METHODS OF COATING POLYMERS AND REDUCTION IN PROTEIN AGGREGATION

Abstract

A method of coating a polymer surface, the method comprising: providing a polymer having a surface, optionally, treating at least a portion of the polymer surface with an oxidising agent, treating at least a portion of the polymer surface with a composition comprising a polysaccharide, oligosaccharide, polyol or mixture thereof, and incubating the treated polymer with the composition for a predetermined time. Also disclosed are polymers comprising such a coating, vessels comprising such coated polymers and medicals devices comprising such polymers.

Claims

1.-25. (canceled)

26. A method of 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 oxidising agent, c) treating at least a portion of the polymer surface with a composition comprising a polysaccharide, oligosaccharide, polyol or mixture thereof, and d) incubating the treated polymer with the composition for a predetermined time to form a coated polymer.

27. The method of claim 26, wherein the polymer comprises a cyclic olefin polymer and/or co-polymer.

28. The method of claim 26, wherein the polysaccharide comprises a hexose derived polysaccharide or oligosaccharide.

29. The method of claim 26, wherein the polysaccharide comprises 20 percent or greater oxidised hexose at the C6 position.

30. The method of claim 26, wherein the polysaccharide is selected from dextrin, dextran polygalacturonic acid, hyaluronic acid, or a combination of two or more of these polysaccharide.

31. The method of claim 26, wherein the oxidising agent comprises a peroxide, optionally comprises hydrogen peroxide, optionally hydrogen peroxide in 30% w/w aqueous solution.

32. The method of claim 26, wherein the predetermined time is in the range of 0.5 mins to 240 mins.

33. The method of claim 26, wherein the step of treating at least a portion of the polymer surface and/or the step of incubating is at a temperature in the range 10 C. to 90 C.

34. The method of claim 26, wherein the composition comprises water.

35. The method of claim 26, wherein the composition comprises an oxidising agent.

36. The method of claim 35, wherein the oxidising agent in the composition comprises a peroxide, O.sub.3, ozonated water, H.sub.2O.sub.2, periodate, hypochlorite, and/or permanganate, optionally comprises hydrogen peroxide, optionally comprises hydrogen peroxide in 30 percent w/w aqueous solution.

37. The method of claim 26, further comprising the step of forming a vessel comprising the coated polymer.

38. The method of claim 37, wherein the vessel is selected from the group consisting of a multi-well plate, a pipette, a bottle, a flask, a vial, an Eppendorf tube, and a culture plate.

39. The method of claim 37, further comprising the step of storing a pharmaceutical proteinaceous composition in the vessel.

40. The method of claim 26, further comprising the step of forming a medical device comprising the coated polymer, wherein the medical device is selected from the group consisting of a dispensing tube, a device comprising a channel, and a syringe.

41. A method of reducing protein or oligonucleotide aggregation and/or adsorption on a polymer surface, the method comprising, a) providing a polymer having a surface coating, the surface coating comprising a polysaccharide oligosaccharide, polyol or mixture thereof directly contacting the surface of the polymer; and b) contacting the surface coating with a proteinaceous composition or a composition comprising an oligonucleotide.

42. The method of claim 41, wherein the proteinaceous composition comprises a pharmaceutical proteinaceous composition comprising a monoclonal antibody composition, or a peptide hormone.

43. A polymer having a coating on at least one surface, the coating comprising a polysaccharide oligosaccharide, polyol or mixture thereof directly contacting the surface of the polymer.

44. The polymer of claim 43, wherein the polymer comprises a cyclic olefin polymer.

45. The polymer of claim 43, wherein the polysaccharide comprises dextrin, polygalacturonic acid, hyaluronic acid, or a combination of two or more of these polysaccharides.

Description

BRIEF DESCRIPTION OF FIGURES

[0198] Embodiments of the present invention will be described in more detail with reference to the accompanying Figures in which:

[0199] FIG. 1. (a) Quantitative determinations of adsorbed BSA-FITC at pristine TOPAS (TW) and ZEONOR (ZW) surfaces retained in the form of a hard layer (black bars) and a soft layer (grey bars). (b) Rinsing protocols developed to tailor assay sensitivity to hard layer (HL) and soft layer (SL).

[0200] FIG. 2. Summary of protein surface coverage determined at pristine and treated surfaces resulting from 2 mg mL.sup.1 BSA-FITC incubation experiments at COP surfaces.

[0201] FIG. 3. Comparison of emission data (MFI) resulting from 2 mg mL.sup.1 BSA-FITC incubation experiments at COP surfaces obtained via microscopy. The pristine surface is used as reference 100% emission.

[0202] FIG. 4. Comparison of emission data (MFI) resulting from 2 mg mL.sup.1 BSA-FITC incubation experiments at COP surfaces obtained via microscopy. The pristine surface is used as reference 100% emission.

[0203] FIG. 5. Summary of protein surface coverage determined at pristine and PGA-treated syringes resulting from 2 mg mL.sup.1 BSA-FITC incubation experiments.

[0204] FIG. 6. Summary of protein surface coverage determined at pristine and PGA-treated syringes resulting from 2 mg mL.sup.1 Insulin-FITC incubation experiments.

[0205] FIG. 7. (a) GATR-FTIR spectra of a Zeonor coupon surface after rinsing with water (ZW) and after treatment in H.sub.2O.sub.2 at 50 C. for 30 min (ZP50). (b) UV-Vis absorbance spectra of a 1 mm Zeonor coupon after rinsing with water only (ZW) and after treatment with H.sub.2O.sub.2 at 50 C. for 30 min (ZP50).

[0206] FIG. 8. (a) GATR-FTIR spectra of a Zeonor coupon surface after rinsing with water (ZW) and after oxidising treatment via exposure to a UV/ozone lamp for 5 (ZU5) and 10 min (ZU10). (b) UV-Vis absorbance spectra of a 1 mm Zeonor coupon after rinsing with water only (ZW), and after and after oxidising treatment via exposure to a UV/ozone lamp for 5 (ZU5) and 10 min (ZU10).

[0207] FIG. 9. Water contact angle measurement obtained at COP coupon surfaces after rinsing in water and undergoing a range of treatment conditions with and without PGA.

[0208] FIG. 10. Comparison between the surface composition of a coupon of TOPAS and syringe type S1, analysed by FTIR.

[0209] FIG. 11. Comparison between the surface composition of a coupon of Zeonor and syringe type S3, analysed by FTIR.

[0210] FIG. 12. Comparison between the surface composition of a coupon of Zeonex and a syringe type S3, analysed by FTIR.

[0211] FIG. 13. Comparison between the surface composition of a coupon of TOPAS and a syringe type S2, analysed by FTIR.

[0212] FIG. 14. Comparison between the surface composition of a coupon of Zeonor and syringe type S2, analysed by FTIR.

[0213] FIG. 15. Comparison between the surface composition of a coupon of Zeonex and syringe type S2, analysed by FTIR.

DETAILED DESCRIPTION

[0214] The studies herein use a fluorescently labelled globular protein, BSA-FITC to monitor the extent of protein surface adsorption at cyclo-olefin polymers (COP) materials. BSA is typically used as an indicator of the ability of a surface to resist unspecific protein adsorption. A second (fluorescently labelled) protein, Insulin-FITC, has been used to confirm the generality of the effect and its applicability to a therapeutic protein.

##STR00001##

[0215] Three types of COP materials were investigated: TOPAS (T) (Topas Advanced Polymer), ZEONOR (Z) and ZEONEX (Zeon Corporation) sourced from commercial suppliers in 1 mm thick coupon form. These materials are used by biodevice manufacturers for the biopharmaceutical industry. Scheme 1 shows a general structure of COP materials of different kinds; structural variations can be achieved via changes in the substituent groups which provide tunable properties. .sup.1 Shin J. Y. et al., Pure and Applied Chemistry, (2005) 77: 801-814..sup.2 Nunes et al. Microfluid Nanofluid (2010) 9:145-161.

[0216] To verify that the results of coupons were generalisable to biomedical devices, studies were conducted using selected syringe biodevices sold for pre-filled biotherapeutics sourced from three different manufacturers: Manufacturers #1-#3. All the syringes are of COP materials, while those manufactured by Manufacturer #1 are siliconized in their internal surface (barrel).

[0217] The adsorption of proteins to surfaces is a complex process; proteins typically undergo complete and/or partial denaturation when adsorbed at surfaces and the strength and nature of the interactions involved in protein adhesion varies.

[0218] FIG. 3a shows quantitative determinations of the amount of BSA-FITC adsorbed at coupons of pristine Topas and Zeonor.

[0219] Coupons (1.25 cm.sup.2) of these two COP materials were immersed in BSA-FITC solutions in phosphate buffer saline solution (PBS) at pH 7 at a concentration of 2 mg mL.sup.1 and incubated for 1 h in the dark to form BSA adlayers at the COP surface. Coupons were then rinsed in (method 1) PBS; or (method 2) in PBS and in elution buffer 1 (EB1=PBS+1% Triton X), as schematically depicted in FIG. 3b. Method 1 is expected to leave the largest amount of protein adsorbed, consisting of both soft and hard adsorbed layers of BSA. Method 2 is expected to remove most of the soft layer. After rinsing via methods 1&2, the adhered BSA-FITC was extracted into a 1 mL volume for quantitation via fluorescence methods. The extraction protocol consisted of incubation for 17 h in EB1 with addition of mercaptoethanol at 1% as a proteolytic agent, in order to fragment the protein and quantitatively release the FITC label into solution. The emission intensity from the extracted solution at 495 nm excitation was used to quantitate the protein via calibration with BSA-FITC standards.

[0220] The present study shows the effects of a surface modification using polysaccharides that shows significant promise in addressing protein adsorption.

[0221] Other work has shown that the protein rejection is observed also on the inner surface of syringes used for biotherapeutics, on COP materials. Protein rejection appears to be general, as it is observed with a general probe globular protein and with a therapeutic protein of smaller size.

Examples

[0222] Surface modification protocols. The surface modification protocols used 1.25 cm.sup.2 coupons of TOPAS (T), ZEONOR (Z) and ZEONEX (ZX); these were subject to two different types of pre-treatment prior to modification with saccharides (id1 # in sample nomenclature): [0223] 1) Rinsing with millipore water (TW, ZW or ZXW) [0224] 2) Mild surface oxidation using hydrogen peroxide 30% at 50 C. (TP50, ZP50 or ZXP50). The pre-treated coupons were subsequently incubated in 1 mg mL.sup.1 solutions of different saccharides to carry out modifications of the surface. Scheme 2 shows the structures of polysaccharides tested in our experiments (id2 # in sample nomenclature): dextran (D), polygalacturonic acid (PGA), hyaluronic acid (H) or no saccharide (NS). The following incubation conditions were tested (id3 # in sample nomenclature): [0225] 1) Saccharide 1 mg mL.sup.1 in deionised water at room temperature for 2 h (W) 2) Saccharide 1 mg mL.sup.1 in deionised water at 50 C.; 4 consecutive incubations of 30 min (total 2 h) (W50X4). [0226] 3) Saccharide 1 mg mL.sup.1 in 30% H.sub.2O.sub.2 at 50 C.; 4 consecutive incubations of 30 min (total 2 h) (P50X4).

[0227] Following the incubation period, all samples were rinsed in deionised water and used for screening the amount of protein adsorption. To identify the treatment undergone by each surface tested, samples are referred by the combination of pre-treatment (id1 #), saccharide (id #2) and modification treatment (id3 #) used, as shown in FIG. 4.

##STR00002##

[0228] Protein adsorption testing protocol. Solutions of BSA-FITC were prepared in phosphate buffer saline solutions (PBS) at pH 7 at concentrations of 2 mg mL.sup.1. Coupons of the COP materials were immersed in BSA-FITC solutions and incubated for 1 h in the dark. The materials were then rinsed in (method 1) PBS and used for either quantitative or qualitative determinations as below:

[0229] a. Quantitative determinations via emission from solution. After rinsing the adhered BSA-FITC was extracted into a 1 mL volume for quantitation via fluorescence methods. The extraction protocol consisted of incubation for 17 h in EB1 with addition of mercaptoethanol at 1% as a proteolytic agent, in order to fragment the protein and quantitatively release the FITC label into solution. The emission intensity from the extracted solution at 470 nm excitation was used to quantitate the protein via calibration with BSA-FITC standards. The protein surface coverage was calculated by normalising the total extracted protein by the exposed COP area during incubation. Error bars in all graphs correspond to 95% C.I.

[0230] b. Qualitative comparisons via fluorescence microscopy. After rinsing the coupons were imaged using upright microscope with 470 nm excitation and a FITC exc/em filter cube to determine the integrated intensity at the COP surface via commercial software. Method 1 makes the method sensitive to both soft and hard adsorbed layers (FIG. 2). The mean fluorescence intensity (MFI) through the emission filter was measured from multiple images and corrected by the background emission (MFI) of the corresponding pristine COP material. Error bars in all graphs correspond to 95% C.I.

[0231] BSA-FITC Adsorption Results on COP Coupons

[0232] FIG. 5 shows results from quantitative determinations of BSA-FITC adsorption at Topas, Zeonor and Zeonex surfaces. The ##-NS-W samples provide controls as it mimics the expected adsorption at e.g. a syringe barrel without any pre-treatment or modification. It is clear that modification with PGA polysaccharides yield the best reductions in the density of protein adsorbates. The best reduction is of 52% and observed for TP50-PGA-P50X4. Table 1 shows a summary of protein rejection results calculated as adsorption relative to the pristine coupon surfaces.

[0233] Protein adsorption changes were also confirmed via qualitative fluorescence microscopy methods as shown in FIG. 6. Emission from the coupon surface detected via microscopy shows that PGA-treatment results in lower emission from adsorbed BSA-FITC on all types of COP coupons tested.

TABLE-US-00001 TABLE 1 Summary of results of protein rejection measurements calculated from average values shown in FIG. 5. TOPAS ZEONOR ZEONEX Polygalacturonic acid 52% 38% 35% Dextran 13% 24% 7% Hyaluronic acid 8%

[0234] FIG. 7 shows the total emission from adsorbed BSA-FITC on the three polymer materials tested after the coupons were treated with PGA alone, with hydrogen peroxide alone or using the combination of PGA and peroxide treatment. It is evident that PGA alone does not result in as significant a reduction as when the surface is also treated with peroxide; whereas peroxide has a largely negative effect on protein rejection unless PGA is added to the treatment solutions.

[0235] Protein Adsorption Results on COP Syringes

[0236] FIG. 8 shows results from quantitative determinations of BSA-FITC adsorption at Manufacturer #1, #2 and #3 COP syringes. The ##-NS-W syringes provide controls as they report the expected adsorption at clean syringe barrels without any pre-treatment or modification. It is clear that whereas pristine syringes display surface coverage of adsorbates that is comparable to that determined on coupon samples, the PGA modifications result in a significant reduction of BSA-FITC adsorption for #1 (79%) and #2 (54%) syringes. #1 syringes do not show significant reduction. However, this is consistent with these devices being siliconized over their inner surface and therefore indicate that COP surfaces are most affected by the polysaccharide treatment directly on their surface without a silica coating. Given the success of the modification protocol on #2 and #3 syringes we expanded the quantitative determinations to a different type of protein, Insulin-FITC, a protein that in its unlabelled form is used for therapeutic applications. FIG. 6 shows results from quantitative determinations with insulin-FITC; it is clear that also with this protein the PGA modification results in a decrease on #2 (83%) and #3 (52%) syringes.

[0237] FIGS. 10 to 15 shows comparisons between the FT-IR spectra of COP materials (as coupons) and the syringe materials (types S1, S2, S3 from manufacturers #1, #2 and #3 respectively) discussed herein.

[0238] Effect of Surface Treatments on COP Materials

[0239] The effect of solution treatments and reactions conditions were investigated using Ge-attenuated total internal reflectance infrared spectroscopy (GATR-FTIR), water contact angle (WCA), and transmittance UV-Vis spectroscopy. FIG. 10a shows GATR-FTIR spectra of a COP coupon before and after exposure to H.sub.2O.sub.2 at 50 C.; the spectra show the appearance of a clear absorbance peak at 1709 cm.sup.1 that is diagnostic of carbonyl functional groups. This indicates that exposure to peroxide at the reaction conditions results in oxidative activation of the COP. This oxidation is however mild and confined to the surface of the material as shown by control UV-Vis absorbance spectra in FIG. 10b, that indicates no change in the bulk optical properties.

[0240] This is to be contrasted with other methods of surface oxidation such as exposure to UV/ozone lamp; this is shown in FIGS. 11a and 11b where the GATR and UV-Vis absorbances of the same type of COP coupon are shown after oxidation via UV/ozone lamp irradiation (10 min). Although the appearance of carbonyl peaks is apparent in the GATR-FTIR spectrum after oxidation, there is a significant increase in the optical absorbance in the UV-Vis absorbance spectrum, that is diagnostic of changes to the bulk structure of the COP polymer. The oxidation with H.sub.2O.sub.2 is therefore relatively mild and does not significantly alter the bulk material.

[0241] WCA measurements were used to monitor changes in hydrophilic character resulting from the surface treatments. FIG. 9 shows WCA values obtained at COP surfaces of the three polymers treated with and without PGA under different conditions. Results indicate that after H.sub.2O.sub.2 exposure alone only slight changes in hydrophilic character are observed; however, exposure to PGA results in significant increases in hydrophilic character.

CONCLUSION

[0242] For COP materials, a process of surface oxidation in combination with immobilization of a polysaccharide reduces still further protein adsorbates.

[0243] Protein rejection appears to be general, as it is observed with a general probe globular protein and with a therapeutic protein of smaller size.

[0244] The disclosures of the published documents referred to herein are incorporated by reference in their entirety.