WO2018215580A1

WO2018215580A1 - Method for sterilizing prefilled plastic syringes containing a vegf antagonist

Info

Publication number: WO2018215580A1
Application number: PCT/EP2018/063611
Authority: WO
Inventors: Bernd Fiedler; Carsten Brockmeyer; Rainer Sigl
Original assignee: Formycon Ag
Priority date: 2017-05-24
Filing date: 2018-05-24
Publication date: 2018-11-29

Abstract

The present invention relates to methods for sterilizing a prefilled plastic syringe containing a liquid formulation of a VEGF antagonist by exposing the prefilled syringe to nitrogen dioxide, ethylen oxide or vaporized hydrogen peroxide. The present invention further relates to a prefilled plastic syringe which is suitable to be sterilized by this method.

Description

Method for sterilizing prefilled plastic syringes containing a VEGF antagonist

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION The surface of prefilled syringes for use in intraocular injection has to be surface sterilized to reduce the risk of contamination during subsequent handling and of bacterial infections of the eye which may occur if the surface of the syringes used for administration is not sufficiently sterilized. However, protein drugs do not tolerate treatment with high temperatures or by radiation which are often used to sterilize medical objects. Hence, the surface of prefilled syringes is typically sterilized with a sterilizing gas or vapor such as ethylene oxide or vaporized hydrogen peroxide.

WO 2008/077155 Al discloses an EO surface sterilization of objects containing biological molecules, while EP 2 453 928 discloses vaporized-hydrogen peroxide surface decontamination of prefilled containers in secondary packaging.

US 2012/0114524A1 discloses vaporized-hydrogen peroxide surface

decontamination of prefilled containers in secondary packaging, while WO 2014/187779 Al discloses a hydrogen peroxide / EO sterilization method for sterilizing the surface of a prefilled syringe.

Plastic syringes adsorb higher amounts of the sterilizing gas or vapor than glass syringes. Consequently, it takes a long time to desorb this sterilizing gas or vapor from the plastic material than from glass, so that the process time for surface sterilization of plastic syringes is prolonged, thereby increasing the risk for the integrity of the drug product. Hence, standard gas or vapor sterilization methods which are typically used for glass syringes are not applicable to plastic syringes and there is a need for methods which allow the efficient surface sterilization of prefilled plastic syringes.

SUMMARY OF THE INVENTION The present inventors have surprisingly found that a prefilled plastic syringe containing a liquid formulation of a VEGF antagonist can be efficiently sterilized with nitrogen dioxide, ethylen oxide or vaporized hydrogen peroxide without affecting the stability of the drug product contained therein. Accordingly, the present invention relates to a method for sterilizing the surface of a prefilled syringe comprising a plastic barrel and containing a liquid formulation of a VEGF antagonist, the method comprising exposing the prefilled syringe to nitrogen dioxide, ethylen oxide or vaporized hydrogen peroxide under conditions such that the surface of the prefilled syringe is sterilized.

In one embodiment the VEGF antagonist retains at least 80% of its activity.

In one embodiment the syringe comprises a gas bubble with a volume which is such that the stopper within the syringe substantially does not move when a vacuum of 2 to 10 mbar is applied to the syringe. In one embodiment the syringe does not comprise a backstop.

The prefilled syringe may be present in a secondary packaging which may be a blister pack or a pouch.

In one embodiment the prefilled syringe is exposed to nitrogen dioxide under the following conditions:

a) temperature between 10°C and 30°C;

b) nitrogen dioxide concentration between 5 and 15 mg/1;

c) relative humidity between 60% and 90%; and

d) a pressure of 400 to 600 mbar

for 30 minutes to 90 minutes. In one embodiment the prefilled syringe is exposed to ethylene oxide under the following conditions:

a) temperature between 20°C and 40°C;

b) ethylene oxide concentration between 300 and 900 mg/1;

c) relative humidity between 40%> and 80%>; and

d) a pressure of 20 to 80 mbar

for 2 to 6 hours.

In one embodiment the prefilled syringe is exposed to vaporized hydrogen peroxide under the following conditions:

a) temperature between 20°C and 40°C;

b) vaporized hydrogen peroxide concentration between 20 and 50%>;

c) relative humidity between 65%> and 95%>; and

d) a pressure of 2 to 20 mbar

for 30 to 90 minutes. The VEGF antagonist may be ranibizumab or aflibercept.

The present invention further relates to a sterilized prefilled syringe comprising a plastic barrel and containing a liquid formulation of a VEGF antagonist, wherein the syringe comprises a gas bubble with a volume which is such that the stopper within the syringe substantially does not move when a vacuum of 2 to 10 mbar is applied to the syringe.

In one embodiment the syringe does not comprise a backstop.

The VEGF antagonist may be ranibizumab or aflibercept.

The present invention further relates to a blister pack or pouch comprising said sterilized prefilled syringe.

DETAILED DESCRIPTION OF THE INVENTION

The present invention as illustratively described in the following may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

The present invention will be described with respect to particular embodiments, but the invention is not limited thereto, but only by the claims. Where the term "comprising" is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term "consisting of is considered to be a preferred embodiment of the term "comprising". If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which preferably consists only of these embodiments. For the purposes of the present invention, the term "obtained" is considered to be a preferred embodiment of the term "obtainable". Where an indefinite or definite article is used when referring to a singular noun, e.g. "a", "an" or "the", this includes a plural of that noun unless something else is specifically stated.

The term "sterilizing" means the process of reducing the amount of at least one biological contaminant present on the syringe surface. In one embodiment, the amount is reduced by at least 10-fold, preferably it is reduced by at least 100-fold, more preferably it is reduced by at least 1 ,000-fold and most preferably it is reduced by at least 10,000-fold. "Terminally sterilizing" means that a final packaged product is sterilized. Within the present invention, the terms "sterilizing" and "terminally sterilizing" are used interchangeably.

The terms "sterilized" or "sterile" mean to refer to a complete absence of microbial life as defined by a probability of nonsterility or a sterility assurance level (SAL). The required SAL for a given product is based on regulatory requirements. For example, required SALs for health care products are defined to be at least 10⁶, i.e. a chance of less than 1 : 1 million of a non-sterile product for aseptically manufactured and terminally sterilized products, respectively.

To determine whether a method is efficient in sterilizing an object such as the surface of a prefilled syringe, biological indicators are distributed in or on the object to be sterilized and after the method has been performed it is determined whether any biological indicators are still detectable. Typically, a biological indicator is a carrier material provided with at least 1 x 10⁶ highly resistant spores of a microorganism such as Bacillus subtilis. For a sterilization method to be effective, it is required that no biological indicators are detectable. A "biological contaminant" is a contaminant that, upon direct or indirect contact with a biological material, may have a deleterious effect on the biological material.

Examples of biological contaminants include viruses; bacteria or bacterial spores; parasites; yeasts; molds; mycoplasmas; and prions. Further, a biological contaminant need not be naturally or accidentally present. For example, a biological contaminant may be Bacillus subtilis spores deliberately placed on the surface of an object to be sterilized in order to monitor the success of the sterilization. A "pre-filled syringe" is a syringe which is supplied by the manufacturer in a filled state, i.e. a measured dose of the drug to be administered is already present in the syringe when it is purchased and ready for administration. In particular, the pharmaceutical composition containing the drug does not have to be drawn from a vial containing the composition by using an empty syringe. The term pre-filled syringe within the meaning of the present invention does not refer to syringes the content of which has been drawn from a vial in a repackaging process.

The components of a pre-filled syringe are known to a skilled person and basically comprise, from the outlet to the rear end, a tip cap or needle shield, a syringe barrel, a stopper located within the syringe barrel and a plunger rod.

The syringe barrel contains a defined volume of the liquid composition which can be expelled from the barrel through an outlet positioned on one end of the barrel when the plunger rod is pushed into and moves along the barrel. The syringe barrel typically has a substantially cylindrical shape. Preferably, the syringe barrel has a low conicality and more preferably the conicality is not more than 5°, preferably not more than 4°, more preferably not more than 3° and most preferably not more than 2°. The outlet may comprise a projection from the outlet end through which extends a channel having a smaller diameter than the rest of the syringe barrel. The outlet may be adapted, for example by a Luer lock type connection, for connection with a needle if no staked needle is used. Preferably, the pre-filled syringe used in the present invention has a small Luer cone channel. The Luer cone channel is the channel which extends through the cone-shaped part of the syringe and is the part of the Luer lock connection through which the liquid formulation within the syringe is expelled. The inner diameter of the Luer cone channel is not more than 2 mm, preferably not more than 1.5 mm, more preferably not more than 1.2 mm and most preferably not more than 1.0 or 0.8 mm. If the syringe comprises a Luer lock type connection, a tip cap is used to seal the barrel which can be removed to allow a needle to be attached to the syringe. This sealing can be achieved by the use of known sealing devices such as the OVS™ system of Vetter Pharma International GmbH. The tip cap is usually made of an elastomer which may comprise a fluoropolymer coating in the interior part which is in contact with the syringe.

In the pre-filled syringe of the present invention the syringe outlet may be firmly connected with a needle so that the pre-filled syringe is supplied with a staked needle and does not need to be assembled prior to use. In this case, the risk of injuries with the needle during assembly of the syringe before injection is reduced. Prior to use the staked needle is typically covered by a needle shield to ensure sterility of the syringe content.

The staked needle can be attached to the pre-filled plastic syringe of the present invention without using an adhesive, since it can be moulded into the syringe. In contrast, an adhesive is required to attach the needle to a glass syringe and can lead to impurities or increased protein oxidation (presentation of Adler at the 2011 PDA Europe "The Universe of Pre-Filled Syringes and Injection Devices", Basel, 7-11 November 2011; presentation of Markovic at the PDA Single Use Systems

Workshop, Bethesda, 22-23 June 2011). For intravitreal administration the needle size is typically 29, 29 ½ or 30 gauge, although 31-, 32, 33- and 34-gauge needles may also be used. The pre-filled syringe may be equipped with a passive needle safety guard to further avoid the danger of needle sticks after injection.

The pre-filled syringe of the present invention comprises a syringe barrel which is made from plastic material. Preferably, the plastic material is selected from cycloolefin polymer and cycloolefin copolymer and more preferably it is a cycloolefin polymer and most preferably it is a cycloolefin polymer known as Crystal Zenith^®.

Cycloolefin copolymers may be produced by chain copolymerization of cyclic monomers such as 8,9,10-trinornorn-2-ene or l,2,3,4,4a,5,8,8a-octahydro-l,4:5,8- dimethanonaphthalene with ethane. Suitable copolymers are those of the Topas™ type which are available in a variety of grades.

Cycloolefin polymers may for example be produced by ring-opening metathesis polymerization of various cyclic monomers followed by hydrogenation. Suitable commercially available containers made of cycloolefin polymer material include containers manufactured from Crystal Zenith™ resin, Zeonor™ and Zeonex™. Such materials have a glass-like transparency, are highly break resistant and provide an excellent moisture barrier. According to the present invention the syringe barrel is silicone-free which means that the inner surface of the syringe barrel has not been treated with silicone oil. Hence, no silicone oil can be detected within the pre-filled syringe of the present invention. The presence and thickness of silicone layers can be determined by known methods such as the rap. ID Layer Explorer^® application which can also be used to measure the amount of silicone oil within the syringe barrel. The amount of silicone oil within the syringe barrel can also be measured by differential weighing methods and quantitation by infrared spectroscopy of the oil diluted in a suitable solvent.

The pre-filled syringe may be uncoated, i.e. the cycloolefm polymer or copolymer material is in direct contact with the liquid composition contained therein and the syringe barrel does not contain any material other than the plastic material of which the syringe is made.

Alternatively, the pre-filled syringe may comprise an internal coating other than a silicone coating. The term "internal coating" is intended to mean a coating on the inner side of the syringe barrel which is in contact with the drug solution, i.e. the liquid composition. Preferably, the internal coating is selected to prevent the entry of gas or vapor into the interior of the prefilled syringe. Examples of such an internal coating include a fluorocarbon film made from a modified ethylene- tetrafluoroethylene copolymer (also called Flurotec^® film, available from West Pharmaceutical Services) and a perfluoropolyether film crosslinked by an

Atmospheric Plasma Immobilization™ process (also called TriboGlide^®, available from TriboFilm Research and described in WO 2005/094214 A2).

Preferably, the pre-filled syringe does not comprise an internal coating. The syringe may also comprise a coating on the outer surface of the syringe which is in contact with the environment such as an oxygen barrier coating.

The syringe barrel is tungsten- free, i.e. it does not contain any traces of tungsten, since it is not necessary to use tungsten in the syringe manufacturing process. Hence, there is no risk of tungsten- induced protein aggregation. In one embodiment the syringe barrel comprises a mark such as a line printed on the syringe barrel which line allows the person injecting the liquid composition to align a pre-determined part of the stopper (such as the tip of the front surface) or plunger rod with the mark. Thereby, any excess liquid composition and potential air bubbles are removed from the syringe barrel, allowing the safe administration of an exact predetermined dosage to the patient.

The syringe barrel has a length of 45 to 65 mm. If the syringe has a nominal maximum fill volume of 1 ml, the length of the syringe barrel is 60 to 65 mm. If the syringe has a nominal maximum fill volume of 0.5 ml, the length of the syringe barrel is 45 to 50 mm. The length of the syringe barrel is the length between the rear end to the outlet to which the needle is attached (but not including the needle, if present).

The syringe barrel has an internal diameter of 4 to 6.5 mm. If the syringe has a nominal maximum fill volume of 1 ml, the internal diameter of the syringe barrel is 5.5 to 6.5 mm. If the syringe has a nominal maximum fill volume of 0.5 ml, the internal diameter of the syringe barrel is 4 to 5 mm.

The wall of the syringe barrel has a thickness of at least 1 mm, preferably of 1 to 3 mm, more preferably of 1.5 to 3 mm and most preferably of 2.4 to 2.8 mm. Due to the thickness of the wall the sterilizing gas cannot enter the interior of the syringe barrel and therefore does not get into contact with the liquid formulation contained within the prefilled syringe.

The plunger rod is pulled and pushed along inside the syringe barrel, allowing the syringe to expel the liquid formulation through the outlet. The plunger rod comprises a stopper contact surface, a rod and a flange (arranged from the outlet end to the rear end). When the plunger rod is moved through the syringe barrel from the rear part towards the outlet by applying pressure to the flange, the stopper contact surface of the plunger rod comes into contact with the rear part of the stopper and moves the stopper through the barrel to expel the liquid composition contained within the syringe through the outlet of the syringe barrel. The stopper contact surface of the plunger rod is preferably substantially flat, i.e. it does not comprise any protrusions for connection to the stopper.

The stopper is located within the syringe barrel between the syringe outlet and the plunger rod. The stopper is typically made of an elastomeric material such as natural or synthetic rubber, which engages an inner surface of the syringe barrel to create a seal that facilitates ejecting the liquid formulation from the syringe when pressure is applied to the flange of the plunger rod and the stopper moves through the syringe barrel. Since the stopper is not mechanically connected to the plunger rod before administration, it is not retractable. The term "non-retractable stopper" therefore is intended to mean that the stopper can only be moved in the direction of the syringe outlet, but not in the opposite direction, i.e. to the rear part of the syringe. It also means that the stopper and the plunger rod are not mechanically connected. Hence, any risk for the contamination of the liquid composition within the syringe is minimized.

The stopper may be coated with a fluoropolymer film such as an ethylene

tetrafluoroethylene (ETFE; marketed as FluroTec^®) barrier film, a fluorinated ethylene propylene (FEP; marketed as Teflon^® FEP) or a polytetrafluoroethylene-like film such as used for an Omniflex stopper at least in that part which comes into contact with the liquid composition contained within the prefilled syringe. This type of coating serves as an effective barrier between the drug and the elastomer, reducing the potential for extractables or leachables which are inherent to all materials. In addition, the coating reduces the occurrence of the reverse process, where the drug product can adsorb or absorb into the plunger rod. The stopper is preferably silicone- free, i.e. at least the surface of the stopper which comes into contact with the drug solution and more preferably the complete stopper has not been coated with silicone oil. Further, the stopper is made from a material which withstands vacuum conditions without dislocation, i.e. the stopper does not move within the syringe barrel when a vacuum is applied. More preferably, the stopper is silicone-free and comprises a coating with ethylene tetrafluoroethylene.

The syringe has a nominal maximum fill volume, i.e. a volume which can be maximally taken up by the syringe, of 0.3 ml to 1.5ml, preferably of 0.5 ml to 1.0 ml, more preferably of 0.5 ml or 1.0 ml and most preferably of 1.0 ml.

The volume of the liquid composition filled into the syringe is about 0.05 ml to about lml, preferably about 0.1 ml to about 0.5 ml, more preferably 0.14 ml to 0.3 ml and most preferably 0.15 ml to 0.2 ml. The skilled person knows that the syringe is usually filled with a volume which is larger than the volume actually administered to the patient to take into account any dead space within the syringe and the needle and the loss due to the preparation of the syringe for injection. Hence, the volume which is actually administered to the patient is between 0.01 ml and 1 ml, preferably between 0.02 and 0.5 ml, more preferably between 0.025 and 0.5 ml, even more preferably between 0.03 ml and

0.05 ml and most preferably the volume which is actually administered to the patient is 0.05 ml.

Ranibizumab is typically administered in a volume of 0.05 ml with a ranibizumab concentration of 6 or 10 mg/ml or in a volume of 0.03 ml or 0.05 ml with a ranibizumab concentration of 10 mg/ml, yielding a delivered amount of 0.3 or 0.5 mg. For aflibercept the administered volume is typically 0.05 ml with an aflibercept concentration of 40 mg/ml, yielding a delivered amount of 2 mg. Additionally, bevacizumab is used off-label for the treatment of ocular diseases. In this case, the administered volume of bevacizumab is 0.05 ml with a bevacizumab concentration of 25 mg/ml, yielding a delivered amount of 1.25 mg.

Hence, in one embodiment the syringe is filled with a volume of the liquid composition of 0.15 ml to 0.2 ml and 0.03 ml to 0.05 ml of the liquid composition are then administered to the patient.

The prefilled syringe of the present invention comprises a gas bubble with a volume which is such that the stopper within the syringe barrel substantially does not move when a vacuum of 2 to 10 mbar is applied to the syringe. It has been found that the stopper within the prior art syringe, i.e. the Lucentis^® prefilled syringe, which has a gas bubble with a volume of about 40 μΐ moves along the syringe barrel when a deep vacuum is applied to the syringe. Due to this movement the stopper may get to the rear end of the syringe so that the syringe loses its sterility.

A stopper does not substantially move within the syringe barrel, if it moves less than 4 mm, preferably less than 3 mm, more preferably less than 2 mm and most preferably less than 1 mm when a vacuum of 2 to 10 mbar is applied to the syringe. In a particular embodiment the stopper does not move at all within the syringe barrel.

The gas bubble has a volume of less than 30 μΐ or 25 μΐ, preferably of less than 20 or 15 μΐ, more preferably of less than 10 μΐ, 8 μΐ or 7 μΐ, even more preferably of less than 6 μΐ or 5 μΐ and most preferably of less than 4 μΐ. In one embodiment, the prefilled syringe of the present invention and to be used in the methods of the present invention does not comprise any gas bubble. It can be determined by visual inspection whether a gas bubble is present in the prefilled syringe or not. The volume of the gas bubble can be calculated based on the measured diameter of the gas bubble using the following formula: V = (⁴/3)·πτ³, wherein r is the radius, i.e. half of the diameter of the gas bubble. The gas bubble which may be formed by air or nitrogen may remain in the syringe after it has been filled with the liquid formulation and the stopper has been introduced. To reduce the volume of the gas bubble or to avoid that a gas bubble remains in the syringe, the stopper is preferably introduced into the syringe barrel while a vacuum is applied to the barrel. To this end, vacuum is applied before the stopper is introduced and when the vacuum has reached its target value, the stopper is drawn into the barrel until it is in contact with the liquid formulation. The lower vacuum is applied, the faster the stopper moves within the syringe barrel and the smaller is the remaining gas bubble. A suitable vacuum for reducing the volume of the air bubble is 30 mbar. Alternatively, a stopper placement tube may be used which has to be brought as close as possible to the liquid formulation within the prefilled syringe to reduce the volume of the gas bubble as far as possible.

The volume of the gas bubble is relevant for the sterilization process, as under the vacuum conditions which are necessary to remove the sterilizing gas or vapor from the prefilled plastic syringe there is a risk that the volume of the gas bubble increases and therefore moves the plunger stopper and plunger rod to the rear end of the syringe barrel which is not sterile. If this movement occurs, the sterility of the drug product may be compromised and sterilizing agent may come into contact with the liquid formulation.

The use of a prefilled plastic syringe comprising a gas bubble with a low volume or which does not comprise a gas bubble is advantageous in the methods described herein, as this type of prefilled syringe can undergo a sterilization process in which a low pressure is applied and the pressure is changed rapidly, thereby enabling the rapid desorption of the sterilizing gas or vapor from the plastic material of the prefilled syringe. This rapid desorption in turn reduces the risk for the activity of the VEGF antagonist present in the prefilled syringe. The use of a prefilled plastic syringe comprising a gas bubble with a low volume or which does not comprise a gas bubble further does not require the use of a backstop with the syringe. A "backstop" reduces the opening diameter of the syringe body and simultaneously enlarges the finger flange with ergonomically shaped wings. The backstop fulfils two functions: first, it prevents the plunger stopper from being pulled out of the syringe and second, it eases the handling of the syringe due to an enlarged finger flange area. The backstop may be made of plastics and connected to the finger flange of the prefilled syringe. The prefilled syringe is filled with the liquid formulation of a VEGF antagonist under aseptic conditions, whether by an automated or manual process. "Aseptic" conditions refer to conditions free of bacterial or microbial contamination. Thus, the contents of the container are sterile and unaffected by surface decontamination methods as described herein. The term "filled" is meant to refer to the placement of contents, such as the liquid formulation, into the syringe in an appropriate amount, such as an appropriate volume or appropriate concentration. The appropriate amount, volume or concentration will vary depending on the nature of the contents and their intended use. The term "VEGF antagonist" refers to a molecule which specifically interacts with VEGF and inhibits one or more of its biological activities, e.g. its mitogenic, angiogenic and/or vascular permeability activity. It is intended to include both anti- VEGF antibodies and antigen-binding fragments thereof and non-antibody VEGF antagonists.

Non-antibody VEGF antagonists include aflibercept, pegaptanib and antibody mimetics. Preferably, the non-antibody VEGF antagonist is aflibercept. Aflibercept which is presently marketed under the name Eylea^® and which is also known as VEGF-trap is a recombinant human soluble VEGF receptor fusion protein in which portions of human VEGF receptors 1 and 2 extracellular domains are fused to the Fc portion of human IgGl (Holash et al. (2002) Proc. Natl. Acad. Sci. USA 99(17): 11393-11398; WO 00/75319 Al). The CAS number of aflibercept is 862111-32-8. It has received a marketing authorization for the treatment of wet age-related macular degeneration, visual impairment due to diabetic macular oedema (DME) and diabetic retinopathy in patients with diabetic macular edema. The present commercial aflibercept formulation contains sodium phosphate, sodium chloride, polysorbate 20, sucrose and water for injection and is supplied in a concentration of 40 mg/ml. In particular, it contains 40 mg/ml Aflibercept, 10 mM sodium phosphate buffer, 40 mM NaCl, 0.03% polysorbate 20, 5%> sucrose; and water for injection. An alternative aflibercept formulation may contain a histidine buffer, sodium chloride, polysorbate 20, sucrose and water for injection and is supplied in a concentration of 40 mg/ml. In particular, it contains 40 mg/ml Aflibercept, 10 mM histidine buffer, 40 mM NaCl, 0.03%) polysorbate 20, 5%> sucrose; and water for injection. The pH of the

commercial and the alternative Aflibercept formulation may be adjusted to 6.2.

Pegaptanib which is presently marketed under the name Macugen^® is a pegylated anti- vascular endothelial growth factor (VEGF) aptamer (Bell et al. (1999) In Vitro Cell Dev Biol Anim. 35(9): 533-42). The CAS number of pegaptanib is 222716-86-1. Antibody mimetics which are VEGF antagonists include binding proteins comprising an ankyrin repeat domain that binds VEGF and inhibits its binding to the receptor, such as DARPin^® MP0112 (see also WO 2010/060748 and WO 2011/135067).

The term "anti- VEGF antibody" refers to an antibody or antibody fragment such as a Fab or a scFV fragment that specifically binds to VEGF and inhibits one or more of its biological activities, e.g. its mitogenic, angiogenic and/or vascular permeability activity. Anti- VEGF antibodies act, e.g., by interfering with the binding of VEGF to a cellular receptor, by interfering with vascular endothelial cell activation after VEGF binding to a cellular receptor, or by killing cells activated by VEGF. Anti- VEGF antibodies include, e.g., antibodies A4.6.1, bevacizumab, ranibizumab, G6, B20, 2C3, and others as described in, for example, WO 98/45331 , US

2003/0190317, US 6,582,959, US 6,703,020, WO 98/45332, WO 96/30046, WO 94/10202, WO 2005/044853, EP 0 666 868 Bl, WO 2009/155724 and Popkov et al. (2004) J. Immunol. Meth. 288: 149-64. Preferably, the anti-VEGF antibody or antigen-binding fragment thereof present in the pharmaceutical composition of the present invention is ranibizumab or bevacizumab. Most preferably, it is ranibizumab or an antigen-binding fragment thereof.

"Ranibizumab" is a humanised monoclonal Fab fragment directed against VEGF-A having the light and heavy chain variable domain sequences of Y0317 as described in SEQ ID Nos. 115 and 116 of WO 98/45331 and Chen et al. (1999) J. Mol. Biol. 293: 865-81. The CAS number of ranibizumab is 347396-82-1. Ranibizumab inhibits endothelial cell proliferation and neovascularisation and has been approved for the treatment of neovascular (wet) age-related macular degeneration (AMD), the treatment of visual impairment due to diabetic macular oedema (DME), the treatment of visual impairment due to macular oedema secondary to retinal vein occlusion (branch RVO or central RVO), or treatment of visual impairment due to choroidal neovascularisation (CNV) secondary to pathologic myopia. Ranibizumab is related to bevacizumab and derived from the same parent mouse antibody as bevacizumab but it is much smaller than the parent molecule and has been affinity matured to provide stronger binding to VEGF-A. Ranibizumab is produced recombinantly in Escherichia coli, e.g. as described in WO 98/45331 A2. The present commercial ranibizumab formulation contains α,α-trehalose dihydrate, histidine hydrochloride monohydrate, histidine, polysorbate 20 and water for injection and is supplied in a concentration of 10 mg/ml. In particular, it contains 6 or 10 mg. Ranibizumab, 100 mg. α,α-trehalose dihydrate; 0.32 mg. L-histidine, 1.66 mg. L-histidine

hydrochloride monohydrate, 0.1 mg Polysorbate 20 and water for injection qs to 1 mL. The pH of the present commercial Ranibizumab formulation may be adjusted to pH 5.5. "Bevacizumab" is a full-length, humanized murine monoclonal antibody that recognizes all iso forms of VEGF and which is the parent antibody of ranibizumab. The CAS number of bevacizumab is 216974-75-3. Bevacizumab inhibits

angiogenesis and is presently approved for the treatment of different cancer types. However, it is also used off-label in ophthalmological diseases such as age-related macular degeneration. The present commercial bevacizumab formulation contains α,α-trehalose dihydrate, sodium phosphate, polysorbate 20 and water for injection and is supplied as a concentrate with a concentration of 25 mg/ml. In particular, it contains 25 mg/ml Bevacizumab, 240 mg α,α-trehalose dihydrate, 23.2 mg sodium phosphate (monobasic, monohydrate), 4.8 mg sodium phosphate (dibasic, anhydrous), 1.6 mg polysorbate 20, and water for Injection, USP to 4 ml.

The antagonist concentration within the pre-filled syringes of the present invention is typically 1-100 mg/ml, preferably 2-75 mg/ml, more preferably 3-50 mg/ml, even more preferably 5 to 30 mg/ml and most preferably 6 or 10 mg/ml. If ranibizumab is contained within the pre-filled syringe of the present invention the ranibizumab concentration is 10 mg/ml. If aflibercept is contained within the pre-filled syringe of the present invention the aflibercept concentration is 40 mg/ml. The pre-filled syringe may contain one or more pharmacologically active agents in addition to the VEGF antagonist. A pharmacologically active agent is able to exert a pharmacological effect when administered to a subject. Preferably, the additional pharmacologically active agent is a PDGF antagonist or an Ang2 antagonist. More preferably, the PDGF antagonist is an anti-PDGF antibody such as rinucumab or an aptamer such as El 0030, marketed as Fovista^®. Most preferably, the PDGF antagonist is E10030 which is described in Green et al. (1996) Biochemistry 35: 14413; US 6,207,816; US 5,731,144; US 5,731,424; and US 6,124,449. Also more preferably, the Ang2 antibody is an anti-Ang2 antibody and most preferably it is nesvacumab. "Secondary packaging" refers to packaging which completely encloses the prefilled plastic syringe and enables sterilization in that it is permeable for sterilizing gases, but is not permeable for biological contaminants. It may consist of one, two or more materials such as plastic wrapping, foil wrapping, paper wrapping or other suitable wrapping which are firmly connected with each other. One type of material which is typically used in secondary packaging are nonwoven flash-spun high-density polyethylene fibers known under the brand name Tyvek^®. Preferably, the secondary packaging is a pouch or a blister pack. A "blister pack" has a cavity or pocket which is usually made from thermoformed plastic and a backing of paperboard or a lidding seal of aluminium foil or plastic. A "pouch" is typically a flexible container made from plastic such as polyethylene (PE), oriented polyamide (OP A) and Tyvek^® and combinations of these materials.

In the methods of the present invention nitrogen dioxide, ethylene oxide or vaporized hydrogen peroxide may be used to sterilize the prefilled syringe comprising a liquid formulation of a VEGF antagonist.

The sterilizing process typically comprises three cycle phases:

1) Pre-conditioning phase

2) Conditioning phase

3) Aeration or post-conditioning phase

In the pre-conditioning phase the syringe is wetted and brought to the sterilization temperature to enable the penetration of sterilizing gases through the secondary packaging. To exchange air with sterilizing gas, the chamber is evacuated using a vacuum pump, before the sterilizing gas is injected.

In the conditioning phase the sterilization parameters temperature, relative humidity, pressure and concentration of the sterilizing gas are kept constant for a time period, before the sterilizing gas is removed from the sterilizing chamber. Finally, at the end of the sterilization cycle the sterilizing gas is washed off by repeatedly adding and removing air or nitrogen. The sterilization process may be influenced by the variables sterilizing gas concentration, relative humidity, pressure, temperature and gas exposure time. These variables have to be chosen so as to ensure that the prefilled syringe is sterilized, but that the activity of the drug product contained therein is not compromised. The gas concentration should be enough to sterilize the product, but not enough to create any problems with residual gas.

Moisture is not only helpful in the transfer of heat to the product, but it also aids in the absorption and desorption of the sterilizing gas into and out of the

product/packaging. Moisture is transferred to the sterilization load during preconditioning and sterilization/conditioning via a controlled steam process.

The higher the temperature, the higher the lethality of the cycle. Heat is transferred to the sterilization load during preconditioning and sterilization/conditioning via a controlled steam process.

The gas exposure time should be the time it takes for the gas to penetrate into the desired area of the devices and the microbiological kill time. The duration is determined at the cycle design/development stage.

If nitrogen dioxide is used for sterilizing the prefilled syringe, the conditioning phase may have the following parameters:

a) temperature between 10°C and 30°C;

b) nitrogen dioxide concentration between 5 and 15 mg/1;

c) relative humidity between 60% and 90%; and d) a pressure of 400 to 600 mbar

for 30 minutes to 90 minutes.

Preferably, the temperature is in the range of 19°C to 26°C, more preferably it is in the range of 20°C to 24°C and most preferably it is 22°C .

Preferably, the nitrogen dioxide concentration is from 7 mg/1 to 13 mg/1, more preferably it is from 8 to 12 mg/1 and most preferably it is from 9 to 11 mg/1. Preferably, the relative humidity is between 68% and 82%, more preferably it is betweeen 70%> and 80%> and most preferably it is 75%.

Preferably, the pressure is 420 to 520 mbar, more preferably it is 450 to 500 mbar and most preferably it is 470 to 480 mbar.

The incubation time is preferably 40 to 80 minutes, more preferably 50 to 70 minutes and most preferably it is 60 minutes.

The pre-conditioning phase for nitrogen dioxide treatment may have the following parameters:

a) temperature between 10°C and 30°C;

b) relative humidity between 65% and 85%; and

d) a pressure of 800 to 1,100 mbar

for 5 to 30 minutes.

Preferably, the temperature in the pre-conditioning phase is in the range of 19°C to 26°C, more preferably it is in the range of 20°C to 24°C and most preferably it is Preferably, the relative humidity in the pre-conditioning phase is between 68% and 82%o, more preferably it is betweeen 70%> and 80%> and most preferably it is 75%.

Preferably, the pressure in the pre-conditioning phase is in the range of 850 to 1,050 mbar, more preferably it is 900 to 1,020 mbar and most preferably it is 980 to 990 mbar.

The incubation time in the pre-conditioning phase is preferably 8 to 25 minutes, more preferably 10 to 22 minutes and most preferably it is 17 minutes.

In the post-conditioning phase a relative humidity between 65%> and 85%, preferably between 68%> and 82%, more preferably it is betweeen 70% and 80% and most preferably of 75% may be applied for a period of 60 minutes to 3 hours, preferably for a period of 70 minutes to 150 minutes, more preferably for a period of 80 minutes to 120 minutes and most preferably for a period of 90 minutes. The above period refers to the sterilant removal time, which may comprise several cycles of adding and removing air to scrub the sterilant.

If ethylene oxide is used for sterilizing the prefilled syringe, the conditioning phase may have the following parameters:

a) temperature between 20°C and 40°C;

b) ethylene oxide concentration between 300 and 900 mg/1;

c) relative humidity between 40% and 80%; and

d) a pressure of 20 to 80 mbar

for 2 to 6 hours.

Preferably, the temperature is in the range of 32°C to 45°C, more preferably it is in the range of 33°C to 45°C and most preferably it is in the range of 35°C to 45°C. Preferably, the ethylene oxide concentration is from 320 mg/1 to 880 mg/1, more preferably it is from 340 to 860 mg/1 and most preferably it is from 400 to 800 mg/1.

Preferably, the relative humidity is between 42% and 78%, more preferably it is betweeen 43% and 76% and most preferably it is between 45% and 75%.

Preferably, the pressure is 22 to 78 mbar, more preferably it is 25 to 75 mbar and most preferably it is 30 to 70 mbar. The incubation time is preferably 2 to 5 hours and most preferably it is 3 to 4 hours.

The pre-conditioning phase for ethylene oxide treatment may have the following parameters:

a) temperature between 20°C and 40°C;

b) relative humidity between 40% and 80%; and

c) a pressure of 20 to 80 mbar

for 30 minutes to 3 hours.

Preferably, the temperature is in the range of 32°C to 45°C, more preferably it is in the range of 33°C to 45°C and most preferably it is in the range of 35°C to 45°C.

Preferably, the relative humidity is between 42% and 78%, more preferably it is betweeen 43% and 76% and most preferably it is between 45% and 75%. Preferably, the pressure is 22 to 78 mbar, more preferably it is 25 to 75 mbar and most preferably it is 30 to 70 mbar.

The incubation time is preferably 40 minutes to 150 minutes and most preferably it is 1 to 2 hours. The post-conditioning phase for ethylene oxide treatment may have the following parameters:

a) temperature between 20°C and 40°C;

b) relative humidity between 40% and 80%; and

c) a pressure of 20 to 80 mbar

for 1 hour to 10 hours.

Preferably, the pressure is 22 to 78 mbar, more preferably it is 25 to 75 mbar and most preferably it is 30 to 70 mbar.

The overall incubation time which may comprise several cycles of adding and removing air is preferably 2 hours to 8 hours, more preferably it is 3 hours to 7 hours and and most preferably it is 4 hours to 6 hours.

Residual levels of ethylene oxide (EO) and/or ethylene chlorohydrine (ECH) may be present after EO sterilization and must be evaluated to assure they meet predefined maximum limits. When determining the suitability of EO for sterilization of pharmaceutical packages, medical devices, prefilled drug vessels (e.g. syringes), etc., it is important to ensure that the levels of residual EO and/or ethylene chlorohydrin (ECH) pose a minimal risk to the patient in normal product use. Moreover when the choice for EO sterilization has been made, irrespective of the provisions of this standard, exposure to EO residuals should be minimized. At the same time, ECH residuals when ECH has been found to be present in the packages or devices sterilized with EO, should also be minimized.

In the embodiments of the current invention, the residuals of both EO and ECH are determined after EO sterilization process. The method used to analyse the EO and ECH residuals is preferably ANSI/AAMI/ISO 10993-7 (Water Extraction).

In another embodiment of the current invention, the EO and ECH residuals are comparable to the EO and ECH residuals of glass vessels after a prolonged time period following the sterilization. The prolonged time period in the current application can be right after the sterilization, right after shipping lag (t=0), 1 month after t=0 (i.e. t=l month), 6 month after t=0 (i.e. t= 6 month), 12 month after t=0 (i.e. t=12 month) or longer time period after t=0. In another embodiment of the current invention, the EO and/or ECH residuals are below a detection limit after a prolonged time period followed the sterilization. The average daily dose of EO to a patient after drug administration shall not exceed 4 mg/syringe, optionally shall not exceed 3mg/syringe, optionally shall not exceed 2mg/syringe, optionally shall not exceed 1 mg/syringe, is preferably lower than 1 μg/syringe, is preferably lower than 0.1 μg/syringe, or is most preferably lower than the detection limit.

In another embodiment, the EO residuals and/or ECH residuals in the first 1 month (or 30 days) are lower than 60 mg/syringe, optionally lower than 30 mg/syringe, optionally lower than 10 mg/syringe, optionally lower than 1 mg/syringe, optionally lower than 0.1 mg/syringe, preferably lower than 1 μg/syringe, preferably lower than 0.1 μg/syringe, most preferably lower than the detection limit.

In another embodiment, the EO residuals and/or ECH residuals in the first 12 months are lower than 60 mg/syringe, optionally lower than 30 mg/syringe, optionally lower than 10 mg/syringe, optionally lower than 1 mg/syringe, optionally lower than 0.1 mg/syringe, preferably lower than 1 μg/syringe, preferably lower than 0.1 μg/syringe, most preferably lower than detection limit. If vaporized hydrogen peroxide is used as sterilizing gas, the conditioning phase may have the following parameters:

a) temperature between 20°C and 40°C;

b) vaporized hydrogen peroxide concentration between 20 and 50%;

c) relative humidity between 65% and 95%>; and

d) a pressure of 2 to 20 mbar

for 30 to 90 minutes.

Preferably, the temperature is in the range of 27°C to 33°C, more preferably it is in the range of 28°C to 32°C and most preferably it is 30°C.

Preferably, the vaporized hydrogen peroxide concentration is from 25% to 45%, more preferably it is from 30% to 40% and most preferably it is 35%.

Preferably, the relative humidity is between 70% and 90%, more preferably it is between 72% and 88% and most preferably it is between 75% and 85%.

Preferably, the pressure is 3 to 15 mbar, more preferably it is 3 to 10 mbar and most preferably it is 4 mbar. The incubation time is preferably 35 minutes to 75 minutes, more preferably 40 minutes to 60 minutes and most preferably it is 50 minutes

The pre-conditioning phase for vaporized hydrogen peroxide treatment may have the following parameters:

a) temperature between 20°C and 40°C; b) relative humidity between 3% and 30%; and

c) a pressure of 30 to 1 ,000 mbar

for 20 minutes to 1 hour. Preferably, the temperature is in the range of 27°C to 33°C, more preferably it is in the range of 28°C to 32°C and most preferably it is 30°C.

Preferably, the relative humidity is between 4% and 25%, more preferably it is betweeen 4% and 22% and most preferably it is between 5% and 20%.

Preferably, the pressure is 50 to 900 mbar, more preferably it is 70 to 850 mbar and most preferably it is 100 to 800 mbar.

The incubation time is preferably 20 minutes to 50 minutes, more preferably it is 30 minutes to 45 minutes and most preferably it is 40 minutes.

The post-conditioning phase for vaporized hydrogen peroxide treatment may have the following parameters:

a) temperature between 20°C and 40°C;

b) relative humidity between 3% and 30%; and

c) a pressure of 2 to 1 ,000 mbar

for 1 hour to 3 hours.

Preferably, the relative humidity is between 7% and 95%, more preferably it is betweeen 8% and 90% and most preferably it is between 10% and 90%. Preferably, the pressure is 3 to 950 mbar and most preferably it is 4 to 900 mbar. The sterilant removal time which may comprise several cycles of adding and removing air is preferably 70 minutes to 160 minutes, more preferably it is 90 minutes to 150 minutes and most preferably it is 120 minutes.

The biological activity of the VEGF antagonist, preferably an anti-VEGF antibody or a VEGF receptor fusion protein and more preferably ranibizumab or aflibercept can be determined by incubating different dilutions of the antagonist which was stored under the conditions described above with human umbilical vein endothelial cells (HUVEC) and VEGF and measuring the VEGF-induced proliferation of the cells in the presence of the antagonist, i.e. by the CellTiter-Blue^® Cell Viability Assay available from Promega, in comparison to cells not incubated with the antagonist. Since the VEGF antagonist inhibits VEGF-induced signal transduction, the VEGF- induced proliferation will be reduced, if biologically active VEGF antagonist is present in the sample.

The VEGF antagonist, preferably the anti-VEGF antibody or VEGF receptor fusion protein and more preferably ranibizumab or aflibercept retains at least 80% of its biological activity after the pre-filled syringe has been subjected to any of the methods of the present invention, such that the VEGF-induced proliferation is inhibited in HUVEC. The VEGF-antagonist, preferably the anti-VEGF antibody or VEGF receptor fusion protein and more preferably ranibizumab or aflibercept retains at least 80% of its biological activity after sterilizing the pre-filled syringe, if the VEGF-induced proliferation is inhibited by at least 50%>, preferably by at least 55% or 60%), more preferably by at least 65%, 70%, 75% or 80%, even more preferably by at least 85%, 87% or 90% and most preferably by at least 92%, 94%, 96%, 98% or 99%.

The drug contained in the pre-filled syringe which is sterilized by the method of the present invention, i.e. the VEGF antagonist, preferably an anti-VEGF antibody, is stable at a temperature of 2 to 8°C for at least six months, preferably for at least 9 months, more preferably for at least one year, particularly preferably for at least 18 months and most preferably for about two years. The drug contained in the pre-filled syringe which is sterilized by the method of the present invention, i.e. the VEGF antagonist, preferably an anti-VEGF antibody or a VEGF receptor fusion protein and more preferably ranibizumab or aflibercept, is stable at room temperature, i.e. a temperature between 20°C and 25°C, for at least three days or one week, preferably for at least two or three weeks, more preferably for about 4 weeks and most preferably for at least three months. The drug contained in the pre-filled syringe which is sterilized by the method of the present invention, i.e. the VEGF antagonist, preferably an anti-VEGF antibody or a VEGF receptor fusion protein and more preferably ranibizumab or aflibercept, is stable at a temperature of about 40°C, for at least four or six hours, preferably for at least 10 or 12 hours, more preferably for at least 18 or 24 hours and most preferably for one or two weeks.

The stability of the drug within the syringe can for example be determined by ion exchange chromatography by which modifications of the drug such as oxidized and deamidated species can be detected or by size exclusion chromatography by which aggregates of the drugs can be detected. A description of such an analysis is provided in the examples section.

The drug, i.e. the VEGF antagonist, preferably the anti-VEGF antibody, is considered stable, if the sum of all impurities comprising aggregates and chemically modified species is less than 2%, preferably less than 1.5%, more preferably less than 1.2% and most preferably less than 1% compared to the amount of non- modified, non-aggregated drug.

While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive. The invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing a claimed invention, from a study of the drawings, the disclosure, and the dependent claims. The detailed description is merely exemplary in nature and is not intended to limit application and uses. The following examples further illustrate the present invention without, however, limiting the scope of the invention thereto. Various changes and modifications can be made by those skilled in the art on the basis of the description of the invention, and such changes and modifications are also included in the present invention.

EXAMPLES

In the following examples 165 μΐ of a solution containing 10 mg/ml ranibizumab, 7.85 mM L-histidine hydrochloride monohydrate. 2.15 mM L-histidine, 10 % (w/v) trehalose dihydrate and 0.01% (w/v) polysorbate 20, pH 5.5, i.e. the components of the commercial ranibizumab formulation, was filled into 0.5 ml cycloolefin polymer syringes which do not contain any silicone and which have a Luer cone with an elastomeric tip cap and a stopper. The elastomeric closures were compatible with the sterilant and gas tight. The volume of the gas bubble within the syringe was about 4 μΐ. Then the prefilled syringe were packed into pouches composed of PET-O/PE and Tyvek.. The samples were then subjected to sterilization as described below and then stored for 0.5, 1 or 3 months at a temperature of 25°C and 60% relative humidity or of 40°C and 75% relative humidity. Further samples were stored at 5°C for 3, 6 or 12 months, before the stability of the ranibizumab antibody was determined.

1. Surface sterilization by ethylene oxide using a deep vacuum process

The pre-conditioning phase for deep vacuum ethylene oxide treatment was performed with the following parameters: a) temperature between 35°C and 45°C;

b) relative humidity between 45% and 75%; and

c) a pressure of 30 to 70 mbar

for 1 to 2 hours.

The conditioning phase for deep vacuum ethylene oxide treatment was performed with the following parameters:

a) temperature between 35°C and 45°C;

b) ethylene oxide concentration between 400 and 800 mg/1;

c) relative humidity between 45% and 75%; and

d) a pressure of 30 to 70 mbar

for 3 to 4 hours.

The post-conditioning phase for deep vacuum ethylene oxide treatment was performed with the fo llo wing parameters :

a) temperature between 35°C and 45°C;

b) relative humidity between 45% and 75%; and

c) a pressure of 30 to 70 mbar

for 3 hours to 3 weeks.

2. Surface sterilization with vaporized hydrogen peroxide

The pre-conditioning phase for vaporized hydrogen peroxide treatment was performed with the following parameters:

a) temperature of 30°C;

b) relative humidity between 5% and 20%; and

c) a pressure of 100 to 800 mbar

for 40 minutes. The conditioning phase for vaporized hydrogen peroxide treatment was performed with the following parameters:

a) temperature between 30°C;

b) hydrogen peroxide concentration of 35%;

c) relative humidity between 5% and 95%; and

d) a pressure of 4 mbar

for 50 minutes.

The post-conditioning phase for vaporized hydrogen peroxide treatment was performed with the fo llo wing parameters :

a) temperature between 30°C;

b) relative humidity between 10% and 90%>; and

c) a pressure of 4 to 900 mbar

for 135 minutes.

3. Surface sterilization by nitrogen dioxide

The pre-conditioning phase for nitrogen dioxide treatment was performed with the following parameters:

a) temperature between 20°C and 24°C;

b) relative humidity between 65% and 85%; and

d) a pressure of 986 to 988 mbar

for 17 minutes. The conditioning phase for nitrogen dioxide treatment was performed with the following parameters:

a) temperature between 20°C and 24°C;

b) nitrogen dioxide concentration between 9 and 11 mg/1;

c) relative humidity between 65% and 85%; and d) a pressure of 472 to 474 mbar

for 59 to 61 minutes.

In the post-conditioning phase for nitrogen dioxide treatment the relative humidity was 65% and 85% and the period was 87 to 93 minutes.

4. Assessment of ranibizumab stability after sterilization

After storage, the samples were analyzed by cation exchange chromatography for the presence of acidic and basic variants of the antibody, by size exclusion

chromatography for the presence of aggregates and by non-reduced SDS page analysis to detect fragmented and modified ranibizumab. a) Cation exchange analysis

The protein samples from the syringes were loaded onto a Dionex, BioLCProPac® WCX-10, 4.0 x 250 mm, 10 μιη column to detect acidic and basic variants of the protein. The protein was eluted with a gradient of mobile phase A (20 mM potassium phosphate buffer, ph 6.0) and mobile phase B (250 mM KC1, 20 mM potassium phosphate buffer, ph 6.0) according to the following Table 2:

Solvent Solvent

Time

composition composition

[min]

[%-B] [mM KC1]

0 0 0

3 0 0

33 50 125

35 50 125

36 0 0

40 0 0 Eluted species were detected and displayed on a graph showing the concentration of the eluted species vs. time. The elution profile showed a main peak with the unmodified protein and some further peaks eluting before and after the main peak, representing acidic and basic variants of the protein, respectively. The total area of all peaks as well as the area of the single peaks was determined. b) Size exclusion chromatography

The protein samples from the syringes were loaded onto a YMC-Pack Dio 1-200, 5 μιη, 20 nm (8.0 x 300 mm) column to detect aggregates of the protein.

The protein was eluted by isocratic elution using 0.1 M potassium phosphate and 0.2 M sodium chloride. Eluted species were detected and displayed on a graph showing the concentration of the eluted species vs. time. The elution profile showed a main peak with the non-aggregated protein and some further peaks of the protein representing aggregated forms of the protein. The area of all peaks was determined. c) Non-reduced SDS-PAGE By non-reduced SDS-PAGE physical modifications such as fragmentation and oligomerization of ranibizumab were determined.

The SDS-PAGE was performed under non-reducing conditions in a 4-12% Tris- Glycine gel. Samples were pre-diluted to 0.4 mg/ml with water and further diluted to 0.2 mg/ml with SDS sample buffer. The samples were incubated at 95°C for 5 min. After the run the gel was rinsed three times with 100 mL deionized water and dyed with Coomassie overnight at room temperature. After discoloration the gel was scanned and analyzed using QuantityOne Software. The running conditions were as follows:

voltage: 125 V

current: 35 mA

power: 5 W

time: 130 min

5. Break-loose and gliding force of the prefilled syringe after sterilization

The syringes stored as described above were tested for their stopper movement forces, i.e. the break loose force and the gliding force. Prior to testing, 27G x 0.5" needles were attached to the luer cone syringes. The testing was performed at a stopper speed of 190 mm/min over a travel length of 10.9 mm in a Tensile testing machine (TH2730, Thumler). 6. Subvisible particles in the prefilled syringe after sterilization

After storage of the sterilized syringes as described above, the light obscuration was determined with the FlowCam PV bench top system (Fluid Imaging Technologies Inc., Maine, USA) using the System software (VisualSpreadsheet software, version 3.4.8) and the fo llo wing parameters :

Mode: Autolmage

Priming Method: manual prime with sample

Flow Rate : 0.100 ml/min

Recalibrations: 0

Stop Reason: Sample Volume Processed

Sample Volume Aspirated: 1.0421 ml

Sample Volume Processed: 1.0392 ml

Fluid Volume Imaged: 0.3421 ml

Frame Rate: 22.00 fps

Magnification: 10X Calibration Factor: 0.6979 Syringe Size: 1.00 ml

Claims

1. Method for sterilizing the surface of a prefilled syringe comprising a plastic barrel and containing a liquid formulation of a VEGF antagonist, the method comprising exposing the prefilled syringe to nitrogen dioxide, ethylen oxide or vaporized hydrogen peroxide under conditions such that the surface of the prefilled syringe is sterilized.

2. Method of claim 1, wherein said VEGF antagonist retains at least 80% of its activity.

3. Method of claim 1 or 2, wherein the syringe comprises a gas bubble with a volume which is such that the stopper within the syringe substantially does not move when a vacuum of 2 to 10 mbar is applied to the syringe.

4. Method of claim 3, wherein the syringe does not comprise a backstop.

5. Method of any one of claims 1 to 4, wherein the prefilled syringe is present in a secondary packaging.

6. Method of claim 5, wherein the secondary packaging is a blister pack or a pouch.

7. Method of any one of claims 1 to 6, wherein the prefilled syringe is exposed to nitrogen dioxide under the following conditions:

a) temperature between 10°C and 30°C;

b) nitrogen dioxide concentration between 5 and 15 mg/1;

c) relative humidity between 60% and 90%>; and

d) a pressure of 400 to 600 mbar

for 30 minutes to 90 minutes.

8. Method of any one of claims 1 to 6, wherein the prefilled syringe is exposed to ethylene oxide under the following conditions:

a) temperature between 20°C and 40°C;

b) ethylene oxide concentration between 300 and 900 mg/1;

c) relative humidity between 40% and 80%; and

d) a pressure of 20 to 80 mbar

for 2 to 6 hours.

9. Method of any one of claims 1 to 6, wherein the prefilled syringe is exposed to vaporized hydrogen peroxide under the following conditions:

a) temperature between 20°C and 40°C;

b) vaporized hydrogen peroxide concentration between 20 and 50%>;

c) relative humidity between 65% and 95%>; and

d) a pressure of 2 to 20 mbar

for 30 to 90 minutes.

10. Method of any one of claims 1 to 9, wherein the VEGF antagonist is ranibizumab or aflibercept.

1 1. Sterilized prefilled syringe comprising a plastic barrel and containing a liquid formulation of a VEGF antagonist, wherein the syringe comprises a gas bubble with a volume which is such that the stopper within the syringe substantially does not move when a vacuum of 2 to 10 mbar is applied to the syringe.

12. Sterilized prefilled syringe of claim 1 1 , wherein the syringe does not comprise a backstop.

13. Sterilized prefilled syringe of claim 1 1 or 12, wherein the VEGF antagonist is ranibizumab or aflibercept.

14. Blister pack or pouch comprising a sterilized prefilled syringe of any one of claims 11 to 13.