's ρ -0.80; Figure 7A, top). However, when the mAbs were divided into IgG1 / IgG2 vs. IgG4 subclasses, subclass-specific behavior emerged. The rank correlation for IgG1 / IgG2 grouping increased significantly (Spearman's ρ -0.89; Figure 7A, middle), while it decreased for the IgG4 subclass (Spearman's ρ -0.76; Figure 7A, bottom). Extending this analysis to predict poor solution behavior, it was observed that using a CS-SINS threshold of 0.35 facilitated the detection of all poorly behaving molecules in IgG1 / IgG2 subclass grouping (Figure 7B). Applying the same criteria to IgG4 grouping revealed that all opaque molecules with low CS-SINS scores belonged to this subclass (Figure 7C), suggesting that the mode of IgG4 self-association leading to opaque behavior may be unique compared to IgG1 / 2 antibodies. More generally, these results comprehensively demonstrate the ability of CS-SINS to identify antibodies with high levels of viscosity and opacity, particularly with respect to IgG1 and IgG2 mAbs, using ultradilute (10 μg / mL) self-associating solution measurements. This assay is robust and readily performable for simultaneous high-throughput screening of a large number (approximately 100–1000) mAb candidates. The CS-SINS assay represents a significant step forward toward identifying developable antibodies with drug-like properties for early detection.

[0085] The above-mentioned detailed description and accompanying examples are illustrative and should not be considered as limitations on the scope of this disclosure as defined solely by the accompanying claims and their equivalents.

[0086] Various modifications and alterations to the embodiments of this disclosure will be obvious to those skilled in the art and will not deviate from the spirit and scope thereof.

Claims

1. The use of a positively charged polymer to stabilize nanoparticles containing a scavenging agent on the surface, wherein the positively charged polymer is polylysine. Uses include conjugating a positively charged polymer to nanoparticles either before, simultaneously with, or after conjugating a scavenger to the nanoparticles.

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

3. Nanoparticles comprising a scavenger and a positively charged polymer on their surface, wherein the positively charged polymer is polylysine.

4. The nanoparticles are those contained in a solution, as described in claim 3.

5. A solution comprising a plurality of nanoparticles as described in claim 3 or 4.

6. A kit comprising nanoparticles and a positively charged polymer, wherein the positively charged polymer is polylysine.

7. The kit according to claim 6, further comprising a solution and / or a scavenger.

8. A method for determining the self-assembly tendency of a target protein, (i) A step of capturing a target protein in the solution defined in claim 5, (ii) Steps to determine the color of the solution Includes, A method wherein the color change of the solution compared to a control solution that does not contain the target protein indicates the self-association tendency of the target protein.

9. A method for screening target proteins at ultradilute concentrations in terms of self-assembly characteristics, Adsorbing a scavenger and a positively charged polymer onto nanoparticles to form a scavenger conjugate; Incubating a scavenger conjugate with a target protein in solution to form a target protein conjugate; Measure the absorbance of the target protein conjugate at multiple wavelengths in the range of 450 nm to approximately 750 nm; Identifying the plasmon wavelength as the wavelength at which the absorbance by the target protein conjugate is maximized; and Screening using changes in plasmon wavelength, Includes, The positively charged polymer is polylysine, by method.

10. A system for screening target proteins at ultradilute concentrations in terms of self-assembly characteristics, Antibody-based scavenger; Polylysine is a positively charged polymer; nanoparticles; solution; Target protein; At least one calibration protein; and spectrophotometer A system that includes this.

11. The use according to claim 1 or 2, wherein the nanoparticles are metal nanoparticles.

12. The use according to claim 11, wherein the metal nanoparticles are gold nanoparticles.

13. The nanoparticles are metal nanoparticles, as described in claim 3 or 4.

14. The nanoparticles according to claim 13, wherein the metal nanoparticles are gold nanoparticles.

15. The solution according to claim 5, wherein the nanoparticles are metal nanoparticles.

16. The solution according to claim 15, wherein the metal nanoparticles are gold nanoparticles.

17. The kit according to claim 6 or 7, wherein the nanoparticles are metal nanoparticles.

18. The kit according to claim 17, wherein the metal nanoparticles are gold nanoparticles.

19. The method according to claim 8 or 9, wherein the nanoparticles are metal nanoparticles.

20. The method according to claim 19, wherein the metal nanoparticles are gold nanoparticles.

21. The system according to claim 10, wherein the nanoparticles are metal nanoparticles.

22. The system according to claim 21, wherein the metal nanoparticles are gold nanoparticles.

23. The positively charged polymer is polylysine, as described in any one of claims 8-9, 19-20. Method of loading.

24. The use according to any one of claims 1-2, 11-12, wherein the capture agent is an antibody.

25. The nanoparticles according to any one of claims 3-4, 13-14, wherein the capture agent is an antibody.

26. The kit according to any one of claims 6-7, 17-18, wherein the capture agent is an antibody.

27. The method according to any one of claims 8 to 9, 19 to 20, or 23, wherein the scavenger is an antibody.

28. The method according to any one of claims 8-9, 19-20, 23, or 27, wherein the target protein is an antibody.

29. The system according to any one of claims 10, 21 to 22, wherein the target protein is an antibody.

30. The use according to any one of claims 1-2, 11-12, or 24, wherein the solution has low ionic strength and / or a pH of 4-6.

5.

31. The solution comprises nanoparticles according to any one of claims 3-4, 13-14, or 25, having low ionic strength and / or a pH of 4-6.

5.

32. The solution is the solution according to any one of claims 5, 15 to 16, having low ionic strength and / or a pH of 4 to 6.

5.

33. The kit according to any one of claims 6-7, 17-18, or 26, wherein the solution has low ionic strength and / or a pH of 4-6.

5.

34. The method according to any one of claims 8-9, 19-20, 23, 27-28, wherein the solution has low ionic strength and / or a pH of 4-6.

5.

35. The system according to any one of claims 10, 21-22, or 29, wherein the solution has low ionic strength and / or a pH of 4 to 6.5.

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