Polymer coating method and method for reducing protein aggregation

Abstract

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

Description

[Technical Field]

[0001] The present invention relates to methods for coating polymer surfaces, the resulting coated polymers, and methods for reducing protein aggregation on polymer surfaces. The present invention also relates to fluid containers, medical devices, and syringes comprising the coated polymers. [Background technology]

[0002] Protein aggregation and denaturation can occur in formulations of proteinaceous compositions contained in devices, causing problems in diagnostics, analysis, and drug delivery. Controlling the formation and denaturation of protein aggregates is problematic.

[0003] Nonspecific protein adsorption is a complex phenomenon. This 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, and vials, 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. Carryover occurs due to nonspecific adsorption of analytes to parts of the analytical system, resulting in bias in both identification and quantitative assays. Therefore, 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. The physicochemical properties of the container material contribute to maintaining the integrity and stability of the drug substance. Protein adsorption to the container surface can lead to a loss of potency of the protein in solution due to changes in concentration, protein denaturation, and / or degradation. Protein aggregation and denaturation of pharmaceutical compositions (antibodies, proteins, and other peptides, such as erythropoietin, interferon gamma, infliximab, etanercept, and adalimumab, all of which can be delivered prefilled in syringes) can also trigger 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 alleviate the problem. Surface modification of protein-contacting materials used in manufacturing and storage, such as ethylene vinyl acetate (EVA) copolymer 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 to the surface that comes into contact with the composition.

[0007] WO 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 in a lumen. The drug contact coating is located on or adjacent to the interior surface of the container so as to be in contact with fluid within the lumen, and is a corrosion-reducing barrier, SiO x C y H z It essentially consists of:

[0008] U.S. Patent Application Publication No. 2015 / 0126941 discloses a filled package including 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 a coating of SiO x (x is 1.5 to 2.9). The protective coating includes a layer of sugars that prevents leaching.

[0009] There is a need to provide a surface of a material that is less susceptible to protein aggregation and denaturation and does not suffer from the problems of the prior art.

[0010] The purpose of the present invention is to address this need. Summary of the Invention

[0011] Accordingly, in a first aspect, the present invention provides a method for coating a polymer surface, the method comprising: (a) providing a polymer having a surface; (b) optionally treating at least a portion of the polymer surface with an oxidizing agent; (c) treating at least a portion of the polymer surface with a composition comprising a polysaccharide, oligosaccharide, polyol, or mixtures thereof; and (d) incubating the treated polymer with the composition for a predetermined period of time.

[0012] 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.

[0013] The composition may be applied over one or more other coating layers (except a layer of silica) already deposited on the polymer surface. Preferably, the polymer surface does not include a silica coating.

[0014] Preferably, however, the composition is applied directly to the polymer surface and does not require an inorganic layer already deposited on the polymer surface. Thus, preferably, the method involves directly treating the polymer surface. Generally, any suitable polymer (e.g., EVA, polyolefin (e.g., polyethylene or polypropylene), polyester (e.g., polyethylene terephthalate), polycarbonate, or any combination or copolymer thereof) can be used in the method, but preferably, the polymer can include a cyclic olefin polymer or copolymer. The polymer (e.g., cyclic olefin polymer) can include at least a portion of recycled polymer.

[0015] 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).

[0016] Although it is believed that many polysaccharides, oligosaccharides, polyols, or mixtures thereof may be useful in this method, polysaccharides may include hexose-derived polysaccharides. The polysaccharides may be polyhydroxylated. Generally, the polysaccharides, when applied to a polymer surface, may 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).

[0017] Preferred polysaccharides are selected from dextran, cellulose, one or more polyols, dextrin, polygalacturonic acid, hyaluronic acid, or a combination of two or more of these polysaccharides.

[0018] The use of these polysaccharides, oligosaccharides, polyols or mixtures thereof is highly advantageous as the inventors have confirmed that they significantly reduce protein aggregation when applied to the surface of a COP.

[0019] The oxidizing agent preferably affects the surface of the polymer but does not adversely affect the bulk of the polymer. The oxidizing agent may include peroxide, optionally hydrogen peroxide, optionally hydrogen peroxide in a 30% w / w aqueous solution. Generally, peroxide and / or other oxidizing agents, such as O, ozonated water, H O with or without a decomposition catalyst (e.g., Cu ions, Fe ions, manganese oxide), periodate, hypochlorite, and / or permanganate, may also be suitable.

[0020] The predetermined time can be in the range of 0.5 minutes to 240 minutes. Any other range of the predetermined time can be 1 minute to 120 minutes, 1 minute to 60 minutes, 1 minute to 30 minutes, 1 minute to 20 minutes, or 1 minute to 10 minutes.

[0021] Treating and / or incubating at least a portion of the polymer surface may be carried out at a temperature ranging from 10°C to 90°C, optionally from 10°C to 70°C.

[0022] Treatment of at least a portion of the polymer surface and / or treatment during incubation may include mechanical, chemical or electromagnetic acceleration of the process, for example by sonication, microwave irradiation, and / or ion catalysis.

[0023] The composition may be an aqueous solution. Thus, the composition may contain water. Where appropriate, one or more co-solvents may be present.

[0024] In some embodiments, the composition may include an oxidizing agent. The oxidizing agent in the composition may include a peroxide, optionally hydrogen peroxide, optionally hydrogen peroxide in a 30% w / w aqueous solution.

[0025] The polymer obtained by this method significantly reduced protein aggregation.

[0026] Thus, in a second aspect, the present invention provides a coated polymer obtainable by coating at least one surface of a polymer according to the method of the first aspect.

[0027] Optionally, the coated polymer does not include a silica coating.

[0028] Thus, in a third aspect, the present invention provides a polymer having a coating on at least one surface, the coating comprising a polysaccharide in direct contact with the surface of the polymer.

[0029] The polymer preferably comprises a cyclic olefin polymer.

[0030] The polysaccharide preferably comprises dextran, cellulose, a polyol (eg, hydrogenated hydrolysate of starch), dextrin, polygalacturonic acid, hyaluronic acid, or a combination of two or more of these polysaccharides.

[0031] The coated polymers of the present invention have an additional advantage in that they enhance the thermal and intrinsic stability of compositions stored in contact with the coated surface (e.g., compared to uncoated surfaces or other materials).

[0032] Thus, in a fourth aspect, the present invention provides the use of a container comprising a coated polymer according to the third aspect for storing a pharmaceutical composition (optionally a peptide composition), thereby enhancing the intrinsic and / or thermal stability of the pharmaceutical composition.

[0033] Thus, in a fifth aspect, the present invention provides a method for reducing aggregation or adsorption of proteins or oligonucleotides on a polymer surface, the method comprising (a) providing a polymer according to the above and second aspects, and (b) contacting the surface with a proteinaceous or oligonucleotide composition. As noted above, this is advantageous as it provides improved storage conditions, for example allowing storage at higher temperatures and / or for longer periods of time than previously possible.

[0034] The proteinaceous composition may comprise a pharmaceutical proteinaceous composition. The pharmaceutical proteinaceous composition may comprise a monoclonal antibody composition or a peptide hormone.

[0035] In some embodiments, the pharmaceutical proteinaceous composition may comprise one or more vaccines (e.g., peptide-containing vaccines), erythropoietin, interferon (α-, β-, and / or γ-interferon), infliximab, etanercept, adalimumab, rituximab, infliximab, trastuzumab, insulin, glucagon, and / or gonadotropins.

[0036] The pharmaceutical composition may include an injectable composition. Examples of injectable compositions include: Abarelix - depot formulation (hormonal drug), AbobotulinumtoxinA injection (Dysport), Acetadote (acetylcysteine ​​injection), Actemra (tocilizumab injection), Acthrel (corticorelin oubain triflutate injection), Actimmune (interferon gamma-1b), Adacel (vaccine), adalimumab (Humira), Adenoscan (adenosine injection), Aldurazyme (laronidase), Alglucerase injection (Ceredase), Alkeran injection (melphalan hydrochloride injection), ALTU-238 (human growth hormone), Arzerra (ofatumumab injection), Avastin (bevacizumab), Azactam injection (aztreonam injection), BayHepB (hepatitis B human immunoglobulin, antibody), BayTet (tetanus immune globulin, antibody), Bexxar (tositumomab) (antibody), Blenoxane (bleomycin sulfate injection, a peptide antibiotic), Botox cosmetic (onabotulinumtoxin A injection, protein) BR3-FC (protein), Briobercept (antibody), BTT-1023 (antibody), Byetta (exenatide, protein), Campath (alemtuzumab, antibody), Canakinumab injection (Ilaris, antibody), Carticel (chondrocyte), Cathflo (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 alfa (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), DMOADs (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), Nephramine (amino acid mixture), Fabrazyme (agalsidase beta, enzyme), Fluarix Quadrivalent (vaccine), Fludara (fludarabine phosphate) (a nucleotide analog derivative), Follitropin alfa injection (Gonal-f RFF, Cinnal-f, Fertilex, Ovaleap, Bemfola, peptide hormone), Follitropin beta injection (Follistim, Follistim AQ cartridge, Puregon, peptide hormone), Follitropin delta injection (Rekovelle, a peptide hormone), Forteo (teriparatide (rDNA-derived) injectable peptide hormone), foscarnet 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, a peptide hormone), Havrix (vaccine), Herceptin (trastuzumab, antibody), hG-CSF (human granulocyte colony-stimulating factor, protein), Humalog (insulin lispro, a 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 (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 for injection (Intron A) (protein); Interferon beta-1b, recombinant, for injection (Betaferon, protein), Iplex (mecasermin linfavert [rDNA-derived] injection) (human growth factor), Iprivask (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 alfa (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, Isophane 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 (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), Thymoglobulin (antithymocyte globulin (rabbit), antibody), Thyrogen (thyrotropin alpha injection, peptide hormone), Trastuzumab-Dml (antibody-drug conjugate), Travasol (amino acid (injection)), Trelstar (triptorelin pamoate injectable suspension, peptide), Twinrix (vaccine), Typhoid Vi-polysaccharide vaccine (Thyphim Vi, vaccine), Urofollitropin 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), may include:

[0037] In a sixth aspect, the present invention provides a container for a fluid comprising a polymer as described above and in the second aspect.

[0038] 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.

[0039] The present invention is particularly useful for medical devices.Accordingly, in a seventh aspect, the present invention provides a medical device comprising a polymer as discussed above and in the second aspect.

[0040] The medical device may be a dispensing tube, channel and / or syringe, for example a disposable syringe.

[0041] In this specification, unless the context indicates otherwise, the cyclic olefin polymer (COP) referred to herein includes cyclic olefin copolymer (COC).The proteinaceous composition referred to herein includes peptides, oligopeptides, and / or polypeptides in the composition, and may include additional components such as excipients (e.g., sugar compounds such as lactose, dextrin, glucose, sucrose, and / or sorbitol), salts, solvents (and / or cosolvents), and other non-proteinaceous active pharmaceutical ingredients, and their formulations.Polysaccharides include oligosaccharides, polyols, or mixtures thereof.

[0042] Embodiments of the present invention will now be described in more detail with reference to the accompanying drawings. [Brief explanation of the drawings]

[0043] [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) rinsing protocols developed to adjust assay sensitivity to hard (HL) and soft (SL) layers. [Figure 2] FIG. 1 shows a summary of the protein surface coverage measured on untreated and treated surfaces obtained from a 2 mg mL BSA-FITC incubation experiment on COP surfaces. [Figure 3] Figure 1 shows a comparison of luminescence data (ΔMFI) obtained from 2 mg mL BSA-FITC incubation experiments on COP surfaces, obtained by microscopy. An untreated surface is used as a reference 100% luminescence. [Figure 4] Figure 1 shows a comparison of luminescence data (ΔMFI) obtained from 2 mg mL BSA-FITC incubation experiments on COP surfaces, obtained by microscopy. An untreated surface is used as a reference 100% luminescence. [Figure 5] FIG. 1 shows a summary of the protein surface coverage measured on untreated and PGA-treated syringes from a 2 mg mL BSA-FITC incubation experiment. [Figure 6] FIG. 1 shows a summary of the protein surface coverage measured on untreated and PGA-treated syringes from a 2 mg mL insulin-FITC incubation experiment. [Figure 7] Figure 1 shows (a) GATR-FTIR spectra of a Zeonor™ coupon surface after rinsing with water (ZW) and after treatment in HO for 30 minutes at 50°C (ZP50), (b) UV-Vis absorbance spectra of a 1 mm Zeonor™ coupon after rinsing with water only (ZW) and after treatment in HO for 30 minutes at 50°C (ZP50). [Figure 8]Figure 1 shows (a) GATR-FTIR spectra of a Zeonor™ coupon surface after rinsing with water (ZW) and after oxidation treatment via 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 after oxidation treatment via exposure to a UV / ozone lamp for 5 minutes (ZU5) and then 10 minutes (ZU10). [Figure 9] FIG. 1 shows water contact angle measurements obtained on COP coupon surfaces after rinsing with water and undergoing various treatment conditions with and without PGA. [Figure 10] FIG. 1 shows a comparison of the surface composition of a TOPAS coupon and syringe type S1 analyzed by FTIR. [Figure 11] FIG. 1 shows a comparison of the surface composition of a Zeonor coupon and a syringe type S3 analyzed by FTIR. [Figure 12] FIG. 1 shows a comparison of the surface composition of a Zeonex coupon and a syringe type S3 analyzed by FTIR. [Figure 13] FIG. 1 shows a comparison of the surface composition of a TOPAS coupon and syringe type S2 analyzed by FTIR. [Figure 14] FIG. 1 shows a comparison of the surface composition of a Zeonor coupon and syringe type S2 analyzed by FTIR. [Figure 15] FIG. 1 shows a comparison of the surface composition of a Zeonex coupon and syringe type S2 analyzed by FTIR. DETAILED DESCRIPTION OF THE INVENTION

[0044] In this study, a fluorescently labeled globular protein, BSA-FITC, is used to monitor the degree of protein surface adsorption on cycloolefin polymer (COP) materials. BSA is typically used as an indicator of a surface's ability to resist nonspecific protein adsorption. A second (fluorescently labeled) protein, insulin-FITC, is used to confirm the generality of the effect and its applicability to therapeutic proteins.

[0045] [ka]

[0046] Scheme 1. General structure of COP materials and examples of polymerization methods. Structural variations can be achieved by the choice of R substituents. Topas™ is obtained by chain polymerization (top route), while Zeonor™ is obtained by ring-opening metathesis (bottom route). 1、2 . 1: Shin JY et al., Pure and Applied Chemistry, (2005)77:801-814.2 2: Nunes et al., Microfluid Nanofluid (2010) 9:145-161)

[0047] Three types of COP materials were investigated. These were TOPAS® (T) (Topas™ Advanced Polymer), ZEONOR® (Z), and ZEONEX® (Zeon Corporation), procured from commercial suppliers in the form of 1 mm thick coupons. These materials are used by biodevice manufacturers for the biopharmaceutical industry. Scheme 1 shows the general structures of the various types of COP materials. Structural variation can be achieved through variation of the substituents, which provides tunable properties.

[0048] To verify that the coupon results are generalizable to biomedical devices, a study was conducted using selected syringe biodevices sold for prefilled biopharmaceuticals supplied by three different manufacturers (manufacturers #1–#3). All syringes were made of COP material, while the syringe manufactured by manufacturer #1 had a siliconized inner surface (barrel).

[0049] 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.

[0050] Figure 3a shows a quantitative measurement of the amount of BSA-FITC adsorbed onto untreated Topas™ and Zeonor™ coupons.

[0051] These two coupons of COP material (1.25 cm 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 hour to form a BSA adhesive layer on the COP surface. The coupons were then rinsed either with PBS (Method 1) or with PBS and elution buffer 1 (EB1 = PBS + 1% Triton X), as shown schematically in Figure 3b. Method 1 is expected to leave the majority of the adsorbed protein, consisting of both a soft and a hard BSA adsorption layer. Method 2 is expected to remove most of the soft layer. After rinsing via Methods 1 and 2, the adhered BSA-FITC was extracted to a 1 mL volume for quantification via fluorescence. The extraction protocol consisted of a 17-hour incubation in EB1 supplemented with 1% mercaptoethanol as a proteolytic agent to fragment the protein and quantitatively release the FITC label into solution. The emission intensity from the extraction solution at 495 nm excitation was used to quantify the protein via calibration with a BSA-FITC standard.

[0052] This study demonstrates the effectiveness of surface modification using polysaccharides, which shows great promise in addressing protein adsorption.

[0053] Other studies have shown that protein rejection is observed on the inner surface of syringes used for biopharmaceuticals, as well as on COP materials. Protein rejection is considered common because it is observed with common probe globular proteins and smaller therapeutic proteins. [Example]

[0054] Surface modification protocol. The surface modification protocol involved the use of 1.25 cm of TOPAS™ (T), ZEONOR™ (Z), and ZEONEX (ZX). 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).

[0055] The pre-treated 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 tested in the experiment (sample nomenclature id2#): dextran (D), polygalacturonic acid (PGA), hyaluronic acid (H), or no saccharide (NS). The following incubation conditions were tested (sample nomenclature id3#): (1) Sugars 1 mg / mL -1 in deionized water at room temperature for 2 hours (W). (2) Sugars 1 mg / mL -1 Incubate in deionized water at 50°C for four consecutive 30-minute periods (total 2 hours) (W50X4). (3) Sugars 1 mg / mL -1 Incubate in 30% H2O2 at 50°C for four consecutive 30 min periods (total 2 h) (P50X4).

[0056] After the incubation period, all samples were rinsed with deionized water and used for protein adsorption screening. To identify the treatments received by each surface tested, samples are referenced by the combination of pretreatment (id1#), sugar (id#2), and modification treatment (id3#) used, as shown in Figure 4.

[0057] [ka]

[0058] Scheme 2. Sugars and abbreviations used as identifiers Protein adsorption test protocol. A solution of BSA-FITC was prepared at 2 mg / mL in phosphate-buffered saline (PBS) at pH 7. -1 The COP material coupons were immersed in the BSA-FITC solution and incubated in the dark for 1 hour. The materials were then rinsed with PBS (Method 1) and used for the following quantitative or qualitative measurements.

[0059] a. Quantification by release from solution. After rinsing, the attached BSA-FITC was extracted into 1 mL for quantification via fluorescence. The extraction protocol consisted of a 17-hour incubation in EB1 supplemented with 1% mercaptoethanol as a proteolytic agent to fragment the protein and quantitatively release the FITC label into solution. Emission intensity from the extraction solution at 470 nm excitation was used to quantify the protein via calibration with a BSA-FITC standard. Protein surface coverage was calculated by normalizing the total amount of extracted protein to the COP area exposed during incubation. Error bars on all graphs correspond to 95% CI.

[0060] b. Qualitative comparison by fluorescence microscopy. After rinsing, the coupons were imaged using an upright microscope with 470 nm excitation and a FITC exc / em filter cube, and the integrated intensity at the COP surface was measured via commercially available software. Method 1 provides good sensitivity for both soft and hard adsorbed layers (Figure 2). 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.

[0061] (BSA-FITC adsorption results for COP coupons) Figure 5 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 protein adsorbate density. The best reduction, 52%, was observed for TP50-PGA-P50X4. Table 1 summarizes the protein removal results, calculated as percent adsorption relative to the untreated coupon surface.

[0062] The changes in protein adsorption were also confirmed by qualitative fluorescence microscopy, as shown in Figure 6. The release from the coupon surfaces detected by microscopy indicates that PGA treatment reduces the release from BSA-FITC adsorbed on all types of COP coupons tested.

[0063] [Table 1]

[0064] Figure 7 shows the total luminescence from adsorbed BSA-FITC on the three polymeric materials tested 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 result in as significant a reduction as when the surface is also treated with peroxide. On the other hand, peroxide significantly negatively impacts protein rejection unless PGA is added to the treatment solution.

[0065] (Protein adsorption results using COP syringe) Figure 8 shows the results of quantitative measurements of BSA-FITC adsorption on COP syringes from manufacturers #1, #2, and #3. The ##-NS-W syringe provides a control to report the adsorption expected on a clean syringe barrel without any pretreatment or modification. While the unused syringes show comparable adsorbate surface coverage to that measured on the coupon samples, it is clear that PGA modification significantly reduces BSA-FITC adsorption on syringes #1 (79%) and #2 (54%). No significant reduction is observed on syringe #1. However, this is consistent with the inner surfaces of these devices being siliconized, thus indicating that the COP surface is most affected by the polysaccharide treatment directly on the surface without a silica coating.

[0066] Given the success of the modification protocol with syringes #2 and #3, quantitative measurements were extended to another type of protein: insulin-FITC. Insulin-FITC is a protein used for therapeutic purposes in unlabeled form. Figure 6 shows the results of quantitative measurements with insulin-FITC. It is clear that PGA modification also reduces the activity of syringes #2 (83%) and #3 (52%) with this protein.

[0067] Figures 10-15 show a comparison of FT-IR spectra between the COP material (as a coupon) and the syringe materials described here (types S1, S2, and S3 from manufacturers #1, #2, and #3, respectively).

[0068] (Effect of surface treatment on COP material) The effects of solution treatment and reaction conditions were investigated using Ge attenuated total internal reflectance infrared spectroscopy (GATR-FTIR), water contact angle (WCA), and transmittance UV-Vis spectroscopy. Figure 10a 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 figure shows the appearance of a distinct absorbance peak at 1000 kJ / cm2. This indicates that COP is oxidatively activated when exposed to peroxide under reaction conditions. However, as shown in the control UV-Vis absorbance spectrum in Figure 10b, this oxidation is mild and limited to the surface of the material, indicating no change in the bulk optical properties.

[0069] This contrasts with other surface oxidation methods, such as exposure to UV / ozone lamps. This is illustrated in Figures 11a and 11b, which show the GATR and UV-Vis absorbance of the same type of COP coupon after oxidation by UV / ozone lamp irradiation (10 min). While the appearance of carbonyl peaks is evident in the GATR-FTIR spectrum after oxidation, the UV-Vis absorbance spectrum shows a significant increase in absorbance, indicating a change in the bulk structure of the COP polymer. Therefore, oxidation by HO is relatively mild, and the bulk material remains unchanged.

[0070] WCA measurements were used to monitor changes in hydrophilicity due to surface treatment. Figure 9 shows the WCA values ​​obtained for COP surfaces of three polymers treated with and without PGA under different conditions. The results show that only minor changes in hydrophilic properties are observed after exposure to H2O2 alone. However, exposure to PGA significantly increases hydrophilicity.

[0071] (Conclusion) For COP materials, the protein adsorption is further reduced by combining the surface oxidation process with the immobilization of polysaccharides.

[0072] Protein rejection is believed to be common because it is observed with common probe globular proteins and therapeutic proteins of smaller size.

[0073] The disclosures of the publications referenced herein are incorporated by reference in their entireties.

Claims

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

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

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

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

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

6. 4. The method of claim 1, wherein the polysaccharide is selected from dextrin, dextran, or a combination of two or more of these polysaccharides.

7. 7. The method of any one of claims 1 to 6, wherein the oxidizing agent comprises a peroxide, optionally comprising hydrogen peroxide, optionally comprising hydrogen peroxide in a 30% w / w aqueous solution.

8. The method of any one of claims 1 to 7, wherein the predetermined time is in the range of 0.5 minutes to 240 minutes.

9. 9. The method according to any one of claims 1 to 8, wherein the treating and / or incubating of at least a portion of the polymer surface is carried out at a temperature in the range of 10°C to 90°C.

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

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

12. The oxidizing agent in the composition is peroxide, O ３ , ozonated water, H ２ O ２ 12. The method of claim 11, comprising: a periodate, a hypochlorite, and / or a permanganate.

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