Method for coating a substrate surface

Abstract

A method of coating a substrate surface comprising providing a substrate having a surface, optionally treating at least a portion of the substrate surface with an oxidizing agent, treating at least a portion of the substrate surface with a composition comprising a polysaccharide, an oligosaccharide, a polyol, or mixtures thereof, and incubating the treated substrate with the composition for a period of time. Also disclosed are substrates comprising such coatings, containers comprising such coated substrates, and medical devices comprising such substrates.

Description

[Technical field]

[0001] The present invention relates to a method for coating a substrate surface, to the coated substrate obtained by the method, and to a method for reducing protein aggregation on a substrate surface. The present invention also relates to fluid containers, medical devices, and syringes comprising the coated substrate. The substrate may be glass, in particular borosilicate glass. [Background technology]

[0002] During the formulation of proteinaceous compositions contained in the devices, protein aggregation and denaturation can occur, which has caused problems in diagnostics, analysis and drug delivery. Controlling the formation and denaturation of protein aggregates has been a challenge.

[0003] Nonspecific protein adsorption is a complex phenomenon: the process is determined by the protein properties (e.g., structure, size, and charge and polarity distribution), the material surface properties (e.g., charge, roughness, and surface energy state), the environmental conditions (e.g., pH, ionic strength, and temperature), and the kinetics of the adsorption process.

[0004] Proteins can bind nonspecifically to the surfaces of materials used during sample preparation, such as pipette tips, sample tubes, well plates, vials, etc., resulting in a loss of experimental accuracy. Regulatory guidelines require that bioanalytical methods be validated not only in terms of linearity, sensitivity, accuracy, precision, selectivity, and stability, but also in terms of carryover contamination. Carryover occurs due to nonspecific adsorption of analytes to parts of the analytical system, thus biasing both qualitative and quantitative assessments. Thus, the linearity, sensitivity, and reproducibility of the analysis are adversely affected.

[0005] Disposable systems are becoming accepted for large-scale storage during manufacturing and processing of recombinant proteins and monoclonal antibodies in liquid and frozen form. The interaction between the container and the drug solution is important, as the physicochemical properties of the container material contribute to maintaining the integrity and stability of the drug substance. Adsorption of proteins to the container surface can lead to loss of potency of the protein in solution due to changes in concentration, denaturation and / or degradation of the protein. Protein aggregation and denaturation of pharmaceutical compositions (antibodies, proteins and other peptides such as erythropoietin, interferon gamma, infliximab, etanercept, adalimumab, etc., all of which can be delivered pre-filled in a syringe) can also cause adverse immune responses, resulting in the withdrawal of some biopharmaceuticals from the market.

[0006] Surface modification of materials used to manufacture medical devices and containers for delivering compositions is one approach to try to alleviate the problem. Surface modification of protein contact materials used in manufacturing and storage, such as ethylene vinyl acetate (EVA) copolymers and low density polyethylene (LDPE), can reduce aggregate formation and protein adsorption, thereby improving product quality and safety. Materials include glass or polymers (e.g., cyclic olefin polymers, COP), which can be modified by applying an inorganic coating on the surface that comes into contact with the composition.

[0007] WO-A-2020 / 092373 discloses a drug container having a thermoplastic wall, a PECVD (plasma enhanced chemical vapor deposition) drug contact coating, and a polypeptide composition contained within a lumen. The drug contact coating is located on or adjacent to the interior surface of the container and in contact with the fluid within the lumen, and is essentially a SiO 2 barrier that reduces corrosion. x C y H z It consists of:

[0008] US-A-2015 / 0126941 discloses a filled package that includes a container, a barrier coating and a protective coating on the container, and a fluid composition contained in the container to extend the shelf life of the package. The barrier coating is SiO x (x is between 1.5 and 2.9). The protective coating includes a layer of sugars that prevents leaching. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2020 / 092373 Brochure [Patent Document 2] US Patent Application Publication No. 2015 / 0126941 Summary of the Invention [Problem to be solved by the invention]

[0010] There is a need to provide materials having surfaces which are less susceptible to adsorption of pharmaceutical compositions (including protein aggregation and denaturation) and which do not suffer from the problems of the prior art.

[0011] The object of the present invention is to address this need. [Means for solving the problem]

[0012] The present invention therefore provides in a first aspect a method of coating a substrate surface, the method comprising the steps of: (a) providing a substrate having a surface; (b) optionally treating at least a portion of the substrate surface with an oxidizing agent; (c) treating at least a portion of the substrate surface with a composition comprising a polysaccharide, oligosaccharide, polyol, or mixtures thereof; and (d) incubating the treated substrate with the composition for a period of time.

[0013] In general, any suitable substrate can be used in the present methods. For example, the substrate can include quartz, glass (e.g., silica-lime glass or borosilicate glass). In other embodiments, the substrate can include one or more polymers (e.g., EVA, polyolefin (e.g., polyethylene or polypropylene), polyester (e.g., polyethylene terephthalate), polycarbonate, or any combination or copolymer thereof), although preferably the substrate includes a cyclic olefin polymer or copolymer. The polymer (e.g., cyclic olefin polymer) can include, at least in part, recycled polymer.

[0014] Cyclic olefin polymers are useful as high temperature polymers with excellent optical properties, good chemical and heat resistance, and excellent dimensional stability. COPs can be made from cyclic olefin monomers such as norbornene, cyclopentadiene (CPD), and / or dicyclopentadiene (DCPD).

[0015] Glass substrates (e.g., borosilicate glass substrates) are useful because they are often used in procedures involving protein or oligonucleotide compositions that are susceptible to protein adsorption and / or aggregation, or oligonucleotide adsorption and / or aggregation.

[0016] Quartz substrates are useful because they can be used in microfluidic and other devices.

[0017] Surprisingly, the use of polysaccharides, oligosaccharides, polyols, or mixtures thereof as coatings can significantly reduce protein adsorption and / or aggregation and can also reduce oligonucleotide adsorption and / or aggregation.

[0018] If the substrate is a polymer, the composition may be applied over one or more other coating layers (except for a silica layer) already deposited on the polymer surface. Preferably, the polymer surface does not include a silica coating.

[0019] The composition can be applied directly to the substrate surface and does not typically require an inorganic layer already deposited on the substrate surface, therefore the method preferably treats the substrate surface directly.

[0020] The polysaccharide may include a hexose-derived polysaccharide, although it is believed that many polysaccharides, oligosaccharides, polyols, or mixtures thereof may be useful in this method. The polysaccharide may be polyhydroxylated. In general, the polysaccharide is preferably applied to a substrate surface to provide a relatively hydrophilic surface (e.g., a water contact angle of less than 80 degrees, less than 70 degrees, less than 60 degrees, less than 50 degrees, or less).

[0021] The polysaccharide is preferably selected from dextran, cellulose, one or more polyols, dextrin, polygalacturonic acid, hyaluronic acid, or a combination of two or more of these polysaccharides.

[0022] The inventors have now discovered that the use of the above polysaccharides, oligosaccharides, polyols or mixtures thereof is highly advantageous in that it significantly reduces protein aggregation when applied to the surface of either a glass or polymer substrate.

[0023] The oxidizing agent preferably affects the surface of the substrate but does not adversely affect the bulk of the substrate. The oxidizing agent may include peroxide, optionally hydrogen peroxide, optionally 30% (w / w) hydrogen peroxide in an aqueous solution. In general, peroxide and / or other oxidizing agents such as O3, hydroxyl radicals, atomic oxygen, ozone water, H2O2 with or without decomposition catalysts (e.g., Cu ions, Fe ions, manganese oxide), periodate, hypochlorite, and / or permanganate may also be applied.

[0024] The predetermined time may be within a range of 0.5 to 240 minutes. Alternatively, the predetermined time may be within a range of 1 to 120 minutes, 1 to 60 minutes, 1 to 30 minutes, 1 to 20 minutes, or 1 to 10 minutes.

[0025] The treatment and / or incubation step of at least a portion of the substrate surface may be carried out at a temperature in the range of 10°C to 90°C, optionally 10°C to 70°C.

[0026] During the treatment and / or incubation steps of at least a portion of the substrate surface, the steps may be mechanically, chemically or electromagnetically accelerated, for example using ultrasonic treatment, microwave or UV irradiation, and / or ionic catalysis.

[0027] The composition may be an aqueous solution. Thus, the composition may comprise water. Where appropriate, one or more co-solvents may be present.

[0028] In one embodiment of the invention, the composition may include an oxidizing agent. The oxidizing agent in the composition may include a peroxide, optionally hydrogen peroxide, optionally 30% (w / w) hydrogen peroxide in an aqueous solution.

[0029] In one embodiment of the invention, the method may further comprise the step of treating the substrate with a basic aqueous solution of pH 7-14, preferably pH 9-14. This may be advantageous as it may improve protein rejection (or lipid, liposome or oligonucleotide rejection) from the substrate surface. This step may be performed after one or more of steps (a), (b), (c) or (d) of the method.

[0030] The matrix obtained by this method significantly reduced protein aggregation.

[0031] Thus, in a second aspect, the present invention provides a coated substrate obtainable by coating at least one surface of a substrate by the method of the first aspect.

[0032] Optionally, the coated substrate does not include a silica coating.

[0033] Thus, in a third aspect, the present invention provides a substrate having a coating on at least one surface thereof, the coating comprising a polysaccharide directly contacting the surface of the substrate.

[0034] The substrate may comprise glass, quartz or a polymer. The glass may comprise borosilicate glass. The polymer may comprise a cyclic olefin polymer.

[0035] The polysaccharides preferably include dextran, cellulose, polyols (eg, hydrogenated hydrolysates of starch), dextrin, polygalacturonic acid, hyaluronic acid, or a combination of two or more of these polysaccharides.

[0036] The coated substrates of the present invention have an additional advantage (e.g., when compared to uncoated surfaces or other materials) in that they enhance the thermal and intrinsic stability of compositions stored in contact with the coated surface.

[0037] Thus, in a fourth aspect, the present invention provides the use of a container comprising a substrate coated according to the third aspect for storing a pharmaceutical composition, optionally a peptide composition, whereby the intrinsic and / or thermal stability of the pharmaceutical composition is enhanced.

[0038] Thus, in a fifth aspect, the present invention provides a method for reducing aggregation or adsorption of lipids or liposomes, proteins or oligonucleotides on a substrate surface, the method comprising the steps of (a) providing a substrate according to the above and second aspects, and (b) contacting the surface with a lipid, liposome-containing composition, proteinaceous composition or oligonucleotide composition.

[0039] As discussed above, this may be advantageous as it provides improved storage conditions, for example allowing storage at higher temperatures and / or for longer periods of time than previously possible.

[0040] Thus, the pharmaceutical composition may comprise a liposome-containing composition, a nucleotide (e.g., oligonucleotide) composition, or a pharmaceutical proteinaceous composition. The pharmaceutical proteinaceous composition may comprise a monoclonal antibody composition, or a peptide hormone.

[0041] In one embodiment of the invention, the pharmaceutical proteinaceous composition may comprise one or more of a vaccine (e.g., a peptide-containing vaccine), erythropoietin, interferon (α-, β-, and / or γ-interferon), infliximab, etanercept, adalimumab, rituximab, infliximab, trastuzumab, insulin, glucagon, and / or gonadotropins.

[0042] The pharmaceutical composition may include an injectable composition. Examples of injectable compositions include the following: Abarelix - depot formulation (hormonal drug), Abobotulinumtoxin A injection (Dysport), Acetadoto (acetylcysteine ​​injection), Actemra (tocilizumab injection), Axrel (corticorelin oubain triflutate injection), Actimune (interferon gamma-1b), Adacel (vaccine), Adalimumab (Humira), Adenoscan (adenosine injection), Aldolazyme (laronidase), Alglucerase injection (Ceredace), Alkeran injection (melphalan hydrochloride injection), ALTU-238 (human growth hormone), Arzera (ofatumumab injection), Avastin (bevacizumab), Azactam injection (aztreonam injection), BayHepB (Hepatitis B human immunoglobulin, antibody), BayTet (tetanus immune globulin, antibody), Beksar (tositumomab) (antibody), Belenoxan (bleomycin sulfate injection, a peptide antibiotic), Cosmetic Botox (Onabotulinumtoxin A for injection, protein), BR3-FC (protein), Briobercept (antibody), BTT-1023 (antibody), Byetta (exenatide, protein), Campath (alemtuzumab, antibody), Canakinumab injection (Ilaris, antibody), Cartilage cells, Casflo (alteplase, protein), Cerezyme (imiglucerase) (enzyme), Certolizumab pegol (Cimzia, antibody), Recombinant choriogonadotropin alpha (r-hCG) injection (Ovidrel, a peptide hormone), Choriogonadotropin (hCG) injection (Pregnyl, Follutein, Profasi, Novare, peptide hormone), Clofarabine injection (Clolar, Evoltra, purine nucleoside), Colistin Methane Injection (Coly-Mycin M), (polypeptide), Corifollitropin alpha (Elonva, a peptide hormone), Copaxone (glatiramer acetate, peptide mixture), Cubicin (daptomycin injection, cyclic lipopeptide), Dacetuzumab (antibody), Darbepoetin alfa (antibody), DDAVP injection (desmopressin acetate hydrate injection peptide hormone), Denosumab injection (Prolia, antibody), DMOAD (Disease-modifying drugs for osteoarthritis, a class of compounds some of which are peptides); Ecallantide injection (Kalbitor, protein), Engerix (vaccine), Enbrel (etanercept, protein), Epratuzumab (antibody), Erbitux (cetuximab, antibody), Erythropoietin (peptide hormone), Essential amino acid injection (Nephramine) (amino acid mixture), Fabrazyme (agalsidase beta, enzyme), Fluarix Quadrivalent (vaccine), Fludara (Fludarabine phosphate) (a nucleotide analogue derivative), Follitropin alfa injection (Gonal-f RFF, Cinnal-f, Fertilex, Ovaleap, Bemfola, peptide hormone), Follistim beta injection (Follistim, Follistim AQ cartridge, Puregon, peptide hormone), Follitropin Delta Injection (Rekovelle, a peptide hormone), Forteo (Teriparatide (rDNA-derived) Injectable Peptide Hormone), Foscamet sodium injection (Foscavir), Fuzeon (enfuvirtide, peptide), GA101 (obinutuzumab, antibody), Ganirelix (Ganirelix acetate injection, peptide), Gardasil (vaccine), GC1008 (fresolimumab, antibody), Gemtuzumab ozogamicin injection (Mylotarg) (antibody drug conjugate), Golimumab injection (Simponi injection, antibody), GlucaGen (glucagon, peptide hormone), Havrix (vaccine), Herceptin (trastuzumab, antibody), hG-CSF (human granulocyte colony-stimulating factor, protein), Humalog (insulin lispro, peptide hormone), Human growth hormone, Humegon (human gonadotropin, peptide hormone), Humulin (insulin and analogs (modified forms of insulin?), peptide hormones); IncobotulinumtoxinA for injection (Xeomin, protein), Increlex (Mecasermin [rDNA derived] injection (human growth factor)), Infanrix (vaccine), Insulin (a peptide hormone), Insulin aspart [rDNA derived] injection (NovoLog) (peptide hormone), Insulin glargine [rDNA derived] injection (Lantus) (peptide hormone), Insulin grildin [rDNA derived] injection (Apidra) (peptide hormone), Interferon Alpha-2b, Recombinant Injectable (Intron A) (Protein); Interferon beta-1b, recombinant, for injection (Betaferon, protein), Iplex (mecasermin rinfavert [rDNA derived] injection) (human growth factor), Iprivasc (Desirudin injection, protein), Istodax (romidepsin injection) (peptide), Kepivance (palifermin, keratinocyte growth factor), Keratinocytes (epidermal cells), KFG (keratinocyte growth factor), Kineret (anakinra, protein), Kinlytic (urokinase injection, enzyme), Kinrix (vaccine), Lente(L) (insulin zinc, peptide hormone), Leptin (peptide hormone), Levemir (insulin analog, peptide hormone), Leukine (sargramostim, protein), Leuprorelin acetate injection (Lupron, peptide), Levothyroxine (amino acid), Lexiscan (regadenoson injection) (nucleoside), Liraglutide injection (Victoza, peptide), Lucentis (ranibizumab injection) (antibody), Lumizyme (alglucosidase alpha, enzyme), Lutropin alpha (LH) injection (Luveris, a peptide hormone), Menactra (vaccine), Menotropin injections (Menopur, Repronex, Pergonal, peptide hormones), MetMab (Onartuzumab, antibody), Miacalcin (polypeptide), mipomersen (Kynamro oligonucleotide), Myozyme (alglucosidase alpha) (enzyme), NEO-GAA (Avalglucosidase alpha, enzyme), Neupogen (filgrastim, protein), Novolin (Novolin R: insulin, Novolin N: insulin isophane, peptide hormone), NeoRecormon (epoetin beta, protein), NPH(N) (Humulin N, Novolin N, Isofen Insulin, Peptide Hormones), Novolin 70 / 30 Innolet (70% NPH, human insulin isophane suspension and 30% regular, human insulin injection) (peptide hormone), Nplate (romiplostim, protein), Octreotide acetate injection (Sandostatin LAR, peptide), Ocrelizumab (Ocrevus, antibody), Orencia (abatacept, antibody), Osteoprotegerin (antibody), Oxytocin injection (Pitocin, a peptide hormone), Panitumumab intravenous injection (Vectibix, antibody), Parathyroid hormone (peptide hormone), Pediarix (vaccine), Peginterferon (peginterferon alpha-2a: Pegasys, peginterferon alpha-2b: PEGintron, Sylatron), Pegfilgrastim (Neulasta, Ristempa, protein), Pegfilgrastim-cbqv (Udenyca, protein), Pertuzumab (2C4, Omnitarg, Perjeta, antibody), Pramlintide acetate injection (Symlin, Symlin pen (management device), peptide hormone), R-Gene 10 (arginine hydrochloride injection) (amino acid), Raptiva (efalizumab, antibody), Recombivarix HB (vaccine), Remicade (infliximab, antibody), Retrovir IV (zidovudine injection) (nucleoside), rhApo2L / TRAIL (Dulanermin, protein), Rituximab (MabThera, Rituxan, Truxima, antibody), Roferon-A (interferon alpha-2a, protein), Somatropin injections (Accretropin, Genotropin, Humatrope, Saizen, Norditropin, Valtropin), Somatropin (rDNA derived) for injection (Nutropin, Nutropin depot, Nutropin AQ, Serostim LQ, Onmitrope, Tev-Tropin), Stelara injection (ustekinumab, antibody), Stemgen (Ancestim, antibodies), telavancin for injection (Vibativ, lipoglycopeptide); Tenecteplase (Metalyse, TNKase, protein), Thymoglobin (antithymocyte globulin (rabbit), antibody), Thyrogen (thyrotropin alpha injection, peptide hormone), Trastuzumab-Dml (antibody-drug conjugate), Travasol (amino acid (for injection)), Trelstar (triptorelin pamoate injectable suspension, peptide), Twinrix (vaccine), Typhoid Vi-polysaccharide vaccine (Thyphim Vi, vaccine), Urofollitropin for injection (Bravelle, Fertinex, Fertinorm, Metrodin, peptide hormone), Ultralente (U) (extended insulin zinc, peptide hormone), Vancomycin hydrochloride (vancomycin hydrochloride injection, glycopeptide), VAQTA (vaccine), Xolair (omalizumab, antibody), Zenapax (daclizumab, an antibody), and / or Zevalin (ibritumomab tiuxetan, antibody), The following can be mentioned.

[0043] In a sixth aspect, the present invention provides a container for a fluid comprising a substrate as described above and in the second aspect.

[0044] The container may be selected from a multi-well plate, a pipette, a bottle, a flask, a vial, an Eppendorf tube, and / or a culture plate.

[0045] The present invention is particularly useful for medical devices. Thus, in a seventh aspect, the present invention provides a medical device comprising a substrate as described above and in the second aspect.

[0046] The medical device may be a tube, for example a dispensing tube, a channel and / or a syringe, for example a disposable syringe.

[0047] In one embodiment, a syringe may be provided, the barrel of the syringe comprising a substrate (coated as described herein), at least one side surface of the substrate being an interior surface of the barrel.

[0048] Advantageously, doing so provides the coating with a significantly reduced slippage force when the syringe plunger is depressed.

[0049] As used herein, unless the context indicates otherwise, cyclic olefin polymers (COPs) herein include cyclic olefin copolymers (COCs). Proteinaceous compositions herein include peptides, oligopeptides, and / or polypeptides in the composition and may include additional ingredients such as excipients (e.g., sugar compounds such as polysorbates, lactose, dextrin, glucose, sucrose, and / or sorbitol), salts, solvents (and / or cosolvents), and other non-proteinaceous active pharmaceutical ingredients and formulations thereof. Polysaccharides include oligosaccharides, polyols, or mixtures thereof. [Brief description of the drawings]

[0050] Embodiments of the invention are explained in more detail with reference to the following figures. [Figure 1](a) Quantitative measurement of adsorbed BSA-FITC on untreated TOPAS™ (TW) and ZEONOR™ (ZW) surfaces retained in the form of hard (black bars) and soft (gray bars) layers, (b) Illustrative diagram showing the rinsing procedure developed to adjust the test sensitivity to hard (HL) and soft (SL) layers. [Diagram 2] FIG. 13 is a graph showing a summary of protein surface coverage measured on untreated and treated surfaces from 2 mg mL BSA-FITC incubation experiments on COP surfaces. [Diagram 3] Graph showing a comparison of luminescence data (ΔMFI) obtained from 2 mg mL-1 BSA-FITC incubation experiments on COP surfaces obtained by microscopy. An untreated surface was used as a reference for 100% luminescence. [Figure 4] Graph showing a comparison of luminescence data (ΔMFI) obtained from 2 mg mL-1 BSA-FITC incubation experiments on COP surfaces obtained by microscopy. An untreated surface was used as a reference for 100% luminescence. [Diagram 5] FIG. 13 is a graph summarizing protein surface coverage measured on untreated and PGA-treated syringes resulting from a 2 mg mL BSA-FITC incubation experiment. [Figure 6] FIG. 13 is a graph summarizing protein surface coverage measured on untreated and PGA-treated syringes resulting from a 2 mg mL insulin-FITC incubation experiment. [Figure 7] FIG. 1 shows (a) GATR-FTIR spectra of a Zeonor® coupon surface after rinsing with water (ZW) and treatment in HO for 30 min at 50° C. (ZP50); (b) UV-Vis absorbance spectra of a 1 mm Zeonor® coupon after rinsing with water only (ZW) and treatment in HO for 30 min at 50° C. (ZP50). [Figure 8]FIG. 13 shows (a) GATR-FTIR spectra of a Zeonor® coupon surface after rinsing with water (ZW) and oxidation treatment by exposure to a UV / ozone lamp for 5 minutes (ZU5) and 10 minutes (ZU10); (b) UV-Vis absorbance spectra of a 1 mm Zeonor® coupon after rinsing with water only (ZW) and oxidation treatment by exposure to a UV / ozone lamp for 5 minutes (ZU5) and 10 minutes (ZU10). [Figure 9] 1 is a graph showing water contact angle measurements taken on COP coupon surfaces after being subjected to various processing conditions with and without a water rinse and PGA. [Figure 10] 1 is a graph showing a comparison of the surface composition of a TOPAS coupon and a syringe type S1 analyzed by FTIR. [Figure 11] 1 is a graph showing a comparison of the surface composition of Zeonor coupons and syringe type S3 analyzed by FTIR. [Figure 12] 1 is a graph showing a comparison of the surface composition of a Zeonex coupon and a syringe type S3 as analyzed by FTIR. [Figure 13] 1 is a graph showing a comparison of the surface composition of a TOPAS coupon and a syringe type S2 analyzed by FTIR. [Figure 14] 1 is a graph showing a comparison of the surface composition of Zeonor coupons and syringe type S2 analyzed by FTIR. [Figure 15] 1 is a graph showing a comparison of the surface composition of a Zeonex coupon and a syringe type S2 as analyzed by FTIR. [Figure 16] FIG. 13 is a graph summarizing protein surface coverage measured on untreated and treated borosilicate glass surfaces resulting from 2 mg mL BSA-FITC incubation experiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] In this study, a fluorescently labeled globular protein, BSA-FITC, was used to observe the degree of protein surface adsorption on the substrate surface.

[0052] The substrates investigated were glass (especially borosilicate glass) and cyclic olefin polymer (COP) materials.

[0053] BSA is commonly used as an indicator of a surface's ability to resist non-specific protein adsorption.

[0054] A second (fluorescently labeled) protein, insulin-FITC, was used to confirm the generality of the effect and its applicability to therapeutic proteins.

[0055] [ka]

[0056] Different structures are obtained by the choice of R substituents: Topas™ is obtained by chain polymerization (upper route), whereas Zeonor™ is obtained by ring-opening metathesis (lower route). (Non-Patent Document 1: Shin JY et al., Pure and Applied Chemistry, (2005)77:801-814) (Non-patent document 2: Nunes et al., Microfluid Nanofluid, (2010)9:145-161)

[0057] Three types of COP materials were investigated: TOPAS® (T) (Topas™ Advanced Polymer), ZEONOR® (Z), and ZEONEX® (Zeon Corporation). The COP materials were procured from a commercial supplier in the form of 1 mm thick coupons. These COP materials are used by biodevice manufacturers for the biopharmaceutical industry. Scheme 1 shows the general structures of the different types of COP materials, where the properties can be tuned and the structures changed by varying the substituents.

[0058] To verify that the coupon results are applicable to biomedical devices, a study was conducted using a selection of syringe biodevices sold for prefilled biopharmaceuticals, supplied by three different manufacturers (manufacturers #1-#3). All syringes were made of COP material, and in the syringes manufactured by manufacturer #1, the inner surface (barrel) was siliconized.

[0059] The adsorption of proteins to surfaces is a complex process: proteins usually undergo complete and / or partial denaturation when adsorbed to a surface, which alters the strength and nature of the interactions involved in protein adhesion.

[0060] FIG. 1(a) shows a quantitative measurement of the amount of BSA-FITC adsorbed onto untreated Topas™ and Zeonor™ coupons.

[0061] Cut pieces or coupons (1.25 cm 2 ) of these two COP materials were 2 ) to 2 mg / mL -1 The coupons were immersed in a BSA-FITC solution in phosphate-buffered saline (PBS) at pH 7 at a concentration of 0.01% and incubated in the dark for 1 h to form a BSA adhesive layer on the COP surface. The coupons were then rinsed in PBS (method 1) or in PBS and elution buffer 1 (EB1 = PBS + 1% Triton X) (method 2) as shown diagrammatically in Figure 1(b). Method 1 is expected to leave most of the adsorbed protein consisting of soft and hard BSA. Method 2 is expected to remove most of the soft layer. After rinsing by methods 1 and 2, the attached BSA-FITC was extracted in a 1 mL volume for quantitative measurement by fluorescence. The extraction procedure consisted of a 17-h incubation in EB1 supplemented with 1% mercaptoethanol as a proteolytic agent to fragment the proteins and quantitatively release the FITC label into the solution. The emission intensity from the extraction solution at 495 nm excitation was used to quantify the proteins by calibration with BSA-FITC standards.

[0062] The present invention demonstrates the effectiveness of surface modification with polysaccharides, which shows great promise in addressing protein adsorption.

[0063] Other studies have shown that protein rejection is also observed on COP materials, i.e., on the inner surface of syringes used for biopharmaceuticals. Protein rejection appears to be common and has been observed in common probe globular proteins and therapeutic proteins of smaller size. EXAMPLES

[0064] <Example: Polymer substrate (substrate)> (Surface modification experiment) In the surface modification experiment, 1.25 cm TOPAS (T), ZEONOR (Z), and ZEONEX (ZX) were used. 2 Coupons were used, which were subjected to two different types of pretreatments before modification with sugars (id1# in sample nomenclature). (1) Rinse with Millipore water (TW, ZW, ZXW). (2) Mild surface oxidation using 30% hydrogen peroxide at 50°C (TP50, ZP50 or ZXP50).

[0065] The pretreated coupons were then soaked in 1 mg mL of various sugars. -1 Surface modification was performed by incubation in solution. Scheme 2 shows the structures of the polysaccharides evaluated in the experiment (id2# in sample nomenclature), namely dextran (D), polygalacturonic acid (PGA), hyaluronic acid (H), or no saccharide (NS). The following incubation conditions were tested (id3# in sample nomenclature): (1) Sugars (1 mg / mL) -1 in deionized water at room temperature for 2 hours (W). (2) Sugars (1 mg / mL) -1 The cells were incubated for 30 minutes in deionized water at 50°C for four consecutive periods (total of 2 hours) (W50X4). (3) Sugars (1 mg / mL) -1 The cells were incubated in 30% H2O2 at 50°C for 30 min for four consecutive cycles (total 2 h) (P50X4).

[0066] After the incubation period, all samples were rinsed with deionized water and used for screening of protein adsorption. As shown in Scheme 2, each sample is referenced by a combination of the pretreatment performed (id1#), the sugar used (id#2), and the modification treatment performed (id3#), identifying the treatment that each sample surface underwent.

[0067] [ka]

[0068] (Protein Adsorption Test Protocol) The solution of BSA-FITC was 2 mg / mL in phosphate-buffered saline (PBS) at pH 7. -1 A coupon of COP material was immersed in the BSA-FITC solution and incubated in the dark for 1 h. The material was then rinsed with PBS (Method 1) and used for the following quantitative or qualitative measurements.

[0069] (a. Quantitative measurement by release from solution) After rinsing the attached BSA-FITC, 1 mL was extracted for quantitative measurement by fluorescence. The extraction procedure consisted of a 17-h incubation in EB1 supplemented with 1% mercaptoethanol as a proteolytic agent to fragment the proteins and quantitatively release the FITC label into solution. Emission intensity from the extraction solution at 470 nm excitation was used to quantify the proteins by calibration with BSA-FITC standards. The surface coverage of the proteins was calculated by normalizing the total amount of extracted protein with the COP area exposed during incubation. Error bars in all graphs correspond to 95% CI.

[0070] (b. Qualitative comparison using a fluorescent microscope.) The rinsed coupons were imaged using an upright microscope with 470 nm excitation and a FITCexc / em filter cube, and the integrated intensity at the COP surface was measured by commercial software. Method 1 provides good sensitivity for both soft and hard adsorbed layers (Figure 1). The mean fluorescence intensity (MFI) through the emission filter was measured from multiple images and corrected by the corresponding background emission of unused COP material (ΔMFI). Error bars in all graphs correspond to 95% CI.

[0071] (BSA-FITC adsorption results of COP coupons) Figure 2 shows the results of quantitative measurements of BSA-FITC adsorption on Topas™, Zeonor™, and Zeonex surfaces. The ##-NS-W sample serves as a control, as it mimics the adsorption expected, for example, in syringe barrels without pretreatment or modification. It is clear that modification with PGA polysaccharides results in the greatest reduction in the density of protein adsorbates. The best reduction, 52%, was observed for TP50-PGA-P50X4. Table A ([Table 1]) gives an overview of the protein removal results, calculated as a percentage of adsorption relative to the untreated coupon surface.

[0072] The changes in protein adsorption were also confirmed by qualitative fluorescence microscopy, as shown in Figure 3. Emissions from the coupon surfaces detected by microscopy indicate that PGA treatment reduces the release from adsorbed BSA-FITC on all types of COP coupons tested.

[0073] [Table 1] Table A summarizes the protein rejection measurement results calculated from the average values ​​shown in FIG.

[0074] Figure 4 shows the total light emission from adsorbed BSA-FITC on the three polymeric materials evaluated after treating the coupons with PGA alone, hydrogen peroxide alone, or a combination of PGA and hydrogen peroxide treatments. It is clear that PGA alone does not cause as great a reduction as when the surfaces are treated in combination with peroxide. On the other hand, peroxide clearly has a significant negative effect on protein rejection unless PGA is added to the treatment solution.

[0075] (Protein adsorption results using COP syringe) Figure 5 shows the results of measurements of the amount of BSA-FITC adsorption on COP syringes from manufacturers #1, #2, and #3. The ##-NS-W syringe provides a control, as it reports the adsorption expected on a clean syringe barrel without any pretreatment or modification. While the unused syringes show comparable surface coverage of the adsorbate to that measured on the coupon samples, it is clear that PGA modification significantly reduces BSA-FITC adsorption on #1 syringe (79%) and #2 syringe (54%). No significant reduction was observed on #1 syringe. However, this is consistent with the inner surface of these devices being siliconized. Thus, it indicates that the COP surface is most effective when subjected to polysaccharide treatment directly on the surface without a silica coating.

[0076] Considering the success of the modification experiments with syringes #2 and #3, quantitative measurements were also performed with another type of protein, insulin-FITC. Insulin-FITC is a protein used for therapeutic applications in an unlabeled form. Figure 6 shows the results of quantitative measurements with insulin-FITC. It is clear that a reduction due to PGA modification was also obtained with syringes #2 (83%) and #3 (52%) for this protein.

[0077] (Effect of surface treatment of COP material) The effects of solution treatment and reaction conditions were investigated using Ge attenuated total internal reflection infrared spectroscopy (GATR-FTIR), water contact angle (WCA), and transmittance UV-Vis spectroscopy. Figure 7(a) shows the GATR-FTIR spectra of a COP coupon before and after exposure to H2O2 at 50 °C. The spectrum shows a peak at 1709 cm, which is diagnostic of carbonyl functional groups. -1 The COP shows the appearance of a clear absorbance peak at 1000 nm, indicating that the COP is oxidatively activated when exposed to peroxide under reaction conditions. However, as shown in the control UV-Vis absorbance spectrum in Figure 7(b), this oxidation is mild and restricted to the surface of the material, with no change in the bulk optical properties.

[0078] This is in contrast to other surface oxidation methods such as exposure to UV / ozone lamps. Figures 8(a) and 8(b) show the GATR and UV-Vis absorbance of the same type of COP coupon after oxidation by UV / ozone lamp irradiation (10 min). The appearance of carbonyl peaks is evident in the GATR-FTIR spectrum after oxidation, while the UV-Vis absorbance spectrum shows a significant increase in absorbance, indicating a change in the bulk structure of the COP polymer. Thus, oxidation by H2O2 is relatively mild and does not significantly change the bulk material.

[0079] WCA measurements were performed to observe the change in hydrophilicity due to surface treatment. Figure 9 shows the WCA values ​​obtained for the COP surfaces of the three polymers treated with and without PGA under different conditions. The measurements show that after exposure to H2O2 alone, only a small change in the hydrophilic properties is observed, whereas exposure to PGA results in a significant increase in hydrophilicity.

[0080] 10-15 show a comparison of the FT-IR spectra of the COP material (as a coupon) and the syringe materials mentioned above (types S1, S2, and S3, supplied by manufacturers #1, #2, and #3, respectively).

[0081] (Conclusion) For COP materials, the protein adsorption can be further reduced by combining a surface oxidation process and immobilization of polysaccharides.

[0082] Protein rejection appears to be common and has been observed in common probe globular proteins and therapeutic proteins of smaller size.

[0083] <Example: Glass substrate (substrate)> The surface coverage of proteins was measured on untreated borosilicate glass and on borosilicate glass modified by two different procedures.

[0084] Untreated borosilicate glass was washed with acetone, isopropanol, and deionized water prior to exposure to proteins.

[0085] In both procedures, the modified borosilicate glass was subjected to an oxidation treatment by immersion in a piranha solution (1H2O2 30%:3H2SO4) for 45 minutes. The oxidation treatment may be replaced by or include a treatment step with an alkaline aqueous solution. The alkaline aqueous solution generally has a pH of 7-14, optionally a pH of 9-14, optionally a pH of 10-14, and a temperature in the range of 40°C to 70°C.

[0086] A second oxidation treatment was then carried out using two different procedures. (1) P50: Borosilicate glass was immersed in 30% H2O2 at 50°C for 30 minutes. (2) U10: Both sides of borosilicate glass were irradiated with a UV ozone lamp for 10 minutes.

[0087] Following the oxidation treatment, the borosilicate glass was functionalized with PGA by immersing it in a 1 mg / mL solution of PGA in 30% H2O2 at 50 °C for 30 min. This treatment was repeated four times for a total of 2 h, changing the PGA solution in peroxide after each treatment (PGA-P50X4).

[0088] The borosilicate glass surface was rinsed with deionized water before exposure to the BSA protein.

[0089] Adsorbed protein was quantitatively measured by emission from solution. After rinsing the attached BSA-FITC was extracted for quantification by fluorescence. The extraction procedure consisted of a 17-h incubation in EB1 supplemented with 1% mercaptoethanol as a proteolytic agent to fragment the proteins and quantitatively release the FITC label into solution. Emission intensity from the extraction solution at 470 nm excitation was used to quantify the proteins by calibration with BSA-FITC standards. The surface coverage of the proteins was calculated by normalizing the total amount of extracted protein with the area exposed during incubation. Error bars in all graphs correspond to 95% CI.

[0090] (Abbreviation) [Table 2]

[0091] <Examples: COP, COC, glass substrate (substrate), and sliding force> (Coupon and syringe coating methods and resistance testing) (solution) (1) PBS buffer: 4.58 g Na2HPO4 and 2.12 g NaH2PO4 in 1 L of Millipore water (2) Sugars in peroxide, concentration 1 mg / mL (SP): 6 mg of sugar in 6 mL of 30% H2O2, 4 times. The SP solution was prepared immediately before the temperature treatment. (3) 2xSSPE buffer: 25 mL of 20xSSPE buffer in 225 mL of Millipore water (4) EB2: 0.5 mL of TritonX-100 and 0.5 mL of mercaptoethanol in 49 mL of 2xSSPE buffer. (5) BF (2 mg / mL): 36 mg of BSA-FITC in 18 mL of PBS buffer.

[0092] (Coating Procedure) (1) The glass / polymer substrate was cleaned according to the following procedure. (a) Millipore water (W): The substrate was rinsed twice with acetone, isopropanol, and Millipore water, changing the water after each rinse. For glass, the rinsing steps may include a pre-rinse with acetone and alcohol (e.g., isopropanol), and a rinse with an aqueous alkaline solution (e.g., NaOH) at pH ≥ 10 and ≤ pH 14, 40°C to 70°C. (b) Hydrogen peroxide 30% T50℃ (P50) or T70℃ (P70): The sample was immersed in H2O2 and placed in a water bath at 50℃ or 70℃ for 15 minutes. (2) The W-NS-W substrate was immersed in water. (3) Substrates with P50 or P70 pretreatment were placed in sugar solution and placed (e.g. in a water bath) at 50° C. for 30 min or at 70° C. for 15 min. The solution may alternatively be sprayed onto the sample. (4) The above procedure was repeated four times, with the sugar solution being replaced each time. (5) The samples were rinsed three times with fresh Millipore water, changing the water after each rinse.

[0093] (Resistance Test) (pH) (1) P50-S-P50X4 polymer or P70-S-P70X4 glass was placed under the following conditions: (a) pH 4: The glass was placed in 1 mL of pH 4 solution at 4° C. for 48 h. (b) pH 10: The glass was placed in 1 mL of pH 10 solution at 4°C for 48 h.

[0094] (temperature) (2) P50-S-P50X4 polymer or P70-S-P70X4 glass was placed under the following conditions: (a) -20°C: The wet glass was placed at -20°C for one week. (b) 4°C: The glass was placed in 1 mL of Millipore water for 1 week. (c) 20°C: The glass was placed in 1 mL of Millipore water for 1 hour. (d) 120°C: The wet glass was placed in an autoclave at 120°C for 20 minutes.

[0095] (Stress / Shear) (3) The P50-S-P50X4 polymer or P70-S-P70X4 glass was placed in 0.5 mL of Millipore water. (4) Shake at 500 rpm for 17 hours.

[0096] (culture) (5) P50-S-P50X4 polymer or P70-PGA-P70X4 glass was placed in 1 mL of BF. (6) Leave it in a dark place at 4°C for one week.

[0097] (storage) (1) P50-S-P50X4 polymer or P70-PGA-P70X4 glass was placed in 1 mL of Millipore water. (2) Leave it in a dark place at 4°C for one week.

[0098] (Quantitative measurement of protein adsorption) In this example, the eluted proteins were detected by using a fluorescent detection method.

[0099] Coupons of the material to be tested were cut to a known surface area. They were then immersed in a solution containing the formulation to be tested (e.g., buffer). A stock solution of the protein-FITC conjugate to be tested (e.g., BSA-FITC) was pipetted in, and the solution was diluted to the protein concentration to be tested (e.g., 2 mg mL -1 ) at 20°C. The coupons were incubated in the dark at the temperature to be tested (e.g., 20°C) for 1 hour to allow the formation of a protein adsorption layer. The coupons were then rinsed with phosphate buffered saline (pH 7) to remove excess / unbound conjugate. The coupons were then incubated for 17 hours in a known volume of elution buffer containing surfactants and proteolytic agents to promote desorption and proteolysis of surface-adsorbed protein-FITC. The fluorescence spectrum of the extracted solution was measured in a cuvette using a fluorometer. λ em,maxThe luminescence intensity at 100 nm was used to determine the protein concentration in the elution volume by calibration with protein-FITC standards. If applicable, the elution solution was diluted with PBS to bring the luminescence within the dynamic linear range, and the dilution factor was used to determine the total protein amount in the extraction volume. Finally, the total protein amount extracted was normalized to the exposed surface area to obtain the protein removal value (Γ protein ,%) was calculated.

[0100] Tables 1 to 4 (Tables 3 to 6) show the test results of COC, COP, and glass coupons at shear / stress (500 rpm, 17 hours), various pH values, various temperatures, and various times. In the tables, "PGA coating" refers to the substrate coated as described above.

[0101] [Table 3] Table 1: Averages calculated for N=3 for COC and COP standards, N=8 for COC and COP shear / stress, N=12 for glass standards, and N=6 for glass standards.

[0102] [Table 4] Table 2: Calculated averages for N=3 for COC and COP at three pHs, N=12 for glass standards and N=6 for glass shear / stress, and N=4 for glasses at pH 4 and pH 10.

[0103] [Table 5] Table 3: Averages calculated for N=15 for COC and COP at 20°C, N=5 for COC and COP at 4°C, N=3 for COC and COP at -20°C, N=12 for glass at 20°C, N=5 for glass at 4°C, N=6 for glass at -20°C, and N=5 for COC and COP at 120°C.

[0104] [Table 6] Table 4: Means calculated for N=15 for COC and COP at t0, N=5 for COC and COP at 1 week and 4 weeks, and N=5 for glass at 1 week

[0105] (Slip force measurement) The sliding force was measured on the coated (PGA coated) or untreated (as a control) inner syringe surface of a syringe barrel equipped with a plunger having a lubricated elastomeric tip. The syringe was filled with the test liquid. The force exerted on the plunger was measured as a function of displacement and the average force was determined.

[0106] Tables 5 and 6 ([Table 7] and [Table 8]) show the results (and standard deviations) of the glide force measurements for coated and untreated syringes made of COP1, COP2, or glass.

[0107] [Table 7] Table 5: Averages calculated for N=3 for untreated COP1 and glass syringes and N=5 for PGA coated COP1 and glass syringes.

[0108] [Table 8] Table 6: Calculated averages for untreated and PGA coated COP syringes with N=5

[0109] (coating roughness and thickness) AFM height profile measurements in air showed a smooth surface topography with Ra = 2.7 ± 0.2 nm. The coating thickness by the trench method was d = 4.5 ± 0.7 nm.

[0110] UHV X-ray photoelectron scattering measurements showed a chemical composition consistent with surface-bound sugar units. The average thickness by substrate attenuation measurements was d = 2.5 nm.

[0111] (References) 1.(a) Gross, T., Ramm, M., Sonntag, H., Unger, W., Weijers, HM, & Adem, EH Surf. Interface Anal. 1992 18, 59;(b) Sawyer, Nesbitt & Secco J. Non-Cryst. Solids, 2012, 358, 290. 2. Jablonski & Zemek Surf. Interface Anal. 2009, 41, 193. 3. Briggs & Beamson Anal. Chem. 1992, 64, 1729. 4. Clare, TL, Clare, BH, Nichols, BM, Abbott, NL & Hamers, RJ Langmuir 2005, 21 (14), 6344. 5. Srinivasan & Nair, Clin.Mater.1990, 6, 277.

[0112] The disclosures of the publications referenced herein are incorporated by reference in their entireties.

Claims

1. 1. A method for coating a surface of a substrate, comprising the steps of: (a) providing a substrate having a surface; (b) optionally treating at least a portion of the substrate surface with an oxidizing agent; (c) treating at least a portion of the substrate surface with a composition comprising a polysaccharide, an oligosaccharide, a polyol, or a mixture thereof; and (d) incubating the treated substrate with the composition for a period of time; A method comprising:

2. The method of claim 1 , wherein the substrate comprises quartz or glass.

3. The method of claim 2 , wherein the substrate comprises borosilicate glass.

4. The method of claim 1 , wherein the substrate comprises a cyclic olefin polymer and / or copolymer.

5. 2. The method of claim 1, wherein the polysaccharide comprises a hexose-derived polysaccharide or oligosaccharide.

6. 2. The method of claim 1, wherein the polysaccharide contains 20% or more oxidized hexoses at the C6 position.

7. 2. The method of claim 1, wherein the polysaccharide is selected from dextrin, dextran polygalacturonic acid, hyaluronic acid, or a combination of two or more of these polysaccharides.

8. 10. The method of claim 1, wherein the oxidizing agent comprises a peroxide, optionally comprising hydrogen peroxide, optionally comprising 30% w / w hydrogen peroxide in an aqueous solution.

9. The method of claim 1 , wherein the predetermined time period is in the range of 0.5 minutes to 240 minutes.

10. 10. The method of claim 1, wherein the treating and / or culturing of at least a portion of the substrate surface is carried out at a temperature in the range of 10°C to 90°C.

11. The method of claim 1 , wherein the composition comprises water.

12. The method of claim 1 , wherein the composition comprises an oxidizing agent.

13. 13. The method of claim 12, wherein the oxidizing agent in the composition is a peroxide, O ３ , ozonated water, H ２ O ２ , periodate, hypochlorite, and / or permanganate, and optionally hydrogen peroxide, optionally 30% w / w hydrogen peroxide in aqueous solution.

14. 10. The method of claim 1, further comprising treating the substrate with a basic aqueous solution having a pH of 9-14.

15. A coated substrate obtained by coating at least one surface of a substrate by the method according to any one of claims 1 to 14.

16. A substrate having a coating on at least one surface, the coating comprising a polysaccharide, an oligosaccharide, a polyol, or a mixture thereof in direct contact with the surface of the substrate.

17. The substrate of claim 15 , wherein the substrate comprises quartz or glass.

18. 20. The substrate of claim 17, wherein the substrate comprises borosilicate glass.

19. The substrate of claim 15, wherein the substrate comprises a cyclic olefin polymer.

20. 16. The substrate of claim 15, wherein the polysaccharide comprises dextrin, polygalacturonic acid, hyaluronic acid, or a combination of two or more of these polysaccharides.

21. A method of using a container, comprising:

16. A method of use, wherein the container has a coated substrate according to claim 15 for storing a pharmaceutical protein composition, thereby enhancing the intrinsic and / or thermal stability of the pharmaceutical protein composition.

22. 16. A method of using a container, said container having a coated substrate according to claim 15 for storing a pharmaceutical composition, thereby enhancing the intrinsic and / or thermal stability of said pharmaceutical proteinaceous composition, said pharmaceutical proteinaceous composition comprising a proteinaceous composition, a liposome-containing composition, or an oligonucleotide-containing composition.

23. 1. A method for reducing aggregation and / or adsorption of proteins or oligonucleotides on a substrate surface, comprising: (a) providing a coated substrate according to claim 15; (b) contacting said surface with a proteinaceous composition or a composition comprising an oligonucleotide; A method comprising:

24. 24. The method of claim 23, wherein the proteinaceous composition comprises a pharmaceutical proteinaceous composition.

25. 24. The method of claim 23, wherein the pharmaceutical proteinaceous composition comprises a monoclonal antibody composition.

26. 24. The method of claim 23, wherein the pharmaceutical proteinaceous composition comprises a peptide hormone.

27. 24. The method of claim 23, wherein the pharmaceutical proteinaceous composition comprises one or more peptides or combinations thereof, any vaccine, erythropoietin, interferon (α-, β-, and / or γ-interferon), infliximab, etanercept, adalimumab, rituximab, infliximab, trastuzumab, insulin, glucagon, and / or gonadotropic hormones.

28. A container comprising the substrate of claim 15.

29. 30. The container of claim 28, wherein the container is selected from a multi-well plate, a pipette, a bottle, a flask, a vial, an Eppendorf tube, and / or a culture plate.

30. A medical device comprising the substrate of claim 15.

31. 31. The medical device of claim 30, wherein the medical device is a dispensing tube, a device with a channel, or a syringe.

32. 20. A syringe having a barrel, the barrel comprising the substrate of claim 15 and the at least one surface of the substrate being an interior surface of the barrel.

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