Therapeutic compounds and methods involving in vitro mammalian skin

Abstract

A method for investigating subcutaneous administration of a therapeutic compound product, comprising the steps of providing an in vitro live mammalian skin sample comprising a dermal layer and an epidermal layer, administering a therapeutic compound product to the interface between the dermal layer and the epidermal layer, and analyzing the skin sample.

Description

[Technical field]

[0001] Embodiments herein relate to methods of using in vitro mammalian skin for the analysis, characterization, development and / or selection of therapeutic compounds. [Background technology]

[0002] Subcutaneous (SubQ) injection is a widely used administration route for pharmaceutical compositions containing therapeutic compounds, such as therapeutic proteins. However, aggregation and poor solubility can be major obstacles to the manufacture and delivery of pharmaceutical compositions of therapeutic compounds. Summary of the Invention [Means for solving the problem]

[0003] According to some embodiments, a method of selecting an administration condition of a therapeutic compound product for subcutaneous administration is described. The method may include providing an in vitro living mammalian skin including a dermal layer and an epidermal layer defining an interface between the dermal layer and the epidermal layer. The method may include administering a therapeutic compound product to the interface under administration conditions. The method may include detecting a physiochemical and / or clinical property of the therapeutic compound product at the interface. The method may include selecting or rejecting the administration condition for further development based on the detected physiochemical and / or clinical property of the therapeutic compound.

[0004] For any of the methods of selecting administration conditions for a therapeutic compound product for subcutaneous administration described herein, the administration conditions include at least one of the formulation, concentration, volume, viscosity, molecular characteristics, route of administration and / or administration device or method of using the administration device (frequency, flow rate, parameters) of said therapeutic compound.

[0005] For any of the methods of selecting administration conditions for a therapeutic compound product for subcutaneous administration described herein, the administration conditions may include formulation additives, buffers, surfactants, or stabilizers.

[0006] For any of the methods of selecting dosing conditions for a therapeutic compound product for subcutaneous administration described herein, the physiochemical and / or clinical properties may include bioavailability, pharmacokinetic properties, aggregation, precipitation, distribution, diffusion rate, retention and / or absorption.

[0007] For any of the methods of selecting administration conditions for a therapeutic compound product for subcutaneous administration described herein, the therapeutic compound product may have acquired physiochemical and / or clinical properties after said administering step.

[0008] For any of the methods of selecting an administration condition for a therapeutic compound product for subcutaneous administration described herein, the method may further include repeating the method with two or more different administration conditions.

[0009] According to some embodiments, a method is described for determining the effect of a molecular property of a therapeutic compound on a physiochemical and / or clinical property of the therapeutic compound after subcutaneous administration. The method may include providing an in vitro living mammalian skin comprising a dermal layer and an epidermal layer, defining an interface between the dermal layer and the epidermal layer. The method may include administering a therapeutic compound product comprising the molecular property to the interface. The method may include detecting the physiochemical and / or clinical property of the therapeutic compound at the interface. By way of example, the physiochemical and / or clinical property may include bioavailability, pharmacokinetic properties, aggregation, precipitation, distribution, diffusion rate and / or absorption.

[0010] According to some embodiments, a method of detecting a change in a molecular characteristic of a therapeutic compound after subcutaneous administration is described. The method may include providing an in vitro live mammalian skin including a dermal layer and an epidermal layer, defining an interface between the dermal layer and the epidermal layer. The method may include administering a therapeutic compound including a characteristic to the interface, the therapeutic compound including a first state of the molecular characteristic. The method may include detecting a second state of the molecular characteristic of the therapeutic compound in the subcutaneous space. The second state may be different from the first state. For any of the methods of detecting a change in a molecular characteristic of a therapeutic compound after subcutaneous administration described herein, the first state and the second state of the molecular characteristic may include any of the concentration, amount, distribution, or distribution at the interface for the therapeutic compound. For any of the methods of detecting a change in a molecular characteristic of a therapeutic compound after subcutaneous administration described herein, the first state of the molecular characteristic has a first value and the second state of the molecular characteristic has a second value that is the same or different from the first state. For any of the methods of detecting a change in a molecular characteristic of a therapeutic compound after subcutaneous administration described herein,

[0011] For any of the methods described herein, the molecular characteristics may include at least one of acidic species, basic species, high molecular weight species, sub-visible particle count, low molecular weight, medium molecular weight, glycosylation (such as non-glycosylated heavy chain or high mannose), non-heavy and light chains, deamidation, deamination, cyclization, oxidation, isomerization, fragmentation / clipping, N-terminal and C-terminal variants, reduced species and partial species, folding structure, surface hydrophobicity, chemical modification, covalent bond, C-terminal amino acid motif PARG or C-terminal amino acid motif PAR-amide.

[0012] For any of the methods described herein, the administering step may include pressing a needle including a cannula against the surface of the dermis layer, thus inserting the needle into the in vitro interface of the skin of a living mammal. The administering step may further include disposing a therapeutic compound product through the needle into the subcutaneous space. In some methods, the needle is pressed against the surface of the dermis at an acute angle. In some methods, the needle includes an opening in fluid communication with the cannula, and the administering step includes directing the opening into the dermis layer. The detecting step may include optical imaging through the dermis layer.

[0013] For any of the methods described herein, the administering step may include soaking, diffusing, or transdermal administration of the therapeutic compound product through the dermis layer and / or into the subcutaneous tissue layer.

[0014] For any of the methods described herein, the in vitro mammalian skin may further comprise subcutaneous tissue defining a subcutaneous space within the interface.

[0015] For any of the methods described herein, the step of providing an in vitro mammalian skin may include immobilizing the in vitro mammalian skin between a proximal substrate and a distal substrate, hi some methods, the proximal substrate and the distal substrate each comprise glass, such as a coverslip.

[0016] For any of the methods described herein, the step of providing an in vitro mammalian skin may include providing a dermal layer of the skin model disposed on a porous substrate containing pores sized to accommodate diffusion of the therapeutic compound product. The step of administering may include placing the porous substrate in fluid communication with a solution containing the therapeutic compound product. The method may further include detecting the presence of subvisible levels of particles in the solution after the step of administering.

[0017] For any of the methods described herein, the in vitro mammalian skin can be in vitro human skin. For any of the methods described herein, the in vitro mammalian skin can be in vitro skin of a non-human primate, such as a cynomolgus monkey. For any of the methods described herein, the in vitro mammalian skin can be of a non-human mammal. For some methods where the in vitro mammalian skin is of a non-human mammal, the method can further include repeating the method using in vitro human skin and comparing the physiochemical and / or clinical properties of the therapeutic compound administered to the in vitro human skin with the physiochemical and / or clinical properties of the therapeutic compound administered to the in vitro mammalian skin that is of a non-human mammal.

[0018] For any of the methods described herein, the detecting step may include imaging and / or analytical testing. Analytical testing may include mass spectrometry, chromatography, electrophoresis, spectroscopy, light obscuration, particle methods (such as nanoparticle / visible / micron-sized resonant mass or Brownian motion), analytical centrifugation, imaging or image characterization, immunoassay, SE-HPLC, rCE-SDS, CEX-HPLC, HIAC, nrCE-SDS, mass spectrometry microscopy, and / or mass spectroscopy. Imaging may include CT scan or magnetic resonance imaging. For any of the methods described herein, the detecting step may include optical imaging. For any of the methods described herein, the detecting step may include obtaining a background signal and subtracting the background signal.

[0019] For any of the methods described herein, the method may further comprise the step of labeling the therapeutic compound with a detectable moiety, such as a fluorophore, a fluorescent dye, a radiolabel, or a quantum dot.

[0020] For any of the methods described herein, the therapeutic compound may comprise an antibody, an antigen-binding antibody fragment, an antibody protein product, a bi-specific T cell engager (BiTE®) molecule, a bispecific antibody, a trispecific antibody, an Fc fusion protein, a recombinant protein, a recombinant virus, a recombinant T cell, a synthetic peptide, an active fragment of a recombinant protein, a nucleic acid, or a virus. For any of the methods described herein, the therapeutic compound may comprise or consist of a therapeutic protein.

[0021] For any of the methods described herein, the method may further include the step of performing an analytical test on the therapeutic compound prior to the step of administering the therapeutic compound to the interface. [Brief description of the drawings]

[0022] [Figure 1A] A series of schematic diagrams illustrating injection and fluorescence-based approaches to characterize the behavior of therapeutic compounds in an in vitro mammalian skin environment according to some embodiments. Figure 1A shows fluorescent labeling of therapeutic compounds, which is suitable for some methods described herein. Of course, some methods described herein may also be performed on unlabeled therapeutic compounds. Figure 1B illustrates imaging options in addition to those shown in Figure 1A. [Figure 1B] A series of schematic diagrams illustrating injection and fluorescence-based approaches to characterize the behavior of therapeutic compounds in an in vitro mammalian skin environment according to some embodiments. Figure 1A shows fluorescent labeling of therapeutic compounds, which is suitable for some methods described herein. Of course, some methods described herein may also be performed on unlabeled therapeutic compounds. Figure 1B illustrates imaging options in addition to those shown in Figure 1A. [Figure 1C] 1 is a flow chart illustrating a method of some embodiments. [Figure 1D]1 is a flow chart illustrating a method of some embodiments. [Figure 1E] 1 is a flow chart illustrating a method of some embodiments. [Figures 2A-2D] 1 is a series of image overlays of mAbs with different pH-sensitive aggregation tendencies injected into mammalian skin in vitro according to some embodiments, with the arrowheads indicating mAb aggregates. [Figure 3A] FIG. 1 is a series of schematic diagrams illustrating exposure and analysis strategies to characterize changes in properties following exposure of in vitro mammalian skin according to some embodiments. [Figure 3B] FIG. 1 is a series of schematic diagrams illustrating exposure and analysis strategies to characterize changes in properties following exposure of in vitro mammalian skin according to some embodiments. [Figure 4A] 1 is a series of graphs showing a comparison of high molecular weight (HMW) species and subvisible particle (SbVP) levels for two formulations of mAb3 exposed to mammalian skin in vitro according to some embodiments. [Figure 4B] 1 is a series of graphs showing a comparison of high molecular weight (HMW) species and subvisible particle (SbVP) levels for two formulations of mAb3 exposed to mammalian skin in vitro according to some embodiments. [Figure 4C] 1 is a series of graphs showing a comparison of high molecular weight (HMW) species and subvisible particle (SbVP) levels for two formulations of mAb3 exposed to mammalian skin in vitro according to some embodiments. [Figure 4D] 1 is a series of graphs showing a comparison of high molecular weight (HMW) species and subvisible particle (SbVP) levels for two formulations of mAb3 exposed to mammalian skin in vitro according to some embodiments. [Figure 5A] 1 is a series of graphs showing a comparison of high molecular weight (HMW) species and subvisible particle (SbVP) levels for several therapeutic proteins in various formulations. [Figure 5B]1 is a series of graphs showing a comparison of high molecular weight (HMW) species and subvisible particle (SbVP) levels for several therapeutic proteins in various formulations. [Figure 5C] 1 is a series of graphs showing a comparison of high molecular weight (HMW) species and subvisible particle (SbVP) levels for several therapeutic proteins in various formulations. [Figure 6] 1 is a series of images of a therapeutic protein solution injected into mammalian skin in vitro according to some embodiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Described herein are methods for analyzing, selecting, and characterizing therapeutic compounds and pharmaceutical compositions comprising same. Previously, evaluation of the interactions of therapeutic compounds and pharmaceutical compositions in a SubQ cavity was limited by the lack of a robust experimental system that adequately captures the biological complexity of human skin. Thus, previously, there was a need for an accurate method for evaluating therapeutic compound properties such as aggregation and solubility in a SubQ environment. Described herein are methods for using in vitro mammalian skin, such as human skin, to model the effects of the skin environment on therapeutic compounds (and pharmaceutical compositions comprising same) in a SubQ cavity. The methods can be used to select administration conditions (such as one or more formulation properties, or a device or route of administration) for a therapeutic compound product, to determine the effect of a molecular property (such as a HMW species) of the therapeutic compound on the physiochemical and / or clinical properties of the therapeutic compound following subcutaneous administration, and / or to detect changes in the molecular property of the therapeutic compound following subcutaneous administration. The methods can include providing an in vitro live mammalian skin including a dermal layer and an epidermal layer that defines an interface between the dermal layer and the epidermal layer. The method can include administering a therapeutic compound product to the interface. The method can include detecting physiochemical and / or clinical properties of the therapeutic compound product at the interface after the therapeutic compound product is administered. The therapeutic compound product may acquire physiochemical and / or clinical properties after administration. Examples of physiochemical and / or clinical properties include bioavailability, pharmacokinetic properties, aggregation, chemical modification, precipitation, distribution, diffusion rate, retention and / or absorption. Advantageously, the methods described herein can faithfully, consistently and robustly determine the effect of the subcutaneous environment on therapeutic compounds and pharmaceutical compositions comprising therapeutic compounds. For example, it is contemplated that the methods described herein can model the subcutaneous environment with greater fidelity than in vitro buffers alone and with greater consistency and reproducibility than live animal models.

[0024] Protein aggregation and poor solubility can be major obstacles in drug manufacturing and delivery. Therapeutically effective doses often require high protein concentrations that are significantly different from physiological conditions, which are achieved by extensively developed formulations. Such formulations generally contain acidic pH (approximately 5.0) and stabilizing additives optimized to maintain the necessary solubility, stability and viscosity profiles of the biological solution that will be delivered to the patient. Upon injection into the skin, various factors within the local environment can affect how the biologic is distributed in the body. As the drug depot forms after injection, initial changes in the solubility and stability of the biologic can be driven by the loss of buffer components and stabilizing additives that diffuse away from the injection site at a faster rate than the biologic. Contact with salts, proteins, cells and extracellular components of the skin environment can result in novel interactions that induce aggregation and can affect the bulk solution behavior of the drug. In vitro skin models provide a useful approach to predict problems with drug distribution in the skin and provide an opportunity to screen conditions that may affect stability, clinical pharmacokinetics and bioavailability.

[0025] In vitro testing plays an increasing role in the development of therapeutic compounds by providing an efficient and cost-effective means to optimize the performance of pharmaceutical compositions and identify potential issues that may affect product quality and efficacy. With the establishment of subcutaneous injection as the preferred route of administration for biologics, there is an increasing need to identify and better understand drug interactions within the cutaneous environment. [1] Tissue engineering allows the production of in vitro mammalian skin that reproduces many of the morphological and functional characteristics of human skin. [2] Can in vitro mammalian skin be produced using open source protocols? [2] or can be purchased from a dealer. [3][4] Laboratory-grown skin models generally consist of three distinct layers of human skin: the epidermis [5] , dermis [6] and subcutaneous tissue [7]) or a model that includes the epidermis and dermis (but lacks the subcutaneous tissue) [8] Additionally, the development of a model that contains all three layers has been described. [9] In the cosmetology and skin care industries, in vitro skin models that focus on the outer layer are used for toxicity and formulation testing.

[10]

[11] However, these models have not generally been used to test the effects of the skin environment on injectable therapeutic compounds.

[0026] The methods described herein use in vitro mammalian skin, such as human skin, to provide insight into the effect of the skin environment on the physicochemical properties of therapeutic compounds and pharmaceutical compositions comprising therapeutic compounds. Some methods described herein include using imaging, such as fluorescence imaging, to characterize the physicochemical and / or clinical properties of therapeutic compounds, such as aggregation tendency and diffusivity upon injection into the skin. Such in vitro techniques allow for the detection of potential aggregation problems and biodistribution challenges within a skin-like environment that may affect drug absorption. Some methods described herein include using analytical measurements (e.g., chromatography-based assays, particle counting methods, and / or mass spectrometry) to characterize changes in molecular properties following subcutaneous administration and / or the effect of molecular properties on the physicochemical and / or clinical properties of therapeutic compounds following subcutaneous administration into in vitro mammalian skin. In vitro mammalian skin can be used in these applications, for example, to optimize therapeutic compounds and pharmaceutical compositions comprising therapeutic compounds, screen formulations, and improve injection strategies for subcutaneously administered biological drugs.

[0027] In vitro mammalian skin In vitro mammalian skin may be used in the methods described herein. As used herein, "in vitro mammalian skin" refers to a living mammalian skin including a dermal layer and an epidermal layer, defining an interface between the dermal layer and the epidermal layer. It may be prepared in an in vitro environment, for example, disposed in a multi-well plate. The in vitro mammalian skin may be disposed on a substrate, such as a glass or polymeric coverslip, or on a porous substrate. Alternatively, the in vitro mammalian skin may be suspended in a medium without a substrate. In some methods, the in vitro mammalian skin may be a substrate comprising an epidermal layer, an epidermal layer, a ... [5] , dermis [6] and subcutaneous tissue [7] These three layers may be provided together or as separate layers. In some methods, the in vitro mammalian skin comprises the epidermis and dermis, but does not include the subcutaneous tissue. [8]

[0028] For any of the methods described herein, the in vitro mammalian skin may further include subcutaneous tissue that defines a subcutaneous space within the interface. Subcutaneous space is understood to refer to the portion of the interface that is further defined at least in part by subcutaneous tissue. Thus, in some embodiment methods, the in vitro mammalian skin interface may further include a subcutaneous space, and the interface may define a subcutaneous space. It is also contemplated that in vitro mammalian skin, including dermal and epidermal layers, may provide a useful model for the subcutaneous environment, even in the absence of subcutaneous tissue.

[0029] By way of example, the in vitro mammalian skin model can be human, non-human primate (such as cynomolgus monkeys) or non-human mammalian (such as mouse, rat, rabbit, dog, goat, sheep or pig) skin. In some methods, the in vitro mammalian skin can be human or non-human primate skin, such as cynomolgus monkeys. In the development of therapeutic compounds, the therapeutic compounds can be tested in animal models (e.g., non-human primates, such as cynomolgus monkeys), for example, in toxicity and / or efficacy tests. Thus, it is contemplated that the in vitro mammalian skin described herein can be useful in the selection and development of therapeutic compounds and pharmaceutical compositions comprising therapeutic compounds prior to animal model testing, for example, in in vitro aggregation, pharmacokinetic and / or toxicity tests using in vitro mammalian skin of the same species as the animal model.

[0030] For any of the methods described herein, the in vitro mammalian skin model can be a non-human mammalian skin. The method can be further repeated using in vitro human skin. The method can include a step of comparing the physiochemical and / or clinical properties of the therapeutic compound administered to the in vitro human skin with the physiochemical and / or clinical properties of the therapeutic compound administered to the in vitro mammalian skin of a non-human mammal. That is, the method can compare the behavior of the therapeutic compound in the in vitro mammalian skin of a non-human mammal with the behavior of the therapeutic compound in the in vitro human skin to confirm the suitability of the non-human mammal for further experiments.

[0031] For any of the methods described herein, the in vitro mammalian skin may comprise or consist of in vitro human skin. For any of the methods described herein, the in vitro mammalian skin may comprise or consist of in vitro skin of a non-human primate, such as a cynomolgus monkey.

[0032] therapeutic compounds As used herein, "therapeutic compound" and variations of this root term have their ordinary and accustomed meaning as would be understood by one of skill in the art in light of this disclosure. It refers to a therapeutic composition that includes an active pharmaceutical ingredient, e.g., a synthetic small molecule, a gene therapy agent, a therapeutic protein, a nucleic acid such as an antisense RNA or siRNA, a virus, or a cell or portion thereof.

[0033] As used herein, "therapeutic protein" and variations of this root term have their normal customary meaning as would be understood by one of skill in the art in light of the present disclosure. It refers to a therapeutic composition comprising a polypeptide macromolecule, e.g., a protein, peptide, or a portion thereof. A therapeutic protein may be a protein for medical use in human subjects, e.g., a candidate for medical use, or a protein approved for medical use by a governmental authority, such as the FDA or EMA. Of course, a therapeutic protein is a type of therapeutic compound. Thus, whenever a therapeutic compound is referred to herein, a therapeutic protein such as those described herein is contemplated.

[0034] In the methods described herein, the therapeutic compound may comprise or consist of a therapeutic protein. In the methods described herein, the therapeutic compound may be selected from the group consisting of an antibody, an antigen-binding antibody fragment, an antibody protein product, a bispecific T-cell engager (BiTE®) molecule (such as a BiTE® molecule containing a half-life extending moiety), a bispecific antibody, a trispecific antibody, an Fc fusion protein, a recombinant protein, a recombinant viral protein, a synthetic peptide, an active fragment of a recombinant protein, a nucleic acid (such as an antisense RNA or siRNA), and a virus.

[0035] "Antibody" has its customary and ordinary meaning as understood by those of skill in the art in light of this disclosure. It refers to an immunoglobulin of any isotype that specifically binds to a target antigen, including, for example, chimeric antibodies, humanized antibodies, and fully human antibodies. By way of example, the antibody may be a monoclonal antibody. Human antibodies can be of any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE. Human IgG antibodies will generally contain two full-length heavy chains and two full-length light chains. An antibody may be derived from only a single source, or may be "chimeric," i.e., different portions of the antibody may be derived from two or more different antibodies from the same or different species. Of course, once an antibody is obtained from a source, it may be subjected to further manipulations, for example to enhance stability and folding. Thus, it will be appreciated that a "human" antibody can be obtained from a source and can be further engineered, for example in the Fc region. An engineered antibody can still be referred to as a type of human antibody. Similarly, unless otherwise specified, variants of human antibodies, such as affinity matured ones, will also be understood to be "human antibodies." In some embodiments, the antibody comprises, consists essentially of, or consists of a human antibody, a humanized antibody, or a chimeric monoclonal antibody.

[0036] The "heavy chain" of an antibody (or antibody protein product) comprises a variable region ("VH") and three constant regions, namely CH1, CH2 and CH3. Exemplary heavy chain constant regions include the constant regions of human IgG1, IgG2, IgG3 and IgG4. The "light chain" of an antibody (or antibody protein product) comprises a variable region ("VL") and a constant region ("CL"). Human light chains include kappa and lambda chains. Exemplary light chain constant regions suitable for the antigen binding proteins described herein include the human lambda constant region and the human kappa constant region.

[0037] In various embodiments, the therapeutic protein is an antibody protein product. As used herein, the term "antibody protein product" refers, in various instances, to any one of several antibody surrogates that are based on the structure of an antibody but are not found in nature. In some embodiments, the antibody protein product has a molecular weight in the range of at least about 12-150 kDa. In certain embodiments, the antibody protein product has a valency (n) ranging from monomer (n=1) to dimer (n=2), trimer (n=3), to tetramer (n=4), if not of higher valency. In some embodiments, the antibody protein product is based on the complete antibody structure and / or mimics an antibody fragment that retains complete antigen-binding ability, such as scFv, Fab, and VHH / VH (described below). The smallest antigen-binding antibody fragment that retains a complete antigen-binding site is the Fv fragment, consisting entirely of the variable (V) region. Soluble and flexible amino acid peptide linkers are used to link the V regions to scFv (single chain fragment variable) fragments to stabilize the molecule, or constant (C) domains are added to the V regions to generate Fab fragments [antigen-binding fragments]. Both scFv and Fab fragments can be easily produced in host cells, e.g. prokaryotic host cells. Other antibody protein products include dimeric and multimeric antibody formats such as diabodies, triabodies and tetrabodies or minibodies (miniAbs), including disulfide bond stabilized scFv (ds-scFv), single chain Fab (scFab), and different formats consisting of scFv linked to oligomerization domains. The smallest fragments are the VHH / VH of camelid heavy chain Abs and single domain Abs (sdAbs). The building block most frequently used to generate new antibody formats is the single variable (V) domain antibody fragment (scFv), which contains the V domains (VH and VL domains) from the heavy and light chains linked by a peptide linker of about 15 amino acid residues. Peptibodies or peptide-Fc fusions are yet another antibody protein product. The structure of a peptibody consists of a biologically active peptide grafted to an Fc domain. Peptibodies have been well described in the art.See, for example, Shimamoto et al., mAbs 4(5):586-591 (2012).

[0038] Therapeutic proteins suitable for the methods described herein can include polypeptides, such as those that bind to one or more of the following: CD proteins, such as CD3, CD4, CD8, CD19, CD20, CD22, CD30, and CD34, including those that interfere with receptor binding; HER receptor family proteins, such as HER2, HER3, HER4, and EGF receptor; cell adhesion molecules, such as LFA-1, MoI, pl50, 95, VLA-4, ICAM-I, VCAM, and alpha v / beta 3 integrin. Growth factors such as vascular endothelial growth factor ("VEGF"), growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, Müllerian inhibitory substance, human macrophage inflammatory protein (MIP-1 alpha), erythropoietin (EPO), nerve growth factors such as NGF-beta, platelet derived growth factor (PDGF), fibroblast growth factors such as aFGF and bFGF, epidermal growth factor (EGF), transforming growth factors (TGFs) such as, in particular, TGF-α and TGF-β, such as TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5, insulin-like growth factor-I and -II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I) and bone morphogenetic factors. Insulin and insulin-related proteins, such as insulin, insulin A chain, insulin B chain, proinsulin and insulin-like growth factor binding proteins. Coagulation and coagulation-related proteins, such as, inter alia, factor VIII, tissue factor, von Willebrand factor, protein C, alpha-1-antitrypsin, plasminogen activators, such as urokinase and tissue plasminogen activator ("t-PA"), bombazine, thrombin and thrombopoietin, (vii) other blood and serum proteins, including, but not limited to, albumin, IgE, and blood group antigens. Colony-stimulating factors and their receptors, such as, inter alia, M-CSF, GM-CSF and G-CSF and their receptors, such as the CSF-1 receptor (c-fms).Receptors and receptor-associated proteins, such as flk2 / flt3 receptor, obesity (OB) receptor, LDL receptor, growth hormone receptor, thrombopoietin receptor ("TPO-R", "c-mpl"), glucagon receptor, interleukin receptor, interferon receptor, T cell receptor, stem cell factor receptor, such as c-Kit and other receptors. Receptor ligands, such as OX40L, which is a ligand for the OX40 receptor. Neurotrophic factors, such as bone-derived neurotrophic factor (BDNF) and neurotrophin-3, -4, -5 or -6 (NT-3, NT-4, NT-5 or NT-6). Relaxin A chain, relaxin B chain and prorelaxin, interferons and interferon receptors, such as interferon-α, -β and -γ and their receptors. Interleukins and interleukin receptors, IL-1 to IL-33 and IL-1 receptor to IL-33 receptor (e.g., IL-8 receptor, among others). Viral antigens, e.g. AIDS envelope viral antigens. Lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor alpha and beta, enkephalinase, RANTES (regulated on activation normally T-cell expressed and secreted), mouse gonadotropin-related peptide, DNAse, inhibin and activin. Integrins, protein A or D, rheumatoid factor, immunotoxins, bone morphogenetic proteins (BMPs), superoxide dismutase, surface membrane proteins, decay accelerating factor (DAF), HIV envelope, transport proteins, homing receptors, addressins, regulatory proteins, immunoadhesins, antibodies. Myostatin, TALL proteins, e.g., TALL-I, amyloid proteins, e.g., but not limited to, amyloid beta protein, thymic stromal lymphopoietin ("TSLP"), RANK ligand ("RANKL" or "OPGL"), c-kit, TNF receptors, e.g., TNF receptor type 1, TRAIL-R2, angiopoietins, and biologically active fragments or analogs or variants of any of the foregoing.

[0039] Examples of therapeutic proteins suitable for the methods described herein include abagovomab, abciximab, actoxumab, adalimumab, afelimomab, afutuzumab, alacizumab, alacizumab pegol, ald518, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab mafenatox, anrukinzumab, apolizumab, arcitumomab, acelizumab, altinumab, atlizumab, atolimumab, tocilizumab, bapineuzumab, basiliximab, bavituximab, bectumomab, belimumab, bemarituzumab, Benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bivatuzumab, bivatuzumab mertansine, blinatumomab, brosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab mertansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, cc49, cedelizumab, certolizumab pegol, cetuximab, sitatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, conatumumab , crenezumab, cr6261, dacetuzumab, daclizumab, darotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorlimomab aritox, drozitumab, durigotumab, dupilumab, ecromeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, elotuzumab, ersilimomab, enabatuzumab, enlimomab pegol, enokizumab, enoticumab, encituximab, epitumomab situxetan, epratuzumab, erenumab, erlizumab, ertumaxomab, etaracizumab, Etrolizumab, evolocumab, exibirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, fbta05, felvizumab, fezakinumab, ficlatuzumab, figitumumab, framvotumab, fontolizumab, foralumab, foravirumab, fresolimumab, fullanumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomilikimab, gs6624, ibalizumab,Ibritumomab tiuxetan, Icrucumab, Igovomab, Imciromab, Imgatuzumab, Incracumab, Indatuximab ravtansine, Infliximab, Intetumumab, Inolimomab, Inotuzumab ozogamicin, Ipilimumab, Iratumumab, Itolizumab, Ixekizumab, Keliximab, Labetuzumab, Lebrikizumab, Remaresomab, Lerdelimumab, Lexatumumab, Ribivirumab, Ligelizumab, Lintuzumab, Lirilumab, Lorvotuzumab mertansine, Lucatumumab, Rumiliximab, Mapatumumab, Maslimomab, Mavrilimumab, matuzumab, mepolizumab, metelitumumab, milatuzumab, minretumomab, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-cd3, nacolomab butafenatox, namilumab, naptumomab estafenatox, narunatumab, natalizumab, nebacumab, necitumumab, nerelimomab, nesbacumab, nimotuzumab, nivolumab, nofetumomab merpentane, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumumab, olokizumab, omalizumab Mab, onartuzumab, oportuzumab monatox, oregovomab, olticumab, otelixizumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, panobacumab, palsatuzumab, pascolizumab, pateclizumab, patritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pintumomab, placumab, ponezumab, priliximab, pritumumab, PRO140, kirisumab, racotumomab, radletumab, rafivirumab, ramucirumab, ranibizumab , raxibacumab, regavirumab, reslizumab, rilotumumab, rituximab, lobatumumab, loredumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, samalizumab, sarilumab, satumomab pendetide, secukinumab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, subizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab,Taplitumomab paptox, tefibazumab, terimomab alitox, tenatumomab, tefibazumab, teneliximab, teplizumab, teprotumumab, tezepelumab, TGN1412, tremelimumab, ticilimumab, tildrakizumab, tigatuzumab, TNX-650, tocilizumab, toralizumab, tositumomab, tralokinumab, trastuzumab, TRBS07, tregalizumab, tuko Antibodies such as tuzumab celmoleukin, tubilumab, ublituximab, urelumab, urtoxazumab, ustekinumab, bapaliximab, batelizumab, vedolizumab, veltuzumab, beparimomab, besenkumab, visilizumab, volociximab, borsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, dillalimumab, or zolimomab alitox.

[0040] In some embodiments, the therapeutic protein is a BiTE® molecule. A BiTE® molecule is an engineered bispecific antigen-binding construct that directs the cytotoxic activity of T cells against cancer cells. It contains two single chain variable fragments (scFv) of different antibodies or amino acid sequences from four different genes fused onto a single peptide chain of about 55 kilodaltons. One of the scFvs binds to T cells via the CD3 receptor, while the other binds to tumor cells via a tumor-specific molecule. Blinatumomab (BLINCYTO®) is an example of a BiTE® molecule specific for CD19. Modified BiTE® molecules, such as those modified to extend half-life (e.g., by including a half-life extending moiety such as an Fc polypeptide or albumin), can also be used in the disclosed methods. In various aspects, the polypeptide is an antigen-binding protein, such as a BiTE® molecule. In some embodiments, the antibody protein product comprises a BiTE® molecule.

[0041] Additional examples of therapeutic compounds include synthetic small molecules such as carfilzomib, cinacalcet HCl, etelcalcetide, ivabradine, sotorasib, imatinib mesylate, bortezomib, bicalutamide, gefitinib, venetoclax and docorbicin, and siRNAs such as olpaciran.

[0042] Of course, therapeutic compounds are usually administered to patients in pharmaceutical compositions, which may also be referred to as formulations. Thus, the methods herein can be used to test the effect of skin environment on not only therapeutic compounds themselves, but also pharmaceutical compositions that contain therapeutic compounds. Thus, whenever a "therapeutic compound" is described herein, it is understood that the methods described herein can also be carried out on the corresponding pharmaceutical composition that contains therapeutic compounds. For example, the methods described herein can be used to investigate the effect of skin environment on different components, amounts and conditions (e.g., pH, viscosity, osmolality and osmolality, etc.) of therapeutic compound formulations, for the development and selection of suitable formulations.

[0043] In some embodiment methods, the therapeutic compound is in a pharmaceutical composition, which may also be referred to as a "formulation." The pharmaceutical composition may be a pharma- ceutically acceptable formulation. The pharmaceutical composition may include the therapeutic compound together with a pharma- ceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative, and / or adjuvant.

[0044] Acceptable pharmaceutical composition materials for the therapeutic compounds described herein are preferably non-toxic to the recipient at the dosages and concentrations employed. In certain embodiments, pharmaceutical compositions may contain formulation materials to alter, maintain or preserve, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium bisulfite); buffers (such as borate buffer, bicarbonate buffer, Tris-HCl, citrate buffer, phosphate buffer, or other organic acid buffers); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, sucrose, mannose, or dextrin); proteins (such as serum albumin, gelatin, or immunoglobulins); colorants, flavorings, and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone and the like); low molecular weight polypeptides; salt forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as Pluronic, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapol, and the like); stability enhancers (such as sucrose or sorbitol); tonicity enhancers (such as alkali metal halides, preferably sodium or potassium chloride, mannitol, sorbitol, and the like); delivery vehicles; diluents; additives and / or pharmaceutical adjuvants.See, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, (AR Gennaro, ed.), 1990, Mack Publishing Company.

[0045] Vehicles or carriers suitable for pharmaceutical compositions can be water for injection, physiological saline or artificial cerebrospinal fluid, optionally supplemented with other materials common in compositions for parenteral administration. Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles. In certain embodiments, the pharmaceutical composition comprises a Tris buffer of about pH 7.0-8.5, or an acetate buffer of about pH 4.0-5.5, and may further comprise sorbitol or a suitable substitute thereof.

[0046] The pharmaceutical composition components are preferably present in concentrations that are acceptable to the site of administration. In certain embodiments, a buffer is used to maintain the composition at or slightly below physiological pH, typically within a pH range of about 4 to about 8. Within this pH range, buffers include about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 8.9, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8 ... These include about 0.7, about 7.8, about 7.9 and about 8.0, as well as ranges between any two of the recited values, such as pH 4.2-8, pH 4.2-7, pH 4.2-6, pH 4.2-5, pH 4.8-8, pH 4.8-7, pH 4.8-6, pH 4.8-5, pH 5-8, pH 5-7, pH 5-6, pH 5.1-8, pH 5.1-7, pH 5.1-6, pH 5.5-8, pH 5.5-7, pH 5.5-6, pH 6-7 or pH 6-8.

[0047] It should be noted that some therapeutic compounds may be self-administered by the subject directly or via an automatic injector (such as an on-body injector), and some may be administered to the subject by another individual, such as a healthcare provider. Thus, the subject therapeutic compounds or pharmaceutical compositions comprising same may be suitable for self-administration by the subject to himself (directly or via a device such as an automatic injector) and / or for administration to the subject by another individual, such as a healthcare provider. For some methods described herein, the pharmaceutical composition comprising the therapeutic compound may be provided in an administration device, such as a pre-filled syringe, an automatic injector, or an IV bag system including a needle in fluid communication with an IV bag.

[0048] Molecular properties It should be understood that the "molecular characteristics" of a therapeutic compound, such as a therapeutic protein, refer to one or more chemical or structural characteristics of the therapeutic protein, which may vary. Examples of molecular characteristics include acidic species, basic species, high molecular weight species, sub-visible particle count, low molecular weight, medium molecular weight, glycosylation (such as non-glycosylated heavy chain or high mannose), non-heavy and light chains, deamidation, deamination, cyclization, oxidation, isomerization, fragmentation / clipping, N-terminal and C-terminal variants, reduced species and partial species, folding structure, surface hydrophobicity, chemical modification, covalent bonds, C-terminal amino acid motif PARG and / or C-terminal amino acid motif PAR-amide. It should be noted, by way of example, that therapeutic proteins and small molecules have been observed to aggregate, and nucleic acids have been observed in multiplexes, which may be briefly considered as a type of "aggregate".

[0049] How to Select Dosage Conditions In some embodiments, a method is described for selecting administration conditions for a therapeutic compound product for subcutaneous administration. As used herein, "administration conditions" refers to at least one parameter that may vary when a therapeutic compound (e.g., as part of a pharmaceutical composition) is administered. Examples of administration conditions include one or more of the formulation components, concentration, volume, pH, temperature, viscosity, osmolality, molecular properties, route of administration, and / or administration device or method of using the administration device (e.g., frequency, flow rate, parameters) of said therapeutic compound. For example, administration conditions may include formulation additives, buffers, surfactants, and / or stabilizers, which may vary depending on the identity and / or concentration of these substances, and / or administration conditions may also include pH. Of course, where applicable, administration conditions may be specified qualitatively (e.g., route of administration) or quantitatively (e.g., as a numerical value or range). For illustration, an example method of elucidating the behavior of a therapeutic compound in an in vitro mammalian skin environment of some embodiments is illustrated in Figures 1A-B.

[0050] An exemplary method of selecting an administration condition for a therapeutic compound product for subcutaneous administration in connection with some embodiments is illustrated in FIG. 1C. The method may include providing an in vitro living mammalian skin including a dermal layer and an epidermal layer defining an interface therebetween (i.e., between the dermal layer and the epidermal layer) 110. The method may further include administering a therapeutic compound product to the interface under administration conditions 120. The method may further include detecting a physiochemical and / or clinical property of the therapeutic compound product at the interface 130. For example, the detecting step may be optical and / or may include analytical testing of the therapeutic compound. The method may further include selecting or rejecting the administration condition for further development based on the detected physiochemical and / or clinical property of the therapeutic compound 140. For example, an administration condition with a lower incidence of HMW species may be selected over an administration condition with a higher incidence of HMW species. "Further development," as used herein, refers to additional characterization, manipulation, and / or testing of a therapeutic compound, such as protein engineering, formulation selection and / or development, animal model testing, clinical trials, manufacturing process development, and / or regulatory submissions. It will be appreciated that one or more of the described method parts may be repeated, omitted, or performed in a different order, as appropriate to the circumstances.

[0051] It will be appreciated that the method of selecting the administration conditions can detect one or more physiochemical and / or clinical properties of the therapeutic compound, including changes in the physiochemical and / or clinical properties. For example, the physiochemical and / or clinical properties can include bioavailability, pharmacokinetic properties, aggregation, precipitation, distribution, diffusion rate, retention and / or absorption. By way of example, the method can detect the absolute amount, the relative amount, changes therein, or the degree of change in one or more physiochemical and / or clinical properties of the therapeutic compound, if applicable and appropriate in the circumstances. The therapeutic compound product may acquire the physiochemical and / or clinical properties after administration.

[0052] In some methods of selecting dosing conditions for a therapeutic compound product for subcutaneous administration, the method is repeated with two or more different dosing conditions, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 different dosing conditions. Optionally, the method can be performed iteratively to optimize the dosing conditions (e.g., iteratively testing a narrower and narrower range of dosing conditions).

[0053] Methods for determining the effect of molecular properties of therapeutic compounds on physiochemical and / or clinical properties - Patents.com In some embodiments, methods are described for determining the effect of the molecular properties of a therapeutic compound on the physiochemical and / or clinical properties of that therapeutic compound following subcutaneous administration.

[0054] In connection with some embodiments, an exemplary method for determining the effect of a molecular signature of a therapeutic compound on the physiochemical and / or clinical properties of the therapeutic compound after subcutaneous administration is illustrated in FIG. ID. The method may include providing an in vitro living mammalian skin including a dermal layer and an epidermal layer defining an interface therebetween (i.e., between the dermal layer and the epidermal layer) 110. The method may further include administering a therapeutic compound product including the molecular signature to the interface 122. It is contemplated that the administered therapeutic compound may be stressed, for example, by exposure to high temperature, repeated freeze-thaw cycles, extreme pH, forced oxidation (e.g., by an oxidizing agent such as H2O2), physiological pH, and / or UV light. Such stress may cause the molecular signature to be present in the therapeutic compound or may increase the relative abundance of the molecular signature compared to a non-stressed control. Thus, in some examples of this method, the therapeutic compound may be stressed prior to administration. The method may include detecting physiochemical and / or clinical properties of the therapeutic compound at the interface. 130 For example, the detecting may be optical and / or may include analytical testing of the therapeutic compound. In some methods of determining the effect of molecular properties of the therapeutic compound on the physiochemical and / or clinical properties of the therapeutic compound, the physiochemical and / or clinical properties include bioavailability, pharmacokinetic properties, aggregation, precipitation, distribution, diffusion rate and / or absorption. It will be appreciated that one or more of the described method portions may be repeated, omitted, or performed in a different order, as appropriate to the circumstances.

[0055] Methods for determining changes in molecular properties In some embodiments, a method for detecting a change in a molecular profile of a therapeutic compound following subcutaneous administration is described.

[0056] In connection with some embodiments, an exemplary method for detecting a change in a molecular characteristic of a therapeutic compound after subcutaneous administration is illustrated in FIG. 1E. The method may include providing an in vitro live mammalian skin including a dermal layer and an epidermal layer defining an interface therebetween (i.e., between the dermal layer and the epidermal layer) 110. The method may include administering a therapeutic compound including a characteristic to the interface, the therapeutic compound including a first state of the molecular characteristic 124. Moving to block 134, the method may further include detecting a second state of the molecular characteristic of the therapeutic compound at the interface 134. Of course, one or more of the described method portions may be repeated, omitted, or performed in a different order, as appropriate to the circumstances. The "first state" and "second state" refer to two states of the molecular characteristic that may be measured. For example, the first state and second state of the molecular characteristic may include any of the concentration, amount, distribution, or distribution at the interface for the therapeutic compound. For example, the first state can refer to a first measurement of the relative amount of high molecular weight (HMW) species prior to administration to the interface (or shortly after administration to the interface), and the second state can refer to a second measurement of the relative amount of high molecular weight (HMW) species after administration to the interface.

[0057] The value (quantitative and / or qualitative) of the first state may be different from the value of the second state, or both values ​​may be the same. In some methods described herein, the first state of the molecular characteristic has a first value, and the second state of the molecular characteristic has a second value that is the same or different from the first state. By way of example, if the first state and the second state are not different (or are not statistically significantly different), it may be determined that there is no change in the molecular characteristic after subcutaneous administration. In some examples of this method, the first state of the molecular characteristic refers to the state of the molecular characteristic before or immediately after administration into the subcutaneous space. The second state of the molecular characteristic refers to the state of the molecular characteristic in the subcutaneous space. The second state may refer to the state of the molecular characteristic after the molecular characteristic has been in the interface for a period of time, e.g., after incubation for several seconds, minutes, or hours. The period of time may be determined before performing this method. In some embodiment methods, the second state is the state of the therapeutic compound after being incubated within the interface for a specified period of time, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 30, 45, or 60 minutes, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 36, 48, 60, 72, 84, 96, or 120 hours. For example, the specified time may be 1 minute or less, 2 minutes or less, 3 minutes or less, 4 minutes or less, 5 minutes or less, 6 minutes or less, 7 minutes or less, 8 minutes or less, 9 minutes or less, 10 minutes or less, 15 minutes or less, 30 minutes or less, 45 minutes or less, or 60 minutes or less, or 1 hour or less, 2 hours or less, 3 hours or less, 4 hours or less, 5 hours or less, 6 hours or less, 7 hours or less, 8 hours or less, 9 hours or less, 10 hours or less, 11 hours or less, 12 hours or less, 18 hours or less, 24 hours or less, 36 hours or less, 48 ​​hours or less, 60 hours or less, 72 hours or less, 84 hours or less, 96 hours or less, 120 hours or less, 144 hours or less, 168 hours or less, 192 hours or less, 216 hours or less, or 240 hours or less. For example, the specified time can be 1-48 hours, 1-96 hours, 1-120 hours, 12-48 hours, 12-96 hours, 12-120 hours, 24-48 hours, 24-96 hours or 24-120 hours. Incubation can be carried out at a specific temperature, for example, 20°C, 25°C, 33°C (which is a typical human skin temperature) or 37°C.In some embodiment methods, the second state is a state of the therapeutic compound at a different temperature than the first state. For example, the first state can be a temperature selected from 20° C., 25° C., 33° C., and 37° C., and the second state can be a different temperature selected from 20° C., 25° C., 33° C., and 37° C., such that the second state is a different temperature than the first state. By way of example, the detecting step can be optical or can include an analytical test of the therapeutic compound as described herein.

[0058] It is believed that exposure to the skin environment, in addition to the effects on bulk solution behavior, may result in changes in critical product quality attributes that affect drug quality and efficacy. Injection site contact with components may result in new interactions that promote modifications (e.g., oligomerization, redox reactions, conformational changes) that affect drug efficacy, immunogenicity, solubility, and stability. Thus, the method may include a step of evaluating and predicting potential changes in molecular properties after sustained exposure to an in vitro mammalian skin environment. The simplicity of this approach allows the method described herein to be used as a platform to optimize formulations and explore strategies to maintain advantageous product quality attributes.

[0059] In some embodiments of the method of detecting a change in a molecular characteristic, the molecular characteristic comprises at least one of acidic species, basic species, high molecular weight species, sub-visible particle count, low molecular weight, medium molecular weight, glycosylation (such as non-glycosylated heavy chain or high mannose), non-heavy and light chains, deamidation, deamination, cyclization, oxidation, isomerization, fragmentation / clipping, N-terminal and C-terminal variants, reduced species and partial species, folding structure, surface hydrophobicity, chemical modification, covalent bond, C-terminal amino acid motif PARG or C-terminal amino acid motif PAR-amide.

[0060] To investigate how molecular properties may change after incubation in the skin environment, therapeutic compounds may be exposed to an in vitro mammalian skin model containing either dermis or subcutaneous tissue. In some embodiment methods, exposure is performed by "basal exposure" or "injection exposure" as illustrated in FIG. 3A. Basal exposure has the advantage of being simple, but it may be more difficult to distinguish minority variants from the majority forms present in the bulk solution. Injection exposure more closely simulates subcutaneous injection, but may pose challenges when diffusion issues exist. In both strategies, supernatants are collected over time and may be evaluated using one or more analytical techniques (e.g., SE-HPLC, rCE-SDS, CEX-HPLC, HIAC, nrCE-SDS, mass spectrometry, mass spectrometry microscopy and / or mass spectroscopy). An important consideration is the compatibility of the medium for both the skin model and the analytical measurements. The medium used must simultaneously maintain the skin model for the duration of the experiment and not interfere with the measurements of interest. For example, the presence of high molecular weight (HMW) species and subvisible particles (SbVPs) in the medium can cause background interference in size exclusion chromatography, high performance liquid chromatography (SEC-HPLC) and high accuracy (HIAC) measurements. Thus, in some methods described herein, the medium can be pre-filtered using a centrifugal filter with a cutoff of 50 KDa or less (e.g., 40 KDa or less, 30 KDa or less, or 25 KDa or less). For example, to quantify the high molecular weight (HMW) species present using SE-HPLC, the medium can be pre-filtered (e.g., using a 50 kDa cutoff centrifugal filter) to reduce the presence of materials that interfere with quantification as shown in the SE-HPLC chromatogram shown in Figure 3B. As an alternative to vendor-supplied media for the skin model, custom media can be used to more closely mimic physiological conditions for the skin environment during subcutaneous injection (e.g., addition of hyaluronic acid, use of blisters / subcutaneous fluid).These custom media cannot be optimized to maintain skin models for long periods of time (>2 days), but may provide more relevant insight into interactions that occur immediately after subcutaneous injection (≦1 day time scale). These custom media may also reduce interference with analytical measurements (interference may be reduced compared to conventional commercial media). For example, serum-free media can be used to minimize or avoid serum proteins from interfering with analytical measurements.

[0061] Further aspects of the method For any of the methods described herein, it will be understood that the method may further comprise or be further characterized by one or more of the additional aspects described herein.

[0062] For any of the methods described herein, the step of providing an in vitro mammalian skin may include immobilizing the in vitro mammalian skin between a proximal substrate and a distal substrate, for example, the proximal substrate and the distal substrate each comprising glass, such as a coverslip.

[0063] For any of the methods described herein, the step of preparing the in vitro mammalian skin may include preparing a dermal layer of the in vitro mammalian skin on a porous substrate that includes pores sized to accommodate diffusion of the therapeutic compound product. Thus, the step of administering the therapeutic compound product may include fluidly connecting the porous substrate with a solution that includes the therapeutic compound product. Examples of porous substrates include polycarbonate and polyester filter membranes. It is contemplated that the porous substrate allows the protein therapeutic product to diffuse into the subcutaneous space of the modeled in vitro mammalian skin, eliminating the need for administration by injection. Thus, in some embodiments, the dermal layer of the in vitro mammalian skin may be placed on a porous substrate, and the therapeutic compound product may be administered by simply contacting the porous substrate with a solution that includes the therapeutic compound product, thus allowing the therapeutic compound to be administered to the interface between the dermal layer and the epidermal layer. For example, the in vitro mammalian skin and the porous substrate may be immersed in the solution. In some embodiments, the method further comprises disposing in vitro mammalian skin on the porous substrate.

[0064] For any of the methods described herein, the administering step may include pressing a needle including a cannula against the surface of the dermis layer, thus inserting the needle into the interface of the in vitro live mammalian skin. The method may further include disposing a therapeutic compound through the needle into the interface. For example, a volume of a therapeutic compound (e.g., as part of a pharmaceutical composition) may be administered to the in vitro mammalian skin. In some embodiment methods, the needle is pressed against the surface of the dermis at an acute angle, for example, at an angle of about 45 degrees or less. It should be understood that in the case of in vitro mammalian skin, pressing the needle at an acute angle can prevent or minimize damage or tearing of the skin. In some embodiment methods, for example, when the skin is relatively thick, the method may include pressing the needle at a perpendicular angle or near a perpendicular angle to the surface of the dermis. In some methods described herein, the needle includes an opening in fluid communication with the cannula, and the administering step includes directing the opening into the dermis layer. In some embodiment methods, the needle remains in place at the interface, and the therapeutic compound is administered until a sufficient volume of the therapeutic compound is administered at the interface. The sufficient volume of the therapeutic compound can be determined by any suitable method, for example, by measuring the bolus of material extruded from the needle at the injection site. For example, the needle can remain in place in the skin model to detect (for example, by optical imaging) the therapeutic compound product described herein, so that in some embodiments, the injection site can be located and monitored throughout the injection period.

[0065] For any of the methods described herein, the in vitro mammalian skin may contain dermis and / or subcutaneous tissue. In the case of such in vitro mammalian skin, the therapeutic compound may be administered, for example, by injection into the in vitro mammalian skin containing dermis and / or subcutaneous tissue (these layers are typically exposed to the therapeutic agent after subcutaneous injection, see FIG. 1A). The reproducibility of this injection may be further enhanced by an injection device that allows fine adjustment of needle puncture, penetration depth and injection volume. Such a device may allow injection to be targeted at a specific location at various angles to accommodate different dimensions of in vitro mammalian skin.

[0066] For any of the methods described herein, the administering step may include soaking, diffusing and / or transdermal administration of the therapeutic compound product through the dermis layer and / or into the subcutaneous tissue layer.

[0067] For any of the methods described herein, the detecting step may include optical imaging through the dermis layer. For any of the methods described herein, the detecting step may include imaging and / or analytical testing.

[0068] For any of the methods described herein, the method may further comprise the step of detecting the presence of sub-visible levels of particles in the solution after said administering step.

[0069] Of course, any of the methods described herein may include analytical testing of the therapeutic compound (or a composition including the therapeutic compound, such as a pharmaceutical composition, or in vitro mammalian skin including the therapeutic compound). By way of example, the analytical testing may include one or more of mass spectrometry, chromatography, electrophoresis, spectroscopy, light obscuration, particle methods (such as nanoparticle / visible / micron sized resonant mass or Brownian motion), analytical centrifugation, imaging or image characterization, immunoassays, SE-HPLC, rCE-SDS, CEX-HPLC, HIAC, nrCE-SDS, mass spectrometry microscopy, and / or mass spectroscopy. Techniques such as mass spectrometry microscopy may investigate the distribution of chemical species in in vitro mammalian skin. In some methods, analytical testing may be performed before administration of the therapeutic compound and / or after administration of the therapeutic compound. For example, the "detecting step" of any of the methods described herein may include analytical testing. For any of the methods described herein, the method may further include performing analytical testing on the therapeutic compound prior to the administering step to ascertain a baseline state of one or more molecular characteristics, for example, prior to administration to an in vitro mammalian skin interface. For any of the methods described herein, the analytical testing may be performed over time. For example, samples containing the therapeutic compound may be collected periodically following administration of the therapeutic compound to the interface, and the samples may be analyzed with one or more analytical techniques described herein. Thus, a change (or lack of change) in one or more molecular characteristics following administration to the skin environment may be determined.

[0070] For detection after administration of the therapeutic compound, several techniques can be applied. In the case of the methods described herein, detection can characterize the distribution of the therapeutic compound in the skin and its ability to diffuse into tissues (FIG. 1A). To elucidate the possibility of precipitation and accumulation in in vitro mammalian skin, the in vitro mammalian skin injected with the labeled therapeutic compound can be visualized using fluorescent imaging (e.g., multiphoton or confocal microscopy). To observe colocalization, the structures and cells of the skin model can also be fluorescently labeled. Since the methods described herein include placing the therapeutic compound at an interface, the imaging techniques described herein can image through the dermal layer. For example, the lasers used in conventional confocal microscopy can damage in vitro mammalian skin, and the signal emitted by some fluorescent dyes may overlap with the background fluorescence of the molecule / substance being visualized. Thus, in some methods described herein, the detecting step includes imaging using multiphoton laser scanning microscopy (also referred to herein as "multiphoton microscopy"). Without being limited to any particular theory, multiphoton microscopy can create a gap between the excitation energy from autofluorescence and the excitation energy from the fluorescent dye, thus minimizing the overlap of the dye with background fluorescence. Furthermore, multiphoton microscopy can minimize damage to in vitro mammalian skin tissue by pulsing photons from different directions so that the tissue does not receive as much energy. Multiphoton microscopy allows non-invasive deep imaging (up to about 1 mm) of thick live tissue specimens with high resolution. Tiling of images in multiphoton microscopy also allows for the observation of a wider field of view. Such images provide direct, qualitative insight into the behavior of the bulk of the therapeutic formulation in the skin environment. Thus, in some embodiment methods, the therapeutic compound is labeled. For example, in some embodiments, the therapeutic compound is labeled with a fluorescent dye that emits a signal that does not overlap with any background fluorescence (e.g., from collagen, which is naturally autofluorescent).Exemplary fluorescent dyes include pH-insensitive dyes, such as ALEXA™ Fluor 488. In other embodiments, the detecting step includes the use of second harmonic generation (SHG) imaging (also known as “frequency doubling”), which is a nonlinear optical imaging method induced by an intense incident laser source, such as a multiphoton laser line. In SHG, two photons of the incident laser are mixed in the tissue and converted into a single photon with twice the energy and frequency levels. SHG imaging is used in the art to visualize macromolecular structures such as extracellular matrix (ECM) components and fibrous collagen (see, for example, Xie et al., Current Protocols in Cytometry, 6.33.1-6.33.11, July 2012). Without being bound to a particular theory, SHG may be able to identify the optimal focal plane for imaging. By way of example, a multiphoton laser wavelength of about 850 nm, such as about 830-870 nm or about 840-860 nm, may be suitable for generating second harmonic collisions for SHG imaging.

[0071] In some examples of the methods described herein, after administration, various quantitative analyses can be applied to the signal from the fluorescently labeled therapeutic compound to characterize the distribution and diffusivity of the biologic after injection. For example, (1) the distribution of the injected biologic can be measured from the intensity profile of the fluorescent signal, (2) Fluorescence Recovery After Photobleaching (FRAP) can be applied to measure the diffusion coefficient of the injected biologic solution, and / or (3) measurement of the fluorescent signal in the medium can provide insight into the diffusion dynamics of the biologic out of the injection site. Exemplary schematics of these techniques are shown in FIG. 1A (bottom panel "C").

[0072] For some examples of the methods described herein, the detecting step includes CT scan or magnetic resonance imaging. For some methods described herein, the detecting step includes optical imaging. The optical imaging may be through the dermis layer. Thus, in any of the methods described herein, the therapeutic compound may be labeled. Of course, to achieve proper detection, only a fraction of the administered therapeutic compound may be labeled. Labeling may facilitate detection of the therapeutic compound and its physiochemical and / or clinical properties. Thus, any of the methods described herein may further include labeling the therapeutic compound with a detectable moiety, such as a fluorophore, a fluorescent dye, a radiolabel, or a quantum dot. The therapeutic compound may be labeled, for example, using an amine-reactive crosslinker (e.g., via an NHS-ester or SDP-ester-linked fluorophore).

[0073] To enhance detection of the therapeutic compound, a background signal (e.g., a signal from in vitro mammalian skin obtained prior to administration of the therapeutic compound) may be subtracted. In some embodiment methods, the detecting step includes obtaining a background signal and subtracting the background signal.

[0074] Further embodiments Described herein are applications that use in vitro mammalian skin to investigate the effects of the skin environment on therapeutic compounds. In some applications, the therapeutic compound (or pharmaceutical composition containing the therapeutic compound) is first exposed to a model of the deeper layers of the skin (dermis or subcutaneous tissue) to simulate the local conditions after subcutaneous injection. Some applications include investigating the aggregation and distribution of biologics after injection into the skin environment. Some applications provide a deeper look into the physicochemical modifications and changes in other molecular properties that occur when the drug product is maintained within the skin environment. Taken together, these approaches provide an opportunity to gain insight into drug-skin interactions associated with subcutaneous injection. The simplicity of these in vitro approaches allows for the implementation of screening strategies that can be used to optimize therapeutic compounds, formulations and injection strategies to improve the quality, efficacy and manufacturability of subcutaneously injected drug products. For example, these methods can be used to develop and select subcutaneous formulations of antibody protein products, such as BiTE® molecules, that contain half-life extending moieties.

[0075] The use of in vitro mammalian skin to assess the effect of the skin environment on a therapeutic compound (or a pharmaceutical composition containing the therapeutic compound) can involve two general steps: (1) drug exposure and (2) signal readout. The details of these two steps can be tailored to suit a particular application.

[0076] For example, the use of the methods described herein can include the evaluation of bulk solution behaviors such as aggregation and diffusion. A labeled therapeutic compound (such as fluorescently labeled) is injected into the skin model. Detection (e.g., imaging such as fluorescence imaging) is applied to directly observe the distribution and aggregation behavior of the therapeutic compound in the in vitro mammalian skin. Protein concentration in the medium outside the in vitro mammalian skin can be monitored to characterize the diffusion of the biologic from the injection site. These techniques can be applied in screening strategies to minimize aggregation and achieve optimal distribution for drug bioavailability.

[0077] For example, applications can include evaluation of property changes (e.g., high molecular weight (HMW), sub-visible particles, and / or chemical changes). In vitro mammalian skin can be used to determine changes in molecular properties of therapeutic compounds that occur following skin exposure. Analytical techniques (e.g., size exclusion chromatography (SEC-HPLC), capillary electrophoresis, cation / anion exchange, mass spectroscopy, HIAC particle counting) can be applied to quantify changes in molecular properties over time. Therapeutic compounds and formulations can be screened and optimized to maintain advantageous property levels and conditions. EXAMPLES

[0078] Example 1: Bulk solution behavior (e.g., aggregation and diffusion) To demonstrate how in vitro mammalian skin can be used to elucidate protein aggregation within the dermal environment, several fluorescently labeled monoclonal antibody (mAb) solutions with various pH-sensitive aggregation tendencies were injected into the skin model and imaged using multiphoton microscopy. As a positive control, a preaggregated solution of mAb3 (prepared by applying an agitation stress) was injected into the skin model (Figure 2A). Preaggregated mAb3 was visualized as bright spots concentrated near the injection site (which can be identified by the dark areas in the image indicating tissue trauma due to injection). No diffuse fluorescence was observed outside of these areas, indicating that aggregation was not reversible and limited the ability of the mAb to diffuse throughout the tissue. To determine whether aggregation could be observed within the skin model, mAb1 (a mAb known to strongly aggregate at neutral pH) was injected into the skin model (Figure 2B). Bright spots concentrated mainly near the injection site were observed, confirming that mAb1 aggregated within the skin model after injection. Diffuse fluorescence was observed, indicating that some mAb1 remained unaggregated and was able to diffuse throughout the skin model. mAb2, a mAb with mild pH sensitivity, was also injected into the skin model (Figure 2C). The presence of diffuse scattering and fewer bright spots indicates that this mAb aggregates only slightly when injected. Finally, no signs of aggregation were observed when mAb3, a mAb with no pH sensitivity, was injected into the skin model (Figure 2D). The results summarized in Figures 2A-D show how the skin model can be used to screen biologic solutions for their propensity to aggregate when injected within the skin environment. Continued development will allow the application of the quantitative analysis shown in Figure 1A (bottom panel "C").

[0079] Example 2: Alteration of molecular properties To demonstrate how the skin model can be used to evaluate changes in properties due to skin exposure, an experiment was performed comparing the levels of high molecular weight (HMW) species and subvisible particles (SbVPs) for two formulations of the same therapeutic protein (mAb3). The concentration of mAb3 in the higher concentration formulation (100 mg / mL, "HC") was twice that present in the lower concentration formulation (50 mg / mL, "LC"). In addition, the two formulations differed in buffer composition (lactate and acetate buffers for the HC and LC formulations, respectively). Both formulations were diluted to the same final concentration (5.0 mg / mL) using culture medium and exposed to the skin model using the "basal exposure" as illustrated in Figure 4A. The culture medium used for dilution was filtered using a 50 kDa cutoff centrifugal filter to remove proteins with sizes that would interfere with quantification of the main and HMW peak species (Figure 3B). Filtered media showed no background interference in SEC-HPLC for either the reference antibody or the BiTE® molecule containing a half-life extending moiety. Thus, it was observed that filtering the media allows the skin model described herein to characterize the changes in HMW and SbVP during exposure. Filtered media also showed a manageable background for HIAC analysis.

[0080] Small aliquots of the exposed biologic solutions were collected over times ranging from 0.1 to 24 hours and analyzed using size exclusion HPLC. The % HMW species detected are compared in the table shown in Figure 4B. For these product lots, the initial level of HMW for the LC formulation drug substance (DS) was slightly higher (0.39%) compared to the HC formulation (0.23%), but upon dilution, the levels of HMW were similar (0.26 and 0.23, respectively). For both formulations, the level of HMW increased slightly over 24 hours, but in a similar manner, reaching final levels of 0.30% and 0.31% for the LC and HC formulations, respectively. The lack of difference in the rate of HMW formation between the LC and HC formulations (Figures 4B and 4C) indicates that the HC formulation does not pose an increased risk of HMW formation compared to the LC formulation. This agreement is highlighted by the overlap of the 95% confidence intervals for each fit (dashed and dotted lines in Figure 4C). It should be noted that the level of HMW is likely concentration sensitive, decreasing as the drug product enters the circulation from the injection site area and is diluted. An example of this effect is seen in the LC formulation, where the level drops from 0.39% in the DS to 0.26% after a 10-fold dilution (50-5 mg / mL, Figure 4B, Table 2). The exposure strategy applied here can be used to reveal changes in the properties of the biologic maintained within the injection site or to represent scenarios of what may occur if proper drug distribution is inhibited. These results showing similarity between the two formulations suggest that there is no additional risk of HMW species in the higher concentration HC formulation compared to the lower concentration LC formulation when administered at the same dose.

[0081] The levels of subvisible particles (2-150 μm SbVP) were monitored after basal exposure as illustrated in Figure 4A. Media was collected over 24 hours and subvisible particles were quantified using a HIAC liquid particle counter. The number of particles detected at each time point, 3, 6 and 24 hours for both the HC and LC formulations, are shown as symbols in Figure 4D. A similar increase was detected for both formulations. Of note, the number of particles detected after 24 hours for the skin model exposed to formulation buffer alone without biologic (square symbols and grey dashed line) was similar to the number of particles reached after 24 hours for the skin model exposed to the biologic. This suggests that the majority of these particles originate from the skin model and / or the media and are unrelated to the biologic. The size distribution of the particles is shown below each data point and is similar for the skin models exposed to both formulations. The agreement of these results suggests that the skin environment had a broadly comparable effect on these two formulations and that there was no added risk of subvisible particle formation between the two formulations of mAb3.

[0082] Example 3: In vitro aggregation and alteration of molecular properties in additional therapeutic proteins To demonstrate the use of an in vitro mammalian skin model to investigate various factors of therapeutic protein solutions that may affect the extent of aggregation and HMW species formation due to exposure to the skin environment, the skin model was exposed to various therapeutic proteins with different targets, therapeutic protein formats, buffer compositions, pH and initial property levels. Four different therapeutic proteins were investigated: (1) an antibody protein product with a half-life extension moiety (Therapeutic protein 1, TP1), (2-3) two antibody protein product molecules, each with specificity for two different targets (Therapeutic protein 2 and Therapeutic protein 3, i.e., TP2 and TP3, respectively), and (4) a mAb (mAb1), which is known to aggregate strongly at neutral pH. For Therapeutic protein 1, two different formulations were used, i.e., a high concentration (HC) formulation (20 mg / mL) and a low concentration (LC) formulation (1 mg / mL), each with different buffer compositions, but formulated at pH 4.2. Additionally, for the HC formulation of Therapeutic protein 1, the protein solution was prepared in two different ways. The proteins were formulated in two different conditions: unstressed and stressed with 5x freeze / thaw (F / T) cycles, the latter of which was known to elevate the levels of HMW species. The other proteins (Therapeutic protein 2, Therapeutic protein 3, mAb1) were formulated at different protein concentrations (0.8, 2.5, and 20 mg / mL, respectively), buffer compositions, and pH (7.0, 6.0, and 5.2, respectively) and were prepared without any additional stress. The in vitro mammalian skin model was basally exposed to these protein solutions by adding them to the culture medium to a final dilution factor of 10x or 2x from their initial formulated concentration.

[0083] The in vitro mammalian skin model was incubated for 24 hours and small aliquots were withdrawn from the medium over the 24 hours to monitor the levels of HMW species and SbVP. The %HMW species detected for these protein solutions when exposed at 2x and 10x dilutions are shown in Figures 5A and 5B, respectively. At both 2x and 10x dilutions, Therapeutic protein 1 in HC formulations prepared with 5xF / T stress had higher initial levels of %HMW compared to the unstressed samples of the same formulations, but plateaued at similar levels after about 6 hours. At 2x dilution, the %HMW levels of Therapeutic protein 1 HC formulations plateaued at a higher level (8%) compared to Therapeutic protein 1 LC formulations (2%). However, at 10x dilution, Therapeutic protein 1 HC formulations plateaued at a similar level to Therapeutic protein 1 LC formulations (2%). This suggests that the final levels of %HMW formed for Therapeutic protein 1 are concentration dependent. At 2x dilution, %HMW increased for both Therapeutic protein 2 and Therapeutic protein 3, with Therapeutic protein 2 increasing at a slightly higher rate despite the lower concentration (0.8 vs. 2.5 mg / mL). This suggests that in some cases, the physicochemical properties of the protein and formulation buffer may have a greater influence on the formation of HMW species than the protein concentration. The %HMW detected for mAb1, known to aggregate at neutral pH, initially decreased and plateaued at 4% at 2x dilution and 2% at 10x dilution. Notably, %HMW generally increased upon exposure to the skin model and media for most proteins, with the exception of mAb1 at both dilutions and HC Therapeutic protein 1 (5xF / T) at 10x dilution. This suggests that the level of %HMW formed within the skin environment may depend on both the nature of the multimerization and the concentration within the skin environment.

[0084] After incubating the therapeutic protein solution with the skin model for 24 hours, the levels of subvisible particles (2-150 μm SbVP) present in the medium were quantified using a HIAC liquid particle counter. Particle concentrations are shown in Figure 5C. For skin models exposed to 20 mg / mL (HC) formulations of Therapeutic Protein 1, 5×F / T and unstressed, at a 2× dilution, approximately 1,000,000 particles / mL were detected by 1.5 hours, with the proportion of larger sized particles increasing over 24 hours. For 1 mg / mL (LC) Therapeutic Protein 1, the number of particles detected at the same 2× dilution was 100-fold lower (approximately 10,000 particles / mL). However, when the skin model was exposed to a 20 mg / mL (HC) Therapeutic Protein 1 formulation at a 10× dilution, similar numbers of particles (approximately 10,000 particles / mL) were measured for both the HC and LC formulations of Therapeutic Protein 1. Without being limited by theory, this suggests that for Therapeutic Protein 1, particle formation is strongly concentration dependent. In the medium from skin models exposed to HC Therapeutic Protein 1 formulation buffer, there were approximately 10,000 particles / mL, indicating that this may be the baseline level of SbVP formed from the skin model / medium system when exposed to formulation buffer. Thus, without being limited by theory, for 20 mg / mL (HC) Therapeutic Protein 1, a 10x dilution may be sufficient within the skin environment to reduce the formation of SbVP due to local interactions at the injection site. For skin models exposed to mAb1, which was known to strongly aggregate at neutral pH, aggregation remained at high levels, with approximately 1,000,000 particles detected at both 2x and 10x dilutions. For skin models exposed to Therapeutic Protein 2 and Therapeutic Protein 3 formulations at 2x dilutions, the levels of SbVP detected in the medium were approximately 3000 and 50,000 particles / mL, respectively, and remained fairly stable over 24 hours. Overall, these results suggest that the propensity to form SbVPs within the skin environment may vary for different therapeutic compound formulations and may be concentration-dependent depending on the nature of the aggregation.This experiment demonstrates how an in vitro mammalian skin model can be used to screen formulation conditions and delivery strategies to reduce the risk of aggregation at the injection site.

[0085] To elucidate the in vitro aggregation of therapeutic proteins injected into an in vitro mammalian skin model, proteins were fluorescently labeled, injected into an in vitro mammalian skin model, and imaged using multiphoton microscopy, as shown in Figures 1A-1B. In this setup, an injection needle was inserted into the skin model and remained embedded for the duration of the experiment. This allowed the injection site to be located and monitored throughout the injection process. A syringe pump was used to control the injection volume and the fluorescently labeled proteins were injected into the skin model, allowing imaging of both collagen structures and the fluorescently labeled proteins within the skin model by multiphoton microscopy. Images of the injection site after 10 μL of various fluorescently labeled proteins were injected are shown in Figure 6. No signs of aggregation were observed near the injection site for the unstressed 20 mg / mL (HC) formulation of Therapeutic protein 1, 1 mg / mL (LC) formulation of Therapeutic protein 1, or 0.8 mg / mL formulation of Therapeutic protein 2, but significant levels of aggregation (bright spots) were observed for the 20 mg / mL mAb1, which was predicted to aggregate strongly at neutral pH. This experiment demonstrates how an in vitro mammalian skin model can be used in conjunction with a fluorescence imaging strategy to detect in vitro aggregation within an in vitro mammalian skin model.

[0086] The examples described herein, among others, illustrate how in vitro mammalian skin can be used by the methods described herein to investigate the effect of the skin environment on the physicochemical properties of subcutaneously injected biologics. The simplicity of the method can facilitate screening strategies to better understand and optimize drug products delivered through the skin. Additional uses of the methods described herein are contemplated, including: (1) using skin models based on subcutaneous tissue layers (which may be more relevant to SubQ exposure); (2) developing devices to improve injection stability; and (3) applying more quantitative analysis of changes in drug distribution and properties. Finally, the use of larger format skin models can be useful to simulate injection of biologic solutions in therapeutically relevant volumes, which will allow testing and optimization of injection strategies (e.g., addition of hyaluronidase) and injection devices.

[0087] References The following documents are incorporated herein by reference in their entireties: 1. Turner MR,Balu-iyer S V.Challenges and Opportunities for the Subcutaneous Delivery of Therapeutic Proteins.J Pharm Sci.2018;107(5):1247-1260.doi:10.1016 / j.xphs.2018.01.007 2. Poumay Y,Dupont F,Marcoux S,Leclercq-Smekens M,Herin M,Coquette AA simple reconstructed human epidermis:Preparation of the culture model and utilization in in vitro studies.Arch Dermatol Res.2004;296(5):203-211.doi:10.1007 / s00403-004-0507-y 3. De Wever BDE,Petersohn D,Mewes KR.Overview of human three-dimensional(3D) skin models used for dermal toxicity assessment(Part 1).Household and Personal Care Today.2013;8(1):18-22. 4. MatTek tissue webpage.https: / / www.mattek.com / product-category / tissue-models / 5. Rosdy M,Clauss LC.Terminal epidermal differentiation of human keratinocytes grown in chemically defined medium on inert filter substrates at the air-liquid interface.J Invest Dermatol.1990;95(4):409-414.doi:10.1111 / 1523-1747.ep12555510 6. Ng KW,Khor HL,Hutmacher DW.In vitro characterization of natural and synthetic dermal matrices cultured with human dermal fibroblasts.Biomaterials.2004;25(14):2807-2818.doi:10.1016 / j.biomaterials.2003.09.058 7. Louis F,Pannetier P,Souguir Z,et al.A biomimetic hydrogel functionalized with adipose ECM components as a microenvironment for the 3D culture of human and murine adipocytes.Biotechnol Bioeng.2017;114(8):1813-1824.doi:10.1002 / bit.26306 8. Bell E,Sher S,Hull B,et al.The reconstitution of living skin.J Invest Dermatol.1983;(81):2-10. 9. Schmidt FF,Nowakowski S,Kluger PJ.Improvement of a Three-Layered in vitro Skin Model for Topical Application of Irritating Substances.Front Bioeng Biotechnol.2020;8(May):1-11.doi:10.3389 / fbioe.2020.00388 10. Alepee N,Tornier C,Robert C,et al.A catch-up validation study on reconstructed human epidermis(SkinEthic TM RHE) for full replacement of the Draize skin irritation test.Toxicol Vitr.2010;24(1):257-266.doi:10.1016 / j.tiv.2009.08.024 11. European THE,Council P,The OF,et al.REGULATION(EC) No 1223 / 2009 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 30 November 2009 on cosmetic products.Off J Eur Union L.2009;342(59).

[0088] All references cited in this specification, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was as if it were set forth in its entirety herein.

[0089] In the context of describing this disclosure (and particularly in the context of the claims which follow), the use of the terms "a," "an," and "the" and similar referents should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise indicated.

[0090] The terms "patient" and "subject" are used interchangeably herein. In general, these terms are understood to refer to a human. In some embodiment methods, the patient or subject is a human.

[0091] Recitation of ranges of values ​​herein, unless otherwise stated herein, is merely intended to serve as a shorthand method of referring individually to each separate value and each endpoint falling within that range, and each separate value and endpoint is incorporated herein as if each separate value was individually set forth herein.

[0092] All methods described herein may be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any examples or exemplary language (e.g., "etc.") provided herein is intended merely to clarify the disclosure and does not impose limitations on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0093] Preferred embodiments of the present disclosure are described herein, including the best mode known to the inventors for carrying out the present disclosure. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading the above description. The inventors expect that such variations will be used by those skilled in the art as appropriate, and the inventors intend the present disclosure to be carried out in other ways than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of elements described herein is encompassed in this disclosure in all possible variations thereof unless otherwise indicated herein or clearly contradicted by context.

Claims

1. providing in vitro living mammalian skin comprising a dermal layer and an epidermal layer, the dermal layer defining an interface between the dermal layer and the epidermal layer; administering a therapeutic compound product to said interface under administration conditions; detecting physiochemical and / or clinical properties of the therapeutic compound product at the interface; and selecting or rejecting the administration regimen for further development based on the detected physiochemical and / or clinical properties of the therapeutic compound. A method for selecting administration conditions for a therapeutic compound product for subcutaneous administration, comprising:

2. 2. The method of claim 1, wherein the administration conditions include at least one of the formulation, concentration, volume, viscosity, molecular properties, route of administration, and / or administration device or method of using the administration device (frequency, flow rate, parameters) of the therapeutic compound.

3. 3. The method of claim 2, wherein the administration conditions include formulation additives, buffers, surfactants, or stabilizers.

4. 2. The method of claim 1, wherein the physicochemical and / or clinical properties include bioavailability, pharmacokinetic properties, aggregation, precipitation, distribution, diffusion rate, retention and / or absorption.

5. 10. The method of claim 1, wherein the therapeutic compound product has acquired the physiochemical and / or clinical properties after the administering step.

6. 10. The method of claim 1, further comprising repeating the method with two or more different administration conditions.

7. providing in vitro living mammalian skin comprising a dermal layer and an epidermal layer, the dermal layer defining an interface between the dermal layer and the epidermal layer; administering a therapeutic compound product containing a molecular signature to the interface; and detecting the physiochemical and / or clinical properties of said therapeutic compound at said interface.

1. A method for determining the effect of the molecular properties of a therapeutic compound on the physiochemical and / or clinical properties of said therapeutic compound after subcutaneous administration, comprising:

8. 8. The method of claim 7, wherein the physicochemical and / or clinical properties include bioavailability, pharmacokinetic properties, aggregation, precipitation, distribution, diffusion rate and / or absorption.

9. providing in vitro living mammalian skin comprising a dermal layer and an epidermal layer, the dermal layer defining an interface between the dermal layer and the epidermal layer; administering a therapeutic compound comprising a property to the interface, the therapeutic compound comprising a first state of a molecular property; and detecting a second state of the molecular signature of the therapeutic compound in the subcutaneous space.

20. A method for detecting a change in a molecular profile of a therapeutic compound following subcutaneous administration, comprising:

10. 10. The method of claim 9, wherein the first state and the second state of the molecular characteristic comprise any of a concentration, an amount, a distribution, or a distribution at the interface with respect to the therapeutic compound.

11. 10. The method of claim 9, wherein the first state of the molecular property has a first value and the second state of the molecular property has a second value that is the same as or different from the first state.

12. 10. The method of claim 9, wherein the molecular characteristics include at least one of acidic species, basic species, high molecular weight species, sub-visible particle count, low molecular weight, medium molecular weight, glycosylation (such as non-glycosylated heavy chain or high mannose), non-heavy and light chain, deamidation, deamination, cyclization, oxidation, isomerization, fragmentation / clipping, N-terminal and C-terminal variants, reduced species and partial species, folded structure, surface hydrophobicity, chemical modification, covalent bond, C-terminal amino acid motif PARG or C-terminal amino acid motif PAR-amide.

13. the administering step comprises: pressing a needle including a cannula against the surface of the dermal layer, thereby inserting the needle into the interface of the in vitro living mammalian skin; and placing the therapeutic compound product through the needle into the subcutaneous space; The method of claim 1 , comprising:

14. 14. The method of claim 13, wherein the needle is pressed against the surface of the dermis at an acute angle.

15. 14. The method of claim 13, wherein the needle includes an opening in fluid communication with the cannula, and the administering step includes directing the opening toward the dermal layer.

16. 16. The method of claim 15, wherein the detecting step comprises optical imaging through the dermal layer.

17. 10. The method of claim 1, wherein the administering step comprises immersion, diffusion, or transdermal administration of the therapeutic compound product through the dermis layer and / or into the subcutaneous tissue layer.

18. The method of claim 1 , wherein the in vitro mammalian skin further comprises subcutaneous tissue defining a subcutaneous space within the interface.

19. The method of claim 1 , wherein the step of providing the in vitro mammalian skin comprises immobilizing the in vitro mammalian skin between a proximal substrate and a distal substrate.

20. 20. The method of claim 19, wherein the proximal substrate and the distal substrate each comprise glass, such as a cover glass.

21. 2. The method of claim 1, wherein the step of preparing the in vitro mammalian skin comprises providing the dermal layer of the skin model disposed on a porous substrate containing pores sized to accommodate diffusion of the therapeutic compound product, and the step of administering comprises fluidly connecting the porous substrate with a solution containing the therapeutic compound product.

22. 22. The method of claim 21, further comprising detecting the presence of sub-visible levels of particles in the solution after the administering step.

23. 23. The method of any one of claims 1 to 22, wherein said in vitro mammalian skin is in vitro human skin.

24. 23. The method of any one of claims 1 to 22, wherein the in vitro mammalian skin is the in vitro skin of a non-human primate, such as a cynomolgus monkey.

25. The method of any one of claims 1 to 22, wherein the in vitro mammalian skin is from a non-human mammal.

26. Repeating the method using in vitro human skin; and comparing the physiochemical and / or clinical properties of the therapeutic compound administered to the in vitro human skin with the physiochemical and / or clinical properties of the therapeutic compound administered to the in vitro mammalian skin of the non-human mammal.

26. The method of claim 25, further comprising:

27. The method of claim 1 , wherein the detecting step comprises imaging and / or analytical testing.

28. 28. The method of claim 27, wherein the analytical test comprises mass spectrometry, chromatography, electrophoresis, spectroscopy, light obscuration, particle methods (such as nanoparticle / visible / micron sized resonant mass or Brownian motion), analytical centrifugation, imaging or image characterization, immunoassay, SE-HPLC, rCE-SDS, CEX-HPLC, HIAC, nrCE-SDS, mass spectrometry microscopy, and / or mass spectroscopy.

29. 28. The method of claim 27, wherein the imaging comprises a CT scan or magnetic resonance imaging.

30. 10. The method of claim 1, further comprising labeling the therapeutic compound with a detectable moiety, such as a fluorophore, fluorescent dye, radiolabel, or quantum dot.

31. 10. The method of claim 1, wherein the therapeutic compound comprises an antibody, an antigen-binding antibody fragment, an antibody protein product, a bispecific T cell engager (BiTE®) molecule, a bispecific antibody, a trispecific antibody, an Fc-fusion protein, a recombinant protein, a recombinant virus, a recombinant T cell, a synthetic peptide, an active fragment of a recombinant protein, a nucleic acid, or a virus.

32. 10. The method of claim 1, wherein the therapeutic compound comprises or consists of a therapeutic protein.

33. 10. The method of claim 1, further comprising the step of performing analytical testing on the therapeutic compound prior to the administering step.

34. The method of claim 1 , wherein the detecting step comprises obtaining a background signal and subtracting the background signal.

35. The method of claim 1 , wherein the detecting step comprises optical imaging.

36. 36. The method of claim 35, wherein the optical imaging comprises multiphoton laser scanning microscopy.

37. 36. The method of claim 35, wherein the optical imaging comprises second harmonic generation (SHG) imaging.

38. 36. The method of claim 35, wherein the step of administering the therapeutic compound comprises placing the therapeutic compound product in the subcutaneous space through the needle, the needle remaining in place in the subcutaneous space during the imaging.

39. 10. The method of claim 1, wherein the in vitro live mammalian skin is provided in a culture medium, the culture medium being filtered prior to the providing step.

40. 40. The method of claim 39, wherein the medium has been filtered through a centrifugal filter having a molecular weight cutoff of 50 KDa or less, e.g., 50 KDa, 40 KDa, 30 KDa, or 25 KDa.

41. The method of claim 1, wherein the molecular characteristics are detected by SEC-HPLC and HIAC.

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