(19) TZZ¥ Z_T (11) EP 3 205 666 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 16.08.2017 Bulletin 2017/33 C07K 14/78 (2006.01) D06N 3/00 (2006.01) D01F 4/00 (2006.01) D01F 11/02 (2006.01) (21) Application number: 17156361.2 (22) Date of filing: 15.02.2017 (84) Designated Contracting States: • Cassingham, Darryl Miles AL AT BE BG CH CY CZ DE DK EE ES FI FR GB Dorset DT9 6RF (GB) GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR (72) Inventors: Designated Extension States: • Purcell, Brendan Patrick BA ME Brooklyn, NY 11225 (US) Designated Validation States: • Williamson, David Thomas MA MD Landenberg, PA 19350 (US) • Marga, Francoise Suzanne (30) Priority: 15.02.2016 US 201662295435 P Brooklyn, NY 11209 (US) • Shofer, Susan J. (71) Applicants: Brooklyn, NY 11231 (US) • Purcell, Brendan Patrick • Cassingham, Darryl Miles Brooklyn, NY 11225 (US) Dorset DT9 6RF (GB) • Williamson, David Thomas Landenberg, PA 19350 (US) (74) Representative: Brevalex • Marga, Francoise Suzanne 56, Boulevard de l’Embouchure Brooklyn, NY 11209 (US) B.P. 27519 • Shofer, Susan J. 31075 Toulouse Cedex 2 (FR) Brooklyn, NY 11231 (US) (54) METHOD FOR MAKING A BIOFABRICATED MATERIAL CONTAINING COLLAGEN FIBRILS (57) Described herein is a method for producing a techniques or by chemical synthesis. The collagen or col- biofabricated material from collagen or collagen-like pro- lagen-like proteins are fibrillated, crosslinked, dehydrat- teins. The collagen or collagen-like proteins are isolated ed and lubricated thus forming the biofabricated material from animal sources or produced by recombinant DNA having a substantially uniform network of collagen fibrils. EP 3 205 666 A1 Printed by Jouve, 75001 PARIS (FR) EP 3 205 666 A1 Description CROSS REFERENCE TO RELATED APPLICATIONS 5 [0001] This application claims priority to U.S. Provisional Application No. 62/295,435 filed February 15, 2016 and which is incorporated by reference in its entirety. This application is related to U.S. Patent Application No. 13/853,001, titled "ENGINEERED LEATHER AND METHODS OF MANUFACTURE THEREOF" and filed on 3/28/2013; U.S. Patent Application No. 14/967,173, titled "ENGINEERED LEATHER AND METHODS OF MANUFACTURE THEREOF" and filed on 12/11/2015; and PCT Patent Application No. PCT/US2015/058794, titled "REINFORCED ENGINEERED BIO- 10 MATERIALS AND METHODS OF MANUFACTURE THEREOF" and filed on 11/3/2015. BACKGROUND OF THE INVENTION Field of the Invention 15 [0002] This invention relates to biofabricated leather materials composed of unbundled and randomly-oriented trimeric collagen fibrils that exhibit superior strength, non-anisotropic properties, and uniformity by comparison to conventional leather products, but which have the look, feel and other aesthetic properties of natural leather. Unlike synthetic leather products composed of plastic resins, the biofabricated leather of the invention is based on collagen, a natural component 20 of leather. Description of Related Art. [0003] Leather. Leather is used in a vast variety of applications, including furniture upholstery, clothing, shoes, luggage, 25 handbag and accessories, and automotive applications. The estimated global trade value in leather is approximately US $100 billion per year (Future Trends in the World Leather Products Industry and Trade, United Nations Industrial Development Organization, Vienna, 2010) and there is a continuing and increasing demand for leather products. New ways to meet this demand are required in view of the economic, environmental and social costs of producing leather. To keep up with technological and aesthetic trends, producers and users of leather products seek new materials exhibiting 30 superior strength, uniformity, processability and fashionable and appealing aesthetic properties that incorporate natural components. [0004] Natural leathers are produced from the skins of animals which require raising livestock. However, the raising of livestock requires enormous amounts of feed, pastureland, water, and fossil fuels. It also produces air and waterway pollution, including production of greenhouse gases like methane. Some states in the United States, such as California, 35 may impose taxes on the amounts of pollutants such as methane produced by livestock. As the costs of raising livestock rise, the cost of leather will rise. [0005] The global leather industry slaughters more than a billion animals per year. Most leather is produced in countries that engage in factory farming, lack animal welfare laws, or in which such laws go largely or completely unenforced. This slaughter under inhumane conditions is objectionable to many socially conscious people. Consequently, there is 40 a demand from consumers with ethical, moral or religious objections to the use of natural leather products for products humanely produced without the mistreatment or slaughter of animals or produced in ways that minimize the number of animals slaughtered. [0006] The handling and processing of animal skins into leather also poses health risks because the handling animal skins can expose workers to anthrax and other pathogens and allergens such as those in leather dust. Factory farming 45 of animals contributes to the spread of influenza (e.g. "bird flu") and other infectious diseases that may eventually mutate and infect humans. Animal derived products are also susceptible to contamination with viruses and prions ("mad cow disease"). For producer and consumer peace of mind, there exists a demand for leather products that do not present these risks. [0007] Natural leather is generally a durable and flexible material created by processing rawhide and skin of an animal, 50 such as cattle hides. This processing typically involves three main parts: preparatory stages, tanning, and retanning. Leather may also be surface coated or embossed. [0008] Numerousways areknown to preparea skin or hide and convert it to leather. These include saltingor refrigerating a hide or skin to preserve it; soaking or rehydrating the hide in an aqueous solution that contains surfactants or other chemicals to remove salt, dirt, debris, blood, and excess fat; defleshing or removing subcutaneous material from the 55 hide; dehairing or unhairing the hide remove most of the hair; liming the hide to loosen fibers and open up collagen bundles allowing it to absorb chemicals; splitting the hide into two or more layers; deliming the hide to remove alkali and lower its pH; bating the hide to complete the deliming process and smooth the grain; degreasing to remove excess fats; frizzing; bleaching; pickling by altering the pH; or depickling, 2 EP 3 205 666 A1 [0009] Once the preparatory stages are complete, the leather is tanned. Leather is tanned to increase its durability compared to untreated hide. Tanning converts proteins in the hide or skin into a stable material that will not putrefy while allowing the leather material to remain flexible. During tanning, the skin structure may be stabilized in an "open" form by reacting some of the collagen with complex ions of chromium or other tanning agents. Depending on the compounds 5 used, the color and texture of the leather may change. [0010] Tanning is generally understood to be the process of treating the skins of animals to produce leather. Tanning may be performed in any number of well-understood ways, including by contacting a skin or hide with a vegetable tanning agent, chromium compound, aldehyde, syntan, synthetic, semisynthetic or natural resin or polymer, or/and tanning natural oil or modified oil. Vegetable tannins include pyrogallol- or pyrocatechin-based tannins, such as valonea, mimosa, 10 ten, tara, oak, pinewood, sumach, quebracho and chestnut tannins; chromium tanning agents include chromium salts like chromium sulfate; aldehyde tanning agents include glutaraldehyde and oxazolidine compounds, syntans include aromatic polymers, polyacrylates, polymethacrylates, copolymers of maleic anhydride and styrene, condensation prod- ucts of formaldehyde with melamine or dicyandiamide, lignins and natural flours. [0011] Chromium is the most commonly used tanning material. The pH of the skin/hide may be adjusted ( e.g., lowered, 15 e.g. to pH 2.8-3.2) to allow penetration of the tanning agent; following penetration the pH may be raised to fix the tanning agent ("basification" to a slightly higher level, e.g., pH 3.8-4.2 for chrome). [0012] After tanning, a leather may be retanned. Retanning refers to the post-tanning treatment that can include coloring (dying), thinning, drying or hydrating, and the like. Examples of retanning techniques include: tanning, wetting (rehydrating), sammying (drying), neutralization (adjusting pH to a less acidic or alkaline state), dyeing, fat liquoring, 20 fixation of unbound chemicals, setting, conditioning, softening, buffing, etc. [0013] A tanned leather product may be mechanically or chemically finished. Mechanical finishing can polish the leather to yield a shiny surface, iron and plate a leather to have a flat, smooth surface, emboss a leather to provide a three dimensional print or pattern, or tumble a leather to provide a more evident grain and smooth surface. Chemical finishing may involve the application of a film, a natural or synthetic coating, or other leather treatment. These may be 25 applied, for example, by spraying, curtain-coating or roller coating. [0014] In animal hide, variations in fibrous collagen organization are observed in animals of different ages or species. These differences affect the physical properties of hides and differences in leather produced from the hides. Variations in collagen organization also occur through the thickness of the hide. The top grain side of hide is composed of a fine network of collagen fibrils while deeper sections (corium) are composed of larger fiber bundles (FIG. 2). The smaller 30 fibril organization of the grain layer gives rise to a soft and smooth leather aesthetic while the larger fiber bundle organ- ization of deeper regions gives rise to a rough and course leather aesthetic. [0015] The porous, fibrous organization of collagen in a hide allows applied molecules to penetrate, stabilize, and lubricate it during leather tanning.