SURFACE COATING TO PREVENT CATION LEACHING

TECHNICAL FIELD

[0001] The invention generally relates to drug holding components of drug delivery systems implementing an injection like process for delivering a drug or drug formulation directly to a patient or through a fluid administration circuit. In some none exclusive implementation, the invention relates to pre-fillable and pre- filled components of such drug delivery systems.

[0002] Many drug delivery systems, like syringes, pre-filled syringes, drug cartridge and needless injectors include an internal chamber for receiving a medicament and a piston, the piston being slidable within the interior chamber. [0003] Such drug delivery systems are often made of glass so two problems are to be solved, the first one is to improve gliding properties in syringe barrels and the second one is to obtain a good stability of the active principles contained in such barrels.

[0004] The siliconization of the inner wall of the chamber allows a smooth movement or gliding of the piston within the chamber. Currently, the silicone oil used for pharmaceutical applications is a polydimethylsiloxane polymer, sprayed inside the barrel.

[0005] Despite the non reactive nature of such silicone oil and glass, it has been found that cations leaching from the silicone oil and the glass could interact with some of the components of the drug or drug formulation and alter the drug properties.

[0006] Moreover silicone oil is an apolar molecule that can mostly generate hydrophobic interactions with other molecules or particles in solutions. When an emulsion with silicone oil comes in contact with proteins, it generates some changes in their structure leading to a loss of protein activity and/or a protein aggregation.

[0007] The aim of the invention is to create surface coatings that prevent cation leaching from glass and stabilize the silicone oil onto the syringe barrel reducing further interactions with proteins stored in the syringe barrel.

BACKGROUND ART

[0008] The current solution developed by the biotech market is a baked silicone syringe with a Flurotec stopper. The baked silicone syringe displays a lower content of silicone by a factor 10 in comparison with a standard sprayed silicone. However, the current solution is not completely satisfactory as there is some limitation in the process. Indeed, only luer syringes can be produced by using this process, secondly, there are some therapeutic proteins that can aggregate when stored in a baked silicone syringe.

[0009] To avoid cation leaching from glass, different surface treatments were proposed. First, in 1981 , a thermal barrier coating adapted to provide a thermally insulating protective barrier on a component was patented by Rolls-Royce (US patent 4,332,618). In this method, the coating was applied to the components by spraying methods and being ductile when exposed to high temperatures. The coating comprises a mixture containing constituents of glass microspheres; a ceramic frit of finely divided particles of alkali silicate titanate glass; and a refractory filler material of finely divided particles (micronized mica; aluminum oxide of mullite). All of the constituents of the mixture were suspended in a high temperature resistant binder material such as potassium silicate, sodium silicate or aluminum orthophosphate.

[0010] In 1991 , Pilkington pic. proposed a coating, which acted as barrier layers to inhibit migration of alkali metal ions from a glass surface (Na leaching between 40 and 700 ng/cm ² ). It acted as color suppressing underlayers for overlying infra-red reflecting or electrically conducting layers. The coating was deposited by pyrolysis of a gaseous mixture of a silane, an unsaturated hydrocarbon and an oxygen-containing gas other than carbon dioxide which does not react with the silane at room temperature on a hot glass surface at a temperature of 600 ⁰ C to 750 ⁰ C (US patent 4,995,89 3 and US 5,165,972). [0011] In 1992, Saint Gobain Vitrage Int, patented antiglare surfaces upon coated substrates as well as barrier surfaces to prevent the migration of alkaline ions out of the glass substrate and a method to reduce or mitigate the speckling or irhdescence effect in a layer formed upon a glass substrate by pyrolytic decomposition of a metal-based powder (US patent 5,093,153).

[0012] In 2001 , Schott patented a method to increase the chemical resistance of the interior surface of the glass body. In order to avoid a disadvantageous de- alkalizing process (mainly sodium element around 10 and 100 ng/cm ² in comparison with 300 ng/cm ² ), the hollow glass body had an interior coating preferably composed by oxides such as SiO2, AI2O3, TiO2. There was a predetermined coating thickness according to the required chemical resistance or working conditions for forming the glass body. The coating is advantageously provided by means of a PICVD process (US patent 6,200,658 B1 ). [0013] In 2002, PPG Industries patented amorphous metal oxide barrier layers of titanium oxide, zirconium oxide and zinc/tin oxide that acted as alkali metal ion barrier layers at thicknesses below 18 nm. The amorphous metal oxide barrier layers are most effective when the density of the layer is equal to or greater than 75% of the crystalline density. The barrier layers prevent migration of alkali metal ions such as sodium ions from glass substrates into a medium e.g. electrolyte of a photochromic cell, liquid material of a liquid crystal display device contacting the glass surface and a photocatalytic coating. The properties of the medium, particularly electroconductive metal oxide coatings, are susceptible to deterioration by the presence of sodium ions migrating from the glass (US patent 6,352,755 B1 ). [0014] In 2003, Schott invented a glass container for the storage of pharmaceutical or diagnostic solutions which behaved in a largely inert manner vis a vis these solutions, i.e., a glass container in which the quantity of ions leached from the glass through the solutions is minimized (US patent 6,537,626 B1 ). [0015] In 2004, in order to avoid deposition of evaporating alkali compounds on an inner surface of a hollow glass body during thermal processing to form a glass container from the hollow glass body, an overpressure is provided in the hollow glass body during the thermal processing. Either rinsing the hollow glass body with a gas, such as air, or at least partially closing the glass body at one end so that sufficiently rapid pressure equilibration is avoided, can provide this overpressure. The glass containers made by these methods are especially suited for food or pharmaceuticals because they have a reduced alkali release from their inner surfaces, for example at most about 2.0 mg/l sodium oxide (US patent 2004/012026 A1 ).

[0016] To improve gliding properties in syringe barrels, different solutions dealing with silicone oil based systems were proposed.

[0017] In 1989, Becton Dickinson proposed a method to reduce high breakout and sustaining forces between slidable surfaces. In this method, a film of lubricant applied at least to one of the surfaces was then subjected to an ionizing plasma. The invention includes articles having slidable surfaces of low breakout and sustaining forces. This treatment was applied on silicone oils (US patents 4,767,414 & 4,822,632). [0018] Becton Dickinson also protected a method for preparing stable coatings of a silicone lubricant on a low surface energy polymeric surface. It included plasma treatment of the surface in an atmosphere of a siloxane monomer. A layer of polysiloxane was deposited on the low energy surface to give a polysiloxane surface. A film of a polysiloxane lubricant having a surface tension substantially the same as or less than the surface energy of the polysiloxane surface was applied to the polysiloxane layer (US 4,844,986).

[0019] In 1994, Becton Dickinson also developed a multi component system. In this system, a first crosslinked basement lubricant was coated onto a syringe barrel. A second lubricant was coated over the crosslinked lubricant. A low viscosity prebasement lubricant was evenly coated onto the inside surface of the syringe barrel and then crosslinked by a plasma to a viscous liquid or substantially solid basement lubricant. A second surface lubricant is then applied over the basement lubricant (US patent 5,338,312).

[0020] In 2002, NovoNordisk proposed a coating system for articles where plastic materials slide against flexible rubber materials. The coating system was a silicone oil based coating having a viscosity of at least 200,000 centistokes. The coating comprises in a preferred embodiment a silicone oil based block or graft copolymer, or segmented copolymer. Such an article was preferably a medical article, such as a container or an injection cylinder and a stopper. The coatings were particularly useful for coating containers for storage and administration of liquid protein solutions, such as insulin formulations (US 6,482,509 B2).

[0021] In 2003, Schott also patented a process for applying a thermally attached lubricating coating on an interior wall of a cylindrical medicinal container. This process can homogenize the applied lubricant on the wall to form a lubricating coating. The lubricant was thermally attached by irradiating the

lubricating coating with infrared radiation selectively in a cylindrical region of the container at elevated temperatures above a maximum operating temperature of the container (US 6,586,039 B2).

SUMMARY OF THE INVENTION

[0022] It is an object of the present invention to inhibit leaching of chemical species from syringe raw material into the said pharmaceutical preparation together to preserve the gliding properties. [0023] It is a further object of the present invention to create a barrier against monovalent cations (Na ⁺ ), divalent cations (Mg ²⁺ ), trivalent (B ³⁺ and Al ³⁺ ) and tetravalent (Si ⁴⁺ ) species coming from the glass and able to interact with the therapeutic proteins

[0024] It is a further object of the present invention to propose a prefilled glass syringe with a staked needle that insures both the protein stability and a good gliding.

[0025] It is a further object of the present invention to avoid the use of tensioactives that are added in the case of baked silicone process and that could also interact with proteins. [0026] One aspect of the present invention comprises a method to prepare a syringe comprising the steps of: a. creating an homogeneous and continuous inner oil layer inside a syringe, b. exposing said inner oil layer to an oxidative plasma gas, said oil being a non reactive oil.

[0027] In a preferred embodiment the method according to the invention further comprises before the step of creating said inner oil layer a preliminary step of oxidative plasma.

[0028] In a further embodiment the method according to the invention comprises a step of sterilization.

[0029] In a further embodiment the non reactive oil is silicon oil.

[0030] In a further embodiment the silicon oil is chosen amongst the alkyl polysiloxane.

[0031] In a further embodiment the alkylpolysiloxane are chosen in the group comprising polydimethylsiloxane, polydiethylesiloxane and polydipropylsiloxane. [0032] In a further embodiment the oil has a viscosity from preferably around 100O cSt. [0033] In a further embodiment the homogeneous and continuous oil layer is 0,5 to 1 ,5 μm

[0034] In a further embodiment the oxidative plasma gas is oxygen, or an oxygen containing gas. [0035] In a further embodiment the oxidative plasma is generated under atmospheric pressure or vacuum.

[0036] In a further embodiment the oxidative plasma is generated by microwaves, audio frequencies, radio-frequencies, low frequencies, DC glow discharge, Corona discharge or arc discharge. [0037] In a further embodiment the syringe is glass based, such as borosilicate [0038] The ionizing plasma was done by using an oxidative gas such as air but pure oxygen or other oxidative gases can also be used.

[0039] The method can be applied or used on on Luer syringes and for syringes in polymers. [0040] Currently, the silicone oil used for pharmaceutical applications is a polydimethylsiloxane polymer. This polymer is directly exposed to a plasma gas after spraying inside the barrel. The plasma is done in a chamber by adding an oxidative gas such as air or oxygen or a mixture of oxygen with an inert gas assuring a barrier effect whereas an outer layer can assure the gliding effect. The inner layer is an effective barrier against monovalent, divalent, thvalent and tetravalent cations extracted from glass.

[0041] Another aspect of the invention is the use of a non-reactive oil exposed to an oxidative plasma treatment as a surface coating of a syringe barrel to be filled with a pharmaceutical preparation, to inhibit leaching of chemical species from syringe raw material into the said pharmaceutical preparation. [0042] In a preferred embodiment the chemical species are cations.

Said cations are monovalent cations such as Na ⁺ , divalent cations such as Mg ⁺⁺ , thvalent cations such as B ⁺⁺⁺ , and Al ⁺⁺⁺ and tetravalent cations such as Si ⁺⁺⁺⁺ . [0043] Another aspect of the invention is to provide a syringe to be filled with a pharmaceutical preparation, characterized in that the barrier effect for all the cation

elements is inferior or equal to 250 ng/cm ² , preferably inferior or equal to 200 ng/cm ² , more preferably inferior or equal to 150 ng/cm ²

[0044] It is a further object of the present invention to provide syringe to be filled with a pharmaceutical preparation, characterized in that the barrier effect for Si ⁺⁺⁺⁺ . cation is inferior or equal to 150 ng/cm ² , preferably inferior or equal to 100 ng/cm ² , more preferably inferior or equal to 90 ng/cm ²

[0045] It is a further object of the present invention to provide syringe to be filled with a pharmaceutical preparation, characterized in that the barrier effect for Al ⁺⁺⁺ cation is inferior or equal to 3,0 ng/cm ² , preferably inferior or equal to 2,0 ng/cm ² , more preferably inferior or equal to 1 ,0 ng/cm ²

[0046] It is a further object of the present invention to provide syringe to be filled with a pharmaceutical preparation, characterized in that the barrier effect for Na ⁺ cation is inferior or equal to 100 ng/cm ² , preferably inferior or equal to 70 ng/cm ² , more preferably inferior or equal to 50 ng/cm ² [0047] It is a further object of the present invention to provide syringe to be filled with a pharmaceutical preparation, characterized in that the barrier effect for Mg ⁺⁺ cation is inferior or equal to 0,2 ng/cm ² , preferably inferior or equal to 0,7 ng/cm ² , more preferably inferior or equal to 0,5 ng/cm ² [0048] It is a further object of the present invention to provide syringe to be filled with a pharmaceutical preparation, characterized in that the barrier effect for B ⁺⁺⁺ cation is inferior or equal to 0,8 ng/cm ² , preferably inferior or equal to 0,7 ng/cm ² , more preferably inferior or equal to 0,5 ng/cm ²

[0049] The effect achieved by the method of the invention is of long duration and syringes retain the advantages of good gliding properties and leaching inhibition of chemical species.

DESCRIPTION OF THE INVENTION

[0050] While this invention is satisfied by embodiments in many different forms, a preferred form is herein described.

[0051] In accordance with the invention the homogeneous and continuous inner oil layer is created inside a syringe. Application of said continuous inner layer may be accomplished by any suitable method, such as, for example dipping,

brushing spraying and the like. The oil layer may be applied in a pure form or in water emulsion with surfactant.

[0052] The non reactive oil chosen amongst the alkylpolysiloxane in some embodiments is a polydimethyl siloxane, such as DOW CORNINGS360 ^® polydimethylsiloxane or NuSiL ^® polydimethylsiloxane having a viscosity ranging from about 100 to about 1 ,000,000 cst.

After the oil layer has been created the syringe is exposed to an oxidative plasma gas. The plasma treatment may be carried out in any plasma generator as for example those described in US3847652 or French patent applications 08FR51900 or 08FR51901.

[0053] The best embodiment is a Hypak ^® syringe formed by an inner layer obtained by CVD technology and an outer layer composed by a sprayed silicone with a viscosity of 1000 cSt plasma treated using an ionizing gas (oxygen) for 15 sec.

Example 1. Barrier effect of the coatings against cations coming from glass

[0054] Different elements compose the borosilicate forming the glass syringes.

The most important ones are listed below : [0055] Silicon (Si) is present in the glass as SiO2 and constitutes the basis of the glass,

[0056] Sodium (Na) is present in the glass as Na2θ and lowers the melting temperature,

[0057] Boron (B) is present in the glass as B ₂ O ₃ and used to get a better thermal resistance,

[0058] Aluminum (Al) is present in the glass as AI ₂ O ₃ in order to get better mechanical properties,

[0059] Magnesium (Mg) is present in the glass as MgO and used to lower the melting temperature.

a) Effect of different coatings on barrier effect

[0060] Different coatings were performed in the syringe barrels Hypak 1 ml long. Raw material was provided by Alcan glass.

o Solid coatings (referred as to C1 to C6) were deposited by using CVD technology with various HMDSO/O2 ratios (Table 1 ). Syringe barrels were inserted in a holder wherein glass syringes are put, the gas containing molecules to coat is introduced and two electrodes that create the electric field whereby a plasma is ignited.

Samples C1 C2 C3 C4 C5 C6 R1

Ratio 1 :0 2:1 1 :1 1 :0.5 1 :0.25 0:1 Uncoated HMDSO/O2 glass

Table 1. Solid coatings obtained by CVD technology

0 Glass barrels were sprayed with silicone oil (Dow Corning DC360

100OcSt PDMS) with a total amount of 0.4 mg/syringe and exposed to an oxygen plasma at various run times. The samples plasma treated silicone were referred as to P1 to P5 (table 2):

P1 P2 P3 P4 P5 R2

Samples

Run time 5s 15s 30s 60s 120s Untreated (Os)

Table 2. Plasma treated silicone (PTS) samples

0 Bilayers composed by an internal CVD coating and a second Plasma Treated Silicone (PTS) layer were performed and referred to as A1 and A1 bis. Samples A2 and A2 bis have only one PTS layer (table 3). The PTS was done on silicone oil (DC360, 1000 cSt) for 15 sec.

Samples A1 A2 Al ois A2bis

First layer HMDSO/O2 1 :0 No HMDSO/02 2: 1 No

Second layer PTS, 15s PTS, 15s PTS, 15s PTS, 15s

[0061] In order to investigate the potential cation extracted from the glass surface, forced extractions were performed. It should be noted that the extraction

method and solvent depended on the element quantification. This is the reason why three different extraction methods were used: [0062] Method 1 for Sodium : Purified water extracts using autoclave: A rigid needle shield was placed on the syringes which were filled with 1 mL of deionized water, stoppered with a Flurotec® plunger stopper and autoclaved twice for 1 hour at 121 O.

[0063] Sodium is analyzed by Flamme - Atomic Absorption Spectrometry (Flamme-AAS), using an air/acetylene flamme, a sodium hallow cathode and a detection at 589nm. An external calibration was performed between 0.3 and 2.0 ppm. Solutions of three syringes were pooled together for analysis.

[0064] Method 2 for Boron, Aluminum and Magnesium: Nitric acid extracts using ultrasonic bath. A rigid needle shield was placed on the syringes, which are filled with 1 mL of 1 % nitric acid, stoppered with a Flurotec® plunger stopper and placed for 2 hours in an ultrasonic bath at room temperature. Boron, Aluminum, Magnesium were analyzed by ICP/MS in one single method, using an argon plasma and a mass spectrometer for the detection, with the specific masses of 11 for Boron, 27 for Aluminum and 24 for Magnesium. An external calibration was performed using one single level at 10ppb for all elements and a check at 5ppb for all elemnts. The calibration was validated between 1 and 10 ppb for Boron, 2 and 10 ppb for Aluminum and 4 and 10 ppb for Magnesium. Solutions of four syringes are pooled together for analysis.

[0065] Method 3 for Silicium : Purified water extracts using autoclave plus 24 hour agitation: A rigid needle shield was placed on the syringes, which were filled with 1 mL of deionized water and stoppered with unsiliconized parylene coated plunger stoppers and autoclaved for 1 hour at 121 O. They w ere then agitated for 24 hours before analysis.

[0066] Silicium was analyzed by ICP/MS using an argon plasma and a mass spectrometer for the detection, with the specific mass of 28 for silicium. Silicium extracted from the glass as well as silicium extracted from the coatings (referred as to C1 to C6 and P1 to P5) were quantified together. An external calibration was performed between 10 and 35 ppb. Solution from a single syringe was diluted in 1 % nitric acid to reach the range of the calibration curve.

[0067] For all the cation elements, the barrier effect measured by analytical tools was then expressed in ng/cm ² .

o Monovalent cations: Sodium (Na ⁺ )

[0068] Results obtained for monovalent cations are summarized in table 4.

Average Relative Barrier effect

System Sample measurement Standard (ng/cm ² ) on 3 replicates deviation (RSD)

C1 <0.5 ppm NA <50

C2 1.2 ppm 8% 120

C3 1.2 ppm 8% 120

Solid coating C4 1.3 ppm 8% 130

C5 0.8 ppm 39% 80

C6 1.3 ppm 0% 130

R1 1.3 ppm 4% 130

P1 1.0 ppm 0% 100

P2 0.9 ppm 6% 90

P3 1.0 ppm 0% 100

PTS

P4 0.5 ppm NA <50

P5 <0.5 ppm NA <50

R2 1.0 ppm 6% 100

A1 =C1 +P2 0.7 ppm 14% 70

A2 =P2 0.9 ppm 22% 90

Bi layer

A1 bis= C2+P2 0.8 ppm 15% 80

A2 bis 1.3 ppm 16% 130

NA: Non Applicable

Table 4. Barrier effect of coatings against monovalent cations

[0069] Only the solid coating C1 provided a filter effect against monovalent sodium cation. It corresponds to a CVD process using pure HMDSO. The presence of oxygen in the reaction chamber decreases the filter effect against Na ⁺ .

[0070] Plasma Treated Silicone (PTS) samples referred as to P1 to P5 provide a filter against monovalent sodium cation for treatment times above 60 seconds. Bilayer A1 composed by an inner coating C1 and an outer layer P2 improves the filter effect against monovalent sodium cation compared to plasma treated silicone alone.

o Divalent cations: Magnesium (Mg ²⁺ )

[0071] Results obtained for divalent cations are summarized in table 5.

Average Relative Barrier effect measurement Standard (ng/cm ² )

System Sample on 3 replicates deviation (RSD)

C1 4 ppb 9% 0.4

C2 7 ppb 19% 0.7

C3 7 ppb 26% 0.7

Solid C4 6 ppb 7% 0.6

C5 7 ppb 46% 0.7

C6 8 ppb 32% 0.8

R1 6 ppb 21% 0.6

P1 <4 ppb NA <0.4

P2 <6 ppb NA <0.6

P3 <4 ppb NA <0.4

PTS

P4 <4 ppb NA <0.4

P5 <5 ppb NA <0.5

R2 <4 ppb NA <0.4

A1 =C1 +P2 5 ppb 25% 0.5

A2 =P2 5 ppb 1 1% 0.5

Bilayer

A1 bis= C2+P2 4ppb 0% 0.4

A2 bis 8 ppb 20% 0.8 NA: Non Applicable.

Table 5. Barrier effect of coatings against divalent cations

[0072] As previously observed for monovalent cations, only the solid coating

C1 provide a filter effect against divalent magnesium cation. It corresponds to a

CVD process using pure HMDSO. The presence of oxygen in the reaction chamber decreases the filter effect against Mg ²⁺ .

[0073] PTS systems are efficient barriers for divalent cations and bilayers can improve the filter effect against divalent magnesium cation compared to solid coatings alone.

o Trivalent cations: Boron (B ³⁺ ) and Aluminum (Al ³⁺ )

[0074] Results obtained for barrier against trivalent cations are summarized in tables 6 and 7.

Average Relative Barrier effect measurement Standard ng/cm ²

System Sample on 3 replicates deviation (RSD)

C1 10 ppb 53% 1

C2 13 ppb 12% 1.3

C3 14 ppb 18% 1.4

Solid C4 18 ppb 3% 1.8

C5 16 ppb 25% 1.6

C6 29 ppb 4% 2.9

R1 34 ppb 21% 3.4

P1 5 ppb 20% 0.5

P2 6 ppb 24% 0.6

P3 7 ppb 16% 0.7

PTS

P4 8 ppb 15% 0.8

P5 1 1 ppb 16% 1.1

R2 12 ppb 32% 1.2

A1 =C1 +P2 23ppb 25% 2.3

A2 =P2 27ppb 22% 2.7

Bi layer

A1 bis= C2+P2 10ppb 0% 1.0

A2 bis 30ppb 33% 3.0

Table 6. Barrier effect of coatings against trivalent cations (B 3+\ )

[0075] As previously observed for monovalent and divalent cations, only the solid coating C1 provide a filter effect against trivalent boron cation. It corresponds to a CVD process using pure HMDSO. The presence of oxygen in the reaction chamber decreases the filter effect against B ³⁺ . PTS systems provide a filter against trivalent boron cation for low time treatments.

Average Relative Barrier effect measurement Standard ng/cm ²

System Sample on 3 replicates deviation (RSD)

C1 23 ppb 23% 2.3

C2 34 ppb 16% 3.4

C3 42 ppb 26% 4.2

Solid C4 50 ppb 16% 5.0

C5 30 ppb 36% 3.0

C6 67 ppb 5% 6.7

R1 64 ppb 12% 6.4

P1 3 ppb 22% 0.3

P2 5 ppb 39% 0.5

P3 9 ppb 18% 0.9

PTS

P4 1 1 ppb 28% 1.1

P5 24 ppb 0% 2.4

R2 23 ppb 35% 2.3

A1 =C1 +P2 43 ppb 35% 4.3

A2 =P2 30 ppb 0% 3.0

Bi layer

A1 bis= C2+P2 20 ppb 0% 2.0

A2 bis 53 ppb 29% 5.3

Table 7. Barrier effect of coatings against trivalent cations (Al 3+ x )

[0076] As previously observed for monovalent and divalent cations, only the solid coating C1 provide a filter effect against trivalent aluminium cation. It corresponds to a CVD process using pure HMDSO. The presence of oxygen in the reaction chamber decreases the filter effect against Al ³⁺ . PTS systems provide a filter against trivalent boron cation for low time treatments.

o Silicium element and tetravalent cation (Si ⁴⁺ )

[0077] Silicium quantified by ICP/MS can come from both the glass or the coating : polymerized HMDSO by CVD or silicone (DC360 10OOcSt - PDMS) oil.

Average Relative Barrier effect measurement Standard ng/cm ²

System Sample on 3 replicates deviation

(RSD)

C1 1.1 ppm 69% 110

C2 1.3 ppm 33% 130

C3 1.1 ppm 6% 110

Solid C4 1.2 ppm 11% 120

C5 1.3 ppm 6% 130

C6 1.0 ppm 29% 100

R1 0.9 ppm 30% 90

P1 1.5 ppm NA 150

P2 1.2 ppm 19% 120

P3 1.1 ppm 16% 110

PTS

P4 0.9 ppm 12% 90

P5 6.1 ppm 8% 610

R2 9.1 ppm 28% 910

Table 8. Barrier effect of coatings against tetravalent cations (Si )

[0078] No real variation observed for solid coatings referred as to C1 to C6. PTS systems provide huge amounts of silicon element from the oil itself (9.1 ppm compared to 0.9 for the reference R1 ). This decreases with the plasma treatment time until a limit between 60 and 120 seconds where the silicone oil is fixed by the plasma treatment.

b) Effect of sterilisation on barrier effect

o Monovalent cations: Sodium (Na ⁺ )

[0079] The ETO sterilization did not affect the barrier effect against monovalent cations (Na+) as shown in Table 9.

Average Relative Barrier effect

System Sample measurement Standard (ng/cm ² ) on 3 replicates deviation (RSD)

P1 0.9 ppm 0 90

P2 0.8 ppm 0 80

PTS with P3 0.7 ppm 0 70 sterilization P4 0.7 ppm 0 70

P5 0.5 ppm 12 50

R2 1.2 ppm 5 120

P1 0.9 ppm 7 90

P2 0.7 ppm 8 70

PTS + E P3 0.7 ppm 0 70 sterilization P4 0.6 ppm 0 60

P5 0.5 ppm 25 50

R2 1.2 ppm 120

Table 9. Effect of sterilization on barrier effect against monovalent cations

Divalent cations: Magnesium (Mg 2+\ )

[0080] The ETO sterilization did not affect the barrier effect against divalent cations (Mg ,2+ ) as shown in Table 10.

Average Relative Barrier effect

System Sample measurement Standard (ng/cm ² ) on 3 replicates deviation (RSD)

P1 <4 ppb NA <0.4

P2 <4 ppb NA <0.4

PTS with P3 <4 ppb NA <0.4 sterilization P4 <4 ppb NA <0.4

P5 <7 ppb NA <0.7

R2 <5 ppb NA <0.5

P1 <4 ppb NA <0.4

P2 <4 ppb NA <0.4

PTS + E P3 <4 ppb NA <0.4 sterilization P4 <4 ppb NA <0.4

P5 <4 ppb NA <0.4

R2 <4 ppb NA <0.4

Table 10. Effect of sterilization on barrier effect against divalent cations (Mg )

Trivalent cations: Boron (B 3+\ ) and Aluminum (A ιl3+\ )

[0081] The ETO sterilization did not affect the barrier effect against trivalent cations (B ³⁺ and Al ³⁺ ) as shown in Tables 11 and 12.

Average Relative Barrier effect

System Sample measurement Standard (ng/cm ² ) on 3 replicates deviation (RSD)

P1 40ppb 0 4

P2 37ppb 16 3.7

PTS with P3 30ppb 0 3 sterilization P4 27ppb 22 2.7

P5 13ppb 43 1.3

R2 53 ppb 11 5.3

P1 37ppb 16 3.7

P2 37 ppb 16 3.7

PTS + E P3 30 ppb 0 3.0 sterilization P4 20 ppb 0 2.0

P5 10 ppb 0 1.0

R2 50 ppb 5.0

Table 11. Effect of sterilization on barrier effect against thvalent cations (B )

Average Relative Barrier effect

System Sample measurement Standard (ng/cm ² ) on 3 replicates deviation (RSD)

P1 50 ppb 0 5

P2 40 ppb 0 4

PTS with P3 27 ppb 22 2.7 sterilization P4 17 ppb 35 1.7

P5 16 ppb 80 1.6

R2 67 ppb 9 6.7

P1 53 ppb 11 5.3

P2 40 ppb 0 4

PTS + E P3 23 ppb 25 2.3 sterilization P4 20 ppb 0 2

P5 8 ppb 27 0.8

R2 60 ppb

Table 12. Effect of sterilization on barrier effect against thvalent cations (Al )

o Silicon element and tetravalent cation

[0082] Silicon element quantified by ICP/MS can come from both the glass or the coating : polymerized HMDSO by CVD or silicone (DC360 100OcSt - PDMS) oil.

[0083] The ETO sterilization did not affect the barrier effect against tetravalent cations (Si ⁴⁺ ) as shown in Table 13.

Average Relative Barrier effect

System Sample measurement Standard (ng/cm ² ) on 3 replicates deviation (RSD)

P1 1.8 ppm 0 180

P2 1.7 ppm 5 170

PTS without P3 2.5 ppm 17 250 sterilization P4 2.9 ppm 6 290

P5 2.8 ppm 37 280

R2 2.4 ppm 18 240

P1 1.9 ppm 16 190

P2 1.4 ppm 22 140

PTS + ETO P3 1.6 ppm 13 160 sterilization P4 1.6 ppm 40 160

P5 2.2 ppm 43 220

R2 1.8 ppm 8 180

Table 13. Effect of sterilization on barrier effect against tetravalent cations (Si )

c) Effect of gas composition on barrier effect

[0084] Different gas were used to treat the silicone oil spray on the glass barrels and different samples were tested and referred as to P : for oxygen plasma at 2s (PO), 5s (P1 ) et 15s (P2)

R2 : reference without plasma

S : plasma with a mixt of oxygen and argon at a ratio of % at 2s (SO), 5s

(S1 ) and 15s (S2)

o Monovalent cations: Sodium (Na ⁺ )

[0085] It is possible to have a barrier effect against monovalent cations by using different oxydative gases (Table 14).

Average Relative Barrier effect

System Sample measurement Standard (ng/cm ² ) on 3 replicates deviation (RSD)

PO 0.6 ppm 9 60

P1 0.6 ppm 10 60

P2 <0.5 ppm NA < 50

PTS -t FTn

R2 0.8 ppm 8 80 sterilization

SO 0.5 ppm 0 50

S1 <0.5 ppm NA < 50

S2 <0.5 ppm NA < 50

Table 14. Effect of gas composition on barrier effect against monovalent cations (Na ⁺ ) o Divalent cations: Magnesium (Mg ²⁺ )

[0086] It is possible to have a barrier effect against divalent cations by using different oxydative gases (Table 15).

Average Relative Barrier effect

System Sample measurement Standard (ng/cm ² ) on 3 replicates deviation (RSD)

PO <4 ppb NA <0.4

P1 <4 ppb NA <0.4

P2 <4 ppb NA <0.4

PTS -t FTn

R2 <4 ppb NA <0.4 sterilization

SO <4 ppb NA <0.4

S1 <6 ppb NA <0.6

S2 <9 ppb NA <0.9

Table 15. Effect of gas composition on barrier effect against divalent cations (Mg ²⁺ )

o Trivalent cations: Boron (B ³⁺ ) and Aluminum (Al ³⁺ )

[0087] It is possible to have a barrier effect against trivalent cations by using different oxydative gases (Tables 16 and 17).

Average Relative Barrier effect

System Sample measurement Standard (ng/cm ² ) on 3 replicates deviation (RSD)

PO 27 ppb 1 1 2.7

P1 23 ppb 12 2.3

P2 22 ppb 3 2.2

PTS -t FTn

R2 30 ppb 0 3.0 sterilization

SO 25 ppb 0 2.5

S1 23 ppb 12 2.3

S2 27 ppb 11 2.7

Table 16. Effect of gas composition on barrier effect against trivalent cations (B )

Average Relative Barrier effect

System Sample measurement Standard (ng/cm ² ) on 3 replicates deviation (RSD)

PO 28 ppb 10 2.8

P1 32 ppb 9 3.2

P2 18 ppb 16 1.8

PTS -t FTn

R2 60 ppb 0 6.0 sterilization

SO 37 ppb 8 3.7

S1 37 ppb 8 3.7

S2 27 ppb 11 2.7

Table 17. Effect of gas composition on barrier effect against trivalent cations (Al )

Silicon element and tetravalent cation

[0088] It is possible to have a barrier effect against tetravalent cations by using different oxydative gases (Table 18).

Average Relative Barrier effect

System Sample measurement Standard (ng/cm ² ) on 3 replicates deviation (RSD)

PO 1.5 ppm 0 150

P1 1.8 ppm 3 180

P2 1.5 ppm 21 150

PTS + FTn

R2 0.9 ppm 5 90 sterilization

SO 2.2 ppm 20 220

S1 1.9 ppm 8 190

S2 1.7 ppm 12 170

Table 18. Effect of gas composition on barrier effect against tetravalent cations (Si ⁴⁺ )

Example 2. Protein stability in the coated syringe barrels

[0089] Syringe barrels previously described in example 1 were filled with different model proteins presented in Table 19. They display different molecular weight and structure. The general data (molecular weight, isoelectric points, number of amino acids) as well as the primary and secondary structure of model proteins were obtained from Protein Data Base.

Number

MW Isoelectric

Proteins of amino (Daltons) point acids

Human insulin (1 HLS) 5,808 5.76 51

BSA (1 e7i) 66,596 5.52 585

Table 19. Selection of proteins

[0090] The secondary structure content are based on the following structures deposited in the Protein Databank (http://www.rcsb.org/pdb/), Protein references are given in bracket and given for a monomer

[0091] Insulin is a polypeptide hormone that regulates glucose metabolism.

[0092] Bovine Serum Albumin (BSA) is a serum albumin that is used to stabilize some enzymes and plays a role in transport of low hydrosoluble proteins.

[0093] The protein stability was studied at 37O and the h ydrodynamic diameter of the protein (nm) was measured by Dynamic Light Scattering (DLS). The measurements were made with a He Ne laser 633 nm at 173° in order to verify the presence of aggregates and to minimize the effects of dust contamination. Each native protein has a specific hydrodynamic diameter that is typically less than 20 nm. A study of the kinetics was performed at 37°C and the hydrodynamic diameter was measured after 7, 15 and 30 days. When proteins formed aggregates, large hydrodynamic diameters were measured. All the experiments were done in isotonic conditions (300 mOsm). [0094] The percentage of intensity was recorded as a function of the hydrodynamic diameter. Several peaks can be observed. Peak 1 corresponds to the hydrodynamic diameter of the native protein and peaks 2 and 3 to the protein aggregates. Values in brackets correspond to the percentage of light intensity for each peak. All the experiments were done in isotonic conditions (300 mOsm). [0095] Prior to the protein contact, the coated stoppers were cleaned 3 times for a period of 5 minutes in ultrasonic bath at 80°C.

Results are summarized in Tables 20 for BSA and Table 21 for insulin.

Day 0 Day 7 Day 14

BSA Size (nm) Int. (%) Size (nm) Int. (%) Size (nm) Int. (%)

Peaki 9.5 100 12.6 89 9.0 100

C1 Peak 2 800 11

Solid Peaki 9.5 100 10.8 92 ND

R1 Peak 2 860 8

Peaki 9.91 100 9.56 97.6 9.47 97.5

P1 Peak 2 4000 2.4 4130 2.5

Peaki 9.91 100 9.65 99.4 9.55 97.6

P2 Peak 2 4300 0.6 4220 2.4

Peaki 9.91 100 9.55 87.3 9.56 75

P3 Peak 2 660 12.7 294 25

PTS

Peaki 9.91 100 9.65 100 9.835 100

P4 Peak 2

Peaki 9.91 100 9.57 97.6 9.55 99.4

P5 Peak 2 4300 2.4 4380 0.6

Peaki 9.91 100 9.84 96 9.57 73

R2 Peak 2 2700 4 > μm 27

Peak i 9.90 85 8.7 62 8.8 55

A1 =C1 +P2 Peak 2 260 15 320 38 215 45

Peak i 9.90 85 9.7 65 9.0 81

Bilayer A2 =P2 Peak 2 260 15 260 35 710 19

Peak i 9.90 85 8.7 68 8.7 57

A1 bis= C2+P2 Peak 2 260 15 580 32 490 43

Table 20. Percentage of native protein (BSA) after stabilization (days) at 37O obtained by DLS.

[0096] Peak 1 corresponds to the hydrodynamic diameter of the native protein and peaks 2 to the protein aggregates. For each peak the diameter (nm) and the percentage of light intensity is mentioned.

[0097] The most stable protein was obtained with 5, 15, 60 and 120 seconds plasma treated barrels cooresponding to samples P1 , P2, P4 and P5. Indeed, the percentage of native protein is more than 73% after 14 days of stabilization.

Day 0 Day 7 Day 14

Insulin Size (nm) Int. (%) Size (nm) Int. (%) Size (nm) Int. (%)

Peaki 6.2 75 ND 5.6 95

C1 Peak 2 275 25 136 5

Solid Peaki 6.2 75 5.5 13 5.5 13

R1 Peak 2 275 25 430 87 200 87

Peaki 6.22 83 5.69 50.7 5.55 50.6

P1 Peak 2 192 17 156 49.3 272 49.4

Peaki 6.22 83 5.67 56 5.55 40.7

P2 Peak 2 192 17 120 44 272 59.3

Peaki 6.22 83 5.51 37.9 5.59 52

P3 Peak 2 192 17 141 62.1 139 48

PTS

Peaki 6.22 83 5.83 63 5.71 61

P4 Peak 2 192 17 130 37 131 39

Peaki 6.22 83 5.8 50.9 5.66 58.5

P5 Peak 2 192 17 120 49.1 130 41.5

Peaki 6.22 83 5.87 41.25 6.6 31.1

R2 Peak 2 192 17 409 58.75 235 68.9

Peaki 5.7 24 5.7 55 5.7 47

A1 =C1+P2 Peak 2 280 76 600 45 110 53

Peaki 5.7 24 5.8 60 5.7 48

Bilayer A2=P2 Peak 2 280 76 720 40 490 52

Peaki 5.7 24 5.7 59 5.8 29

A1 bis= C2+P2 Peak 2 280 76 580 41 310 61

Table 21. Percentage of native protein (Insulin) after stabilization (days) at 37O obtained by DLS. Peak 1 corresponds to the hydrodynamic diameter of the native protein and peaks 2 to the protein aggregates. For each peak the diameter (nm) and the percentage of light intensity is mentioned.

[0098] The most stable protein was obtained with 5, 15, 30, 60 and 120 seconds plasma treated barrels corresponding to samples P1 , P2, P3, P4 and P5. Indeed, the percentage of native protein is more than 31.1 % after 14 days. In double layers, the protein stability increases as a function of time.

Example 3. Functionality of the coated syringe barrels

[0099] Tests (Activation Gliding Force tests) were performed to determine the necessary forces to move a piston inside a treated or untreated syringe barrel. These tests were performed using a LLOYD-CB190 tensile testing machine dynamometer using NEXYGEN operating software, according to two test protocols outlined briefly below.

[00100] Activation Gliding Force (AGF) tests were applied on containers filled with 1 ml_ of demineralised water and each plugged with one piston. Each container-piston system was tested 24 times in order to ensure the reproducibility and to validate the results. To prepare the 24 syringes for a system, and particularly to insert the piston in the container, a SPV machine was used.

[00101] These gliding tests made it possible to establish the value of various friction forces referenced B, S and F, respectively: - the friction force B is the force required, under static conditions, to break the contact at the contact region between the piston and the container,

- the friction force S is the force required, under dynamic conditions, to move the piston in the container. The friction force S is measured half way of the piston travel. In order to measure the friction force S, the barrel was filled with water,

- and the friction force F is the force required, again in dynamic mode, to move the piston when it reaches the end of its travel in the container. Just like the friction force S, the friction force F is measured with a container initially filled with water.

a) Effect of different coatings on functionality

[00102] The gliding properties were measured on the different systems described in example 1. Here, the barrels were associated with a Parylene C coated stopper (4023/50 West company, bromobutyl). The results obtained were as followed (Table 22):

System Sample B

C1 >10 >10 >10

C2 > 10 > 10 > 10

C3 > 10 > 10 > 10

Solid coating C4 >10 >10 >10

C5 > 10 > 10 > 10

C6 > 10 > 10 > 10

R1 > 10 > 10 > 10

P1 2.5 (± 0.2) 4.5 (± 0.2) 5 (± 0.3)

P2 3.5 (± 0.5) 4.7 (± 0.3) 5.5 (± 0.3)

P3 5.8 (± 0.4) 5.1 (± 0.4) 6.9 (± 0.3)

PTS

P4 6.1 (± 0.4) 6.3 (± 0.3) 7.1 (± 0.4)

P5 7.3 (± 0.5) 6.8 (± 0.3) 7.7 (± 0.3)

R2 2.1 (± 0.2) 4.6 (± 0.3) 4.9 (± 0.3)

A1 =C1 +P2 3.2 (± 0.6) 4.6 (± 0.2) 4.6 (± 0.3)

Bi layer A2 =P2 2.9 (± 0.2) 4.5 (± 0.2) 4.6 (± 0.2)

A1 bis= C2+P2 4.6 (± 1.1 ) 5.4 (± 0.7) 6.0 (± 1.0) Table 22. Activation Gliding Forces( in N)

[00103] The friction forces were higher than 10 N on all solid systems (referred as to C 1 to C6).

[00104] The friction force in the static mode was the lowest on the reference R2 and after 5s of plasma treatment (typically less than 2.5 N on P1 ), intermediary after 15 s of treatment (3.5N on P2) and reached 6N after 30s of treatment on P3. The sustainable force was less than 5N for the reference R2 or after 5 or 15 s of plasma treatment on P1 and P2 whereas it grew up to 5.1 N or more than 6N after 30s of treatment on P3, P4 and P5.

[00105] Last, the friction force in a dynamic mode was always less than 6N for the reference and for short durations of treatment (5 and 15s) whereas it was more than 6N for longer treatment durations (30, 60 and 120s). [00106] On bilayers associating an inner solid coating (C1 or C2) and an outer layer plasma treated silicone A2, the friction forces were similar to those measured on simple PTS system P2. Indeed, the friction forces B, S and F were less than 6N.

b) Effect of a long plasma duration on functionality

[00107] Long plasma duration was tested on silicone oil typically 10 min of treatment on samples P6 (table 23). Two durations between the step of silicone spray and plasma treatments were assessed after 30 min and 20 days. The forces obtained after 2 min (P5) or 10 min (P6) of treatment are similar in both cases.

System Sample B F

Pϊ 4.6 (0.3) 6.2 (0.2)

Time between P2 6.3 (0.4) 6.7 (0.3) silicone spray P3 6.3 (0.4) 6.6 (0.1 ) and plasma P5 7.0 (0.6) 7.1 (0.3)

20 days P6 8.0 (0.8) 7.8 (0.4)

R2 3.1 (0.3) 5.3 (0.3)

Pl 5.8 (0.6) 5.5 (0.1 )

Time between ^~ P2 5.8 (0.6) 5.7 (0.3) silicone spray ^~ P3 6.6 (0.5) 5.9 (0.2) and plasma ^~ P5 7.9 (0.2) 6.2 (0.1 )

< 30 min ^~ P6 9.9 (0.8) 6.1 (0.2)

^~ R2 4.2 (0.4) 5.3 (0.4)

Table 23. Effect of long plasma duration on functionality

c). Effect of sterilization on functionality

[00108] Silicone spraying was performed with the bench (LDN) (Table 24).

System Sample B

P1 5.5 (0.4) 6.4 (0.3)

P2 6.4 (0.4) 6.8(0.3)

PTS with P3 6.6 (0.4) 6.7(0.4) sterilization P4 7.1 (0.6) 6.9 (0.5)

P5 7.9 (0.7) 7.8 (0.5)

R2 3.1 (0.3) 5.3 (0.3)

P1 5.2 (0.4) 6.2 (0.4)

P2 6.0 (0.4) 6.8(0.3)

PTS + ETO P3 6.5 (0.4) 7.1 (0.7) sterilization P4 6.9 (0.5) 7.2 (0.5)

P5 7.4 (0.6) 8.3(0.6)

R2 2.5 (0.5) 4.6 (0.6)

Table 24.Effect of sterilization on functionality

[00109] Similar gliding forces were obtained before and after ETO sterilization on PTS systems P1 , P2, P3, P4 and P5.

d). Effect of the spray homogeneity and gas composition on functionality

[00110] Different gas were used to treat the silicone oil spray on the glass barrels and different samples were tested and referred as to

P : for oxygen plasma at 2s (PO), 5s (P1 ) et 15s (P2)

R2 : reference without plasma

S : plasma with a mixt of oxygen and argon at a ratio of % at 2s (SO), 5s (S1 ) and

15s (S2) [00111] Two programs of silicone spray were assessed on barrels with LDN bench and the functionality was assessed for both programs added by a Plasma treatment (Tables 8 and 9). LDN program 04

System Sample B

PO 4.6 (0.3) 6.1 (0.3)

P1 6.0 (0.5) 6.5 (0.3)

P2 7.0 (0.5) 6.8 (0.3)

R2 3.3 (0.4) 5.2 (0.3)

PTS without

SO 4.2 (0.3) 6.4 (0.3) sterilization

S1 5.1 (0.4) 6.6 (0.4)

S2 6.5 (0.7) 6.8 (0.4)

T1 5.1 (0.6) 6.6 (0.4)

T2 6.4 (0.5) 7.6 (0.6)

SO 4.8 (0.4) 6. 7 (0.3)

PTS + ETO

T1 5.1 (0.5) 6. 8 (0.4) sterilization

T2 6.4 (0.7) 7. 6 (0.4)

Table 25. Functionality after one program of LDN

o LDN programme 12

System Sample B

PO 5.1 (0.4) 6.6 (0.4)

P1 6.7 (0.8) 6.8 (0.4)

P2 7.5 (0.7) 7.1 (0.4)

PTS withni it

R2 3.5 (0.4) 5.5 (0.2) inn

SO 4.6 (0.4) 6.7 (0.4)

S1 5.8 (0.7) 7.0 (0.4)

S2 6.8 (0.6) 7.3 (0.4)

Table 26. Functionality after a second program of LDN