# US 2025/0147008 A1 · Methods comprising therapeutic compounds and in vitro mammalian skin

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- Number: US 2025/0147008 A1
- Document ID: US20250147008A1
- Status: Published application
- Kind: A1
- Application number: 18/684,262
- Filed: 2022-08-19
- Published: 2025-05-08
- Earliest claimed: 2021-08-20
- Assignees: Amgen Inc.
- Applicants: Amgen Inc.
- Inventors: Marisa K. JOUBERT; Nathan H. JOH; Joshua M. TOKUDA; John FERBAS; Jiansong XIE; Dong XIANG
- Web page: https://patentgrep.com/patent/US20250147008A1

## CPC

- G01N 33/15: Medicinal preparations {; Physical properties thereof, e.g. dissolubility}
- G01N 33/5082: {Supracellular entities, e.g. tissue, organisms}
- C07K 16/00: Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
- C12N 5/0698: {Skin equivalents}
- G01N 33/5044: {involving specific cell types}
- C07K 2317/94: Stability, e.g. half-life, pH, temperature or enzyme-resistance
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

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

## Claims

### Claim 1

A method of selecting a condition of administration for a therapeutic compound product for subcutaneous administration, the method comprising;
providing a live in vitro mammalian skin comprising a dermal layer and epidermal layer that define an interface therebetween;
administering a therapeutic compound product to the interface under a condition of administration;
detecting a physiochemical and/or clinical characteristic of the therapeutic compound product in the interface; and
selecting or rejecting the condition of administration for further development based on the detected physiochemical and/or clinical characteristic of the therapeutic compound.

### Claim 2 (depends on 1)

The method of claim 1, wherein the condition of administration comprises at least one of formulation, concentration, volume, viscosity, a molecular attribute of said therapeutic compound, route of administration, and/or administration apparatus or method of using an administration apparatus.

### Claim 3 (depends on 2)

The method of claim 2, wherein the condition of administration comprises an excipient, buffer, surfactant, or stabilizer of a formulation.

### Claim 4 (depends on 1)

The method of claim 1, wherein the physiochemical and/or clinical characteristic comprises bioavailability, pharmacokinetic property, aggregation, precipitation, distribution, diffusion rate, retention and/or absorption.

### Claim 5 (depends on 1)

The method of claim 1, wherein the therapeutic compound product acquired the physiochemical and/or clinical characteristic after said administering.

### Claim 6 (depends on 1)

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

### Claim 7

A method of determining an impact of a molecular attribute of a therapeutic compound on a physiochemical and/or clinical characteristic of the therapeutic compound after subcutaneous administration, the method comprising;
providing a live in vitro mammalian skin comprising a dermal layer and epidermal layer that define an interface therebetween;
administering a therapeutic compound product comprising a molecular attribute to the interface; and
detecting a physiochemical and/or clinical characteristic of the therapeutic compound in the interface.

### Claim 8 (depends on 7)

The method of claim 7, wherein the physiochemical and/or clinical characteristic comprises bioavailability, pharmacokinetic property, aggregation, precipitation, distribution, diffusion rate, and/or absorption.

### Claim 9

A method of detecting a change in a molecular attribute of a therapeutic compound after subcutaneous administration, the method comprising;
providing a live in vitro mammalian skin comprising a dermal layer and epidermal layer that define an interface therebetween;
administering the therapeutic compound comprising the attribute to the interface, wherein the therapeutic compound comprises a first state of a molecular attribute; and
detecting a second state of the molecular attribute of the therapeutic compound in the subcutaneous space.

### Claim 10 (depends on 9)

The method of claim 9, wherein the first state and second state of the molecular attribute comprise any of concentration, quantity, distribution on the therapeutic compound, or distribution in the interface.

### Claim 11 (depends on 9)

The method of claim 9, wherein the first state of the molecular attribute has a first value, and wherein the second state of the molecular attribute has a second value that is the same or different than the first state.

### Claim 12 (depends on 9)

The method of claim 9, wherein the molecular attribute comprises at least one of acidic species, basic species, high molecular weight species, subvisible particle number, low molecular weight, middle molecular weight, glycosylation, non-heavy chain and light chain, deamidation, deamination, cyclization, oxidation, isomerization, fragmentation/clipping, N-terminal and C-terminal variants, reduced and partial species, folded structure, surface hydrophobicity, chemical modification, covalent bond, a C-terminal amino acid motif PARG, or a C-terminal amino acid motif PAR-Amide.

### Claim 13 (depends on 1)

The method of claim 1, wherein administering comprises:
pressing a needle comprising a cannula to a surface of the dermal layer, thereby inserting the needle in the interface of the live in vitro mammalian skin; and
disposing the therapeutic compound product in the subcutaneous space via the needle.

### Claim 14 (depends on 13)

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

### Claim 15 (depends on 13)

The method of claim 13, wherein the needle comprises an opening in fluid communication with the cannula, wherein administering comprises orienting the opening toward the dermal layer.

### Claim 16 (depends on 15)

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

### Claim 17 (depends on 1)

The method of claim 1, wherein administering comprises soaking, diffusion, or transdermal administration of the therapeutic compound product through the dermal layer and/or into the hypodermal layer.

### Claim 18 (depends on 1)

The method of claim 1, wherein the in vitro mammalian skin further comprises a hypodermis that defines a subcutaneous space within the interface

### Claim 19 (depends on 1)

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

### Claim 20 (depends on 19)

The method of claim 19, wherein the proximal substrate and the distal substrate each comprise glass.

### Claim 21 (depends on 1)

The method of claim 1, wherein providing the in vitro mammalian skin comprises providing the dermal layer of the skin model disposed on a porous substrate that comprises pores sized to accommodate diffusion of the therapeutic compound product, and wherein said administering comprises placing the porous substrate in fluid communication with a solution comprising the therapeutic compound product.

### Claim 22 (depends on 21)

The method of claim 21, further comprising detecting a presence of level of subvisible particles in the solution after said administering.

### Claim 23 (depends on 1)

The method of claim 1, wherein the in vitro mammalian skin is an in vitro human skin.

### Claim 24 (depends on 1)

The method of claim 1, wherein the in vitro mammalian skin is an in vitro skin of a non-human primate, such as a cynomolgus monkey.

### Claim 25 (depends on 1)

The method of claim 1, wherein the in vitro mammalian skin is of a non-human mammal.

### Claim 26 (depends on 25)

The method of claim 25, further comprising:
repeating the method using an in vitro human skin; and
comparing physiochemical and/or clinical characteristics of the therapeutic compound administered to the in vitro human skin with those of the therapeutic compound administered to the in vitro mammalian skin is of the non-human mammal.

### Claim 27 (depends on 1)

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

### Claim 28 (depends on 27)

The method of claim 27, wherein the analytical testing comprises mass spectrometry, chromatography, electrophoresis, spectroscopy, light obscuration, a particle method, analytical centrifugation, imaging or imaging characterization, immunoassay, SE-HPLC, rCE-SDS, CEX-HPLC, HIAC, nrCE-SDS, mass spectrometry microscopy, and/or mass spectroscopy.

### Claim 29 (depends on 27)

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

### Claim 30 (depends on 1)

The method of claim 1, further comprising labeling the therapeutic compound with a detectable moiety selected from a fluorophore, fluorescent dye, radiolabel, and quantum dot.

### Claim 31 (depends on 1)

The method of claim 1, wherein the therapeutic compound comprises 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.

### Claim 32 (depends on 1)

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

### Claim 33 (depends on 1)

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

### Claim 34 (depends on 1)

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

### Claim 35 (depends on 1)

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

### Claim 36 (depends on 35)

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

### Claim 37 (depends on 35)

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

### Claim 38 (depends on 35)

The method of claim 35, wherein administering the therapeutic compound comprises disposing the therapeutic compound product in the subcutaneous space via the needle, and wherein the needle remains disposed in the subcutaneous space during said imaging.

### Claim 39 (depends on 1)

The method of claim 1, wherein the live in vitro mammalian skin is provided in media, and wherein the media has been filtered prior to said providing.

### Claim 40 (depends on 39)

The method of claim 39, wherein the media has been filtered with a centrifuge filter having a molecular weight cutoff of no more than 50K Da.

### Claim 41 (depends on 9)

The method of claim 9, wherein the molecular attribute is detected by SEC-HPLC and HIAC.