AU2018201120A1 - Syringe - Google Patents

Abstract

The present invention relates to a syringe, particularly to a small volume syringe such as a syringe suitable for ophthalmic injections. Fig 1 4o s 4% Fig2 Fg Fig 3 4-45 Fig 4 Fig 5 '

Description

The present invention relates to a syringe, particularly to a small volume syringe such as a syringe suitable for ophthalmic injections.

2018201120 15 Feb 2018

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Figi

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2018201120 15 Feb 2018

SYRINGE

TECHNICAL FIELD

The present invention relates to a syringe, particularly to a small volume syringe such as a syringe suitable for ophthalmic injections.

BACKGROUND ART

Many medicaments are delivered to a patient in a syringe from which the user can dispense the medicament. If medicament is delivered to a patient in a syringe it is often to enable the patient, or a caregiver, to inject the medicament. It is important for patient safety and medicament integrity that the syringe and the contents of that syringe are sufficiently sterile to avoid .0 infection, or other, risks for patients. Sterilisation can be achieved by terminal sterilisation in which the assembled product, typically already in its associated packaging, is sterilised using heat or a sterilising gas.

For small volume syringes, for example those for injections into the eye in which it is intended that about 0.1ml or less of liquid is to be injected the sterilisation can pose difficulties that are not necessarily associated with larger syringes. Changes in pressure, internal or external to the syringe, can cause parts of the syringe to move unpredictably, which may alter sealing characteristics and potentially compromise sterility. Incorrect handling of the syringe can also pose risks to product sterility.

Furthermore, certain therapeutics such as biologic molecules are particularly sensitive to sterilisation, be it cold gas sterilisation, thermal sterilisation, or irradiation. Thus, a careful balancing act is required to ensure that while a suitable level of sterilisation is carried out, the syringe remains suitably sealed, such that the therapeutic is not compromised. Of course, the syringe must also remain easy to use, in that the force required to depress the plunger to administer the medicament must not be too high.

There is therefore a need for a new syringe construct which provides a robust seal for its content, but which maintains ease of use,

DISCLOSURE OF THE INVENTION

The present invention provides a pre-tilled syringe, the syringe comprising a body, a stopper and a plunger, the body comprising an outlet at an outlet end and the stopper being arranged within the body such that a front surface of the stopper and the body define a variable volume chamber

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2018201120 15 Feb 2018 from which a fluid can be expelled though the outlet, the plunger comprising a plunger contact surface at a first end and a rod extending between the plunger contact surface and a rear portion, the plunger contact surface arranged to contact the stopper, such that the plunger can be used to force the stopper towards the outlet end of the body, reducing the volume of the variable volume 5 chamber, characterised in that the fluid comprises an ophthalmic solution. In one embodiment, the ophthalmic solution comprises a VEGF-antagonist.

In one embodiment, the syringe is suitable for ophthalmic injections, more particularly intravitreal injections, and as such has a suitably small volume. The syringe may also be silicone oil free, or substantially silicone oil free, or may comprise a low level of silicone oil as lubricant. 0 In one embodiment, despite the low silicone oil level, the stopper break loose and slide force is less than 20N.

For ophthalmic injections, it is particularly important for the ophthalmic solution to have particularly low particle content. In one embodiment, the syringe meets US Pharmacopeia standard 789 (USP789).

Syringe

The body of the syringe may be a substantially cylindrical shell, or may include a substantially cylindrical bore with a non circular outer shape. The outlet end of the body includes an outlet through which a fluid housed within the variable volume chamber can be expelled as the volume of said chamber is reduced. The outlet may comprise a projection from the outlet end through

Ϊ0 which extends a channel having a smaller diameter than that of the variable volume chamber. The outlet may be adapted, for example via a luer lock type connection, for connection to a needle or other accessory such as a sealing device which is able to seal the variable volume chamber, but can be operated, or removed, to unseal the variable volume chamber and allow connection of the syringe to another accessory, such as a needle. Such a connection may be made directly between the syringe and accessory, or via the sealing device. The body extends along a first axis from the outlet end to a rear end.

The body may he made from a plastic material (e.g. a cyclic olefin polymer) or from glass and may include indicia on a surface thereof to act as an injection guide. In one embodiment the body may comprise a priming mark. This allows the physician to align a pre-determined part of the stopper (such as the tip of the front surface or one of the circumferential ribs, discussed later) or plunger with the mark, thus expelling excess ophthalmic solution and any air bubbles from the

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2018201120 15 Feb 2018 syringe. The priming process ensures that an exact, pre-determined dosage is administered to the patient.

The stopper may be made from rubber, silicone or other suitable resiliently deformable material. The stopper may be substantially cylindrical and the stopper may include one or more 5 circumferential ribs around an outer surface of the stopper,, the stopper and ribs being dimensioned such that the ribs form a substantially fluid tight seal with an internal surface of the syringe body. The front surface of the stopper may be any suitable shape, for example substantially planar, substantially conical or of a domed shape. The rear surface of the stopper may include a substantially central recess. Such a central recess could be used to connect a 0 plunger to the stopper using a snap fit feature or thread connection in a known manner. The stopper may be substantially rotationally symmetric about an axis through the stopper.

The plunger comprises a plunger contact surface and extending from that a rod extends from the plunger contact surface to a rear portion. The rear portion may include a user contact portion adapted to be contacted by a user during an injection event. The user contact portion may comprise a substantially disc shaped portion, the radius of the disc extending substantially perpendicular to the axis along which the rod extends. The user contact portion could be any suitable shape. The axis along which the rod extends may be the first axis, or may be substantially parallel with the first axis.

The syringe may include a backstop arranged at a rear portion of the body. The backstop may be .0 removable from the syringe. If the syringe body includes terminal flanges at the end opposite the outlet end the backstop may be configured to substantially sandwich terminal flanges of the body as this prevent movement of the backstop in a direction parallel to the first axis.

The rod may comprise at least one rod shoulder directed away from the outlet end and the backstop may include a backstop shoulder directed towards the outlet end to cooperate with the rod shoulder to substantially prevent movement of the rod away from the outlet end when the backstop shoulder and rod shoulder are in contact. Restriction of the movement of the rod away from the outlet end can help to maintain sterility during terminal sterilisation operations, or other operations in which the pressure within the variable volume chamber or outside the chamber may change. During such operations any gas trapped within the variable volume chamber, or bubbles that may form in a liquid therein, may change in volume and thereby cause the stopper to move. Movement of the stopper away from the outlet could result in the breaching of a sterility zone created by the stopper. This is particularly important for low volume syringes

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2018201120 15 Feb 2018 where there are much lower tolerances in the component sizes and less flexibility in the stopper. The term sterility zone as used herein is used to refer to the area within the syringe that is sealed by the stopper from access from either end of the syringe. This may be the area between a seal of the stopper, for example a circumferential rib, closest to the outlet and a seal of the stopper, 5 for example a circumferential rib, furthest from the outlet. The distance between these two seals defines the sterility zone of the stopper since the stopper is installed into the syringe barrel in a sterile environment.

To further assist in maintaining sterility during the operations noted above the stopper may comprise at a front circumferential rib and a rear circumferential rib and those ribs may be 0 separated in a direction along the first axis by at least 3mm, by at least 3.5 mm, by at least 3.75mm or by 4mm or more. One or more additional ribs (for example 2, 3, 4 or 5 additional ribs, or between 1-10,2-8, 3-6 or 4-5 additional ribs) may be arranged between the front and rear ribs, in one embodiment there are a total of three circumferential ribs.

A stopper with such an enhanced sterility zone can also provide protection for the injectable 5 medicament during a terminal sterilisation process. More ribs on the stopper, or a greater distance between the front and rear ribs can reduce the potential exposure of the medicament to the sterilising agent. However, increasing the number of ribs can increase the friction between the stopper and syringe body, reducing ease of use. While this may be overcome by increasing the siliconisation of the syringe, such an increase in silicone oil levels is particularly undesirable :0 for syringes for ophthalmic use.

The rod shoulder may be arranged within the external diameter of the rod, or may be arranged outside the external diameter of the rod. By providing a shoulder that extends beyond the external diameter of the rod, but still fits within the body, the shoulder can help to stabilise the movement of the rod within the body by reducing movement of the rod perpendicular to the first axis. The rod shoulder may comprise any suitable shoulder forming elements on the rod, but in one embodiment the rod shoulder comprises a substantially disc shaped portion on the rod.

In one embodiment of the syringe, when arranged with the plunger contact surface in contact with the stopper and the variable volume chamber is at its intended maximum volume there is a clearance of no more than about 2mm between the rod shoulder and backstop shoulder. In some embodiments there is a clearance of less than about 1.5 mm and in some less than about 1mm. This distance is selected to substantially limit or prevent excessive rearward (away from the outlet end) movement of the stopper.

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In one embodiment the variable volume chamber has an internal diameter greater than 5mm or 6mm, or less than 3mm or 4mm. The internal diameter may be between 3mm and 6mm, or between 4mm and 5mm, in another embodiment the syringe is dimensioned so as to have a nominal maximum fill volume of between about 0.1ml and about 1.5ml, In certain embodiments the nominal maximum fill volume is between about 0.5ml and about 1ml. In certain embodiments the nominal maximum fill volume is about 0.5ml or about 1 ml, or about 1.5ml.

The length of the body of the syringe may be less than 70mm, less than 60mm or less than 50mm. In one embodiment the length of the syringe body is between 45mm and 50mm.

In one embodiment, the syringe is filled with between about 0.01ml and about 1.5ml (for example between about 0.05ml and about 1ml, between about 0.1ml and about 0.5ml, between about 0.15ml and about 0.175ml) of a VEGF antagonist solution. In one embodiment, the syringe is filled with 0.165ml of a VEGF antagonist solution. Of course, typically a syringe is filled with more than the desired dose to be administered to the patient, to take into account wastage due to “dead space” within the syringe and needle. There may also be a certain amount of wastage when the syringe is primed by the physician, so that it is ready to inject the patient.

Thus, in one embodiment, the syringe is filled with a dosage volume (i.e, the volume of medicament intended for delivery to the patent) of between about 0.01ml and about 1.5mi (e.g. between about 0.05ml and about 1ml, between about 0.1ml and about 0.5ml) of a VEGF antagonist solution. In one embodiment, the dosage volume is between about 0.03ml and about 0.05ml. For example, for Lucentis, the dosage volume is 0,05ml or 0.03ml (0.5mg or 0.3mg) of a lOmg/ml injectable medicament solution; for Eylea. the dosage volume is 0.05ml of a 4Qmg/ml injectable medicament solution. Although unapproved for Ophthalmic indications, bevacizumab is used off-label in such ophthalmic indications at a concentration of 25mg/ml; typically at a dosage volume of 0.05ml (1.25mg). In one embodiment, the extractable volume from the syringe (that is the amount of product obtainable from the syringe following filling, taking into account loss due to dead space in the syringe and needle) is about 0.09ml.

In one embodiment the length of the syringe body is between about 45mm and about 50mm, the internal diameter is between about 4mm and about 5mm, the fill volume is between about 0.12 and about 0.3ml and the dosage volume is between about 0.03ml and about 0.05ml.

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As the syringe contains a medicament solution, the outlet may be reversibly seated to maintain sterility of the medicament. This sealing may be achieved through the use of a sealing device as is known in the art. For example the OVS™ system which is available from Vetter Pharma International GmbH.

It is typical to siliconise the syringe in order to allow ease of use, i.e. to apply silicone oil to the inside of the barrel, which decreases the force required to move the stopper. However, for ophthalmic use, it is desirable to decrease the likelihood of silicone oil droplets being injected into the eye. With multiple injections, the amount of silicone droplets can build up in the eye, causing potential adverse effects, including “floaters’' and an increase in intra-ocular pressure.

Furthermore, silicone oil can cause proteins to aggregate. A typical 1ml syringe comprises 10080()pg silicone oil in the barrel, though a survey of manufacturers reported that 500-1000pg was typically used in pre-filled syringes (Badkar et al. 2011, AAPS PharmaSciTech, 12(2):564-572). Thus, in one embodiment, a syringe according to the invention comprises less than about 800pg (i.e. about less than about 500pg, less than about 300pg, less than about 200pg, less than about

100pg, less than about 75pg, less than about 50pg, less than about 25pg, less than about 15pg, less than about lOpg) silicone oil in the barrel. If the syringe comprises a low level of silicone oil, this may be more than about lpg, more than about 3pg, more than about 5pg, more than about 7pg or more than about 10pg silicone oil in the barrel. Thus, in one embodiment, the syringe may comprise about Ipg-about 500pg, about 3pg-abont 200pg, about 5pg-about lOOpg or about lOpg-about 50pg silicone oil in the barrel. Methods for measuring the amount of silicone oil in such a syringe barrel are known in the art and include, for example, differential weighing methods and quantitation by infrared-spectroscopy of the oil diluted in a suitable solvent. Various types of silicone oil are available, but typically either DC36O (Dow Corning®; with a viscosity of lOOOcP) or DC365 emulsion (Dow Corning®; DC360 oil with a viscosity of 25 350cP) are used for syringe siliconisation. In one embodiment, the pre-filled syringe of the invention comprises DC365 emulsion.

During testing it was surprisingly found that, for syringes having small dimensions, such as those discussed above, and particularly those described in conjunction with the Figures below, the break loose and sliding forces for the stopper within the syringe are substantially unaffected by reducing the siliconisation levels far below the current standard to the levels discussed here. This is in contrast to conventional thinking that would suggest that if you decrease the silicone oil level, the forces required would increase (see e.g. Schoenknecht. AAPS National Biotechnology Conference 2007 - Abstract no. NBC07-000488, which indicates that while 400pg silicone oil is

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2018201120 15 Feb 2018 acceptable, usability improves when increased to 800pg). Having too great a force required to move the stopper can cause problems during use for some users, for example accurate dose setting or smooth dose delivery may be made more difficult if significant strength is required to move, and/or keep in motion, the stopper. Smooth administration is particularly important in sensitive tissues such as the eye, where movement of the syringe during administration could cause local tissue damage. Break loose and slide forces for pre-filled syringes known in the art are typically in the region of less than 20N, but where the pre-filled syringes contain about lQOpg-about 800pg silicone oil. In one embodiment the glide/slide force for the stopper within the pre-filled syringe is less than about 1 IN Or less than 9N, less than 7N, less than 5N or .0 between about 3N to 5N, In one embodiment, the break loose force is less than about 1 IN or less than 9N, less than 7N, less than 5N or between about 2N to 5N. Note that such measurements are for a filled syringe, rather than an empty syringe. The forces are typically measured at a stopper travelling speed of l90mm/min. In one embodiment, the forces are measured with a 30G x 0.5 inch needle attached to the syringe. In one embodiment, the syringe has a nominal maximal fill .5 volume of between about 0.5ml and 1ml, contains less than about lOOpg silicone oil and has a break loose force between about 2N to 5N.

In one embodiment the syringe barrel has an internal coating of silicone oil that has an average thickness of about 450nm or less (i.e. 400nm or less, 350nm or less, 3Q0nm or less, 200nm or less, lOOnm or less, 50nm or less, 20nm or less). Methods to measure the thickness of silicone oil in a syringe are known in the art and include the rap.ID Layer Explorer® Application, which can also be used to measure the mass of silicone oil inside a syringe barrel.

In one embodiment, the syringe is silicone oil free, or substantially silicone oil free. Such low silicone oil levels can be achieved by using uncoated syringe barrels and/or by avoiding the use of silicone oil as a lubricant for product contacting machine parts, or pumps in the syringe assembly and fill line. A further way to reduce silicone oil and inorganic silica levels in a prefilled syringe is to avoid the use of silicone tubing in filling lines, for example between storage tanks and pumps.

The syringe according to the invention may also meet certain requirements for particulate content. In one embodiment, tlie ophthalmic solution comprises no more than 2 particles >5Qpm in diameter per ml. In one embodiment, the ophthalmic solution comprises no more than 5 particles >25pm in diameter per ml. In one embodiment, the ophthalmic solution comprises no more than 50 particles >I0pm in diameter per ml. In one embodiment, the ophthalmic solution

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2018201120 15 Feb 2018 comprises no more than 2 particles >50qm in diameter per ml, no more than 5 particles >25pm in diameter per ml and no more than 50 particles >10pm in diameter per ml. In one embodiment, a syringe according to the invention meets USP789 (United States Pharmacopoeia: Particulate

Matter in Ophthalmic Solutions), in one embodiment the syringe has low levels of silicone oil sufficient for the syringe to meet USP789.

VEGF Antagonists Antibody VEGF antagonists

VEGF is a well-characterised signal protein which stimulates angiogenesis. Two antibody VEGF antagonists have been approved for human use, namely ranibizumab (Lucentis©) and 0 bevacizumab (Avastin®).

Non-Antibody VEGF antagonists

In one aspect of the invention, the non-antibody VEGF antagonist is an immunoadhesin. One such immuoadhesin is aflibercept (Eylea®), which has recently been approved for human use and is also known as VEGF-trap (Holash et al. (2002) PNAS USA 99:11393-98; Riely & Miller (2007) Clin Cancer Res 13:4623-7s). Aflibercept is the preferred non-antibody VEGF antagonist for use with the invention. Aflibercept is a recombinant human soluble VEGF receptor fusion protein consisting of portions of human VEGF receptors 1 and 2 extracellular domains fused to the Fc portion of human IgG 1. It is a dimeric glycoprotein with a protein molecular weight of 97 kilodaltons (kDa) and contains glycosylation, constituting an additional 15% of the total

Ό molecular mass, resulting in a total molecular weight of 115 kDa. It is conveniently produced as a glycoprotein by expression in recombinant CHO K1 cells. Each monomer can have the following amino acid sequence (SEQ ID NO: 1):

SDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPKITVTLKKFPLDTLIPDGKRIIwnSRKGFIISNATY

KEIGLLTCEATWGHLYKTNYLTHRQTNTIWWLSPSHGIELSVGEKLVLNCTARTELNVGIDFNWEYPS

SKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSGLMTKKNSTFVRVHEKDKTHTCPP

CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNST YRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL.TKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG and disulfide bridges can be formed between residues 30-79, 124-185, 246-306 and 352-410 within each monomer, and between residues 211-211 and 214-214 between the monomers.

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Another non-antibody VEGF antagonist immunoadhesin currently in pre-clinical development is a recombinant human soluble VEGF receptor fusion protein similar to VEGF-trap containing extracellular ligand-binding domains 3 and 4 from VEGFR2/KDR, and domain 2 from VEGFRl/Flt-1; these domains are fused to a human IgG Fc protein fragment (Li et aL, 2011

Molecular Vision 17:797-803). This antagonist binds to isoforms VEGF-A, VEGF-B and VEGFC. The molecule is prepared using two different production processes resulting in different glycosylation patterns on the final proteins. The two glycoforms are referred to as KH902 (conbercept) and KH906. The fusion protein can have the following amino acid sequence (SEQ IDNO:2):

WSYroTGVLLCALLSCLLLTGSSSGGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDT

LIPDGKRIIM3SRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVVI>SPSHGIELSVGEK LVLNCTARTELNVGIDFNWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAASSG LMTKKNSTFVRVHEKPFVAFGSGMESLVEATVGERVRLPAKYLGYPPPEIKWYKNGIPLESKHTIKAGHVL TIMEVSERDTGNYTVILTNPISKEKQSHWSLATVYVPPGPGDKTHTCPLCPAPELLGGPSVFLFPPKPKDT

LMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAXTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKC

KVSNKALPAPIEKTISKAKGGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK atppvldsdgsfflyskltvdksrwqqgnvfscsvmhealhnhytqkslslspgk and, like VEGF-trap, can be present as a dimer. This fusion protein and related molecules are further characterized in EPI767546.

Ϊ0 Other non-antibody VEGF antagonists include antibody mimetics (e.g. Affibody® molecules, affilins, affitins, anticalins, avimers, Kunitz domain peptides, and monobodies) with VEGF antagonist activity, This includes recombinant binding proteins comprising an ankyrin repeat domain that binds VEGF-A and prevents it from binding to VEGFR-2. One example for such a molecule is DARPin® MP0112. The ankyrin binding domain may have the following amino acid sequence (SEQ ID NO: 3):

gsdlgkklleaaragqddevrilmangadvntadstgwtplhlavpwghleivevllkygadvnakdfqgw

TPLHLAAAIGHQEIVEVLLKNGADVNAQDKFGKTAFDISIDNGNEDLAEILQKAA

Recombinant binding proteins comprising an ankyrin repeat domain that binds VEGF-A and prevents it from binding to VEGFR-2 are described in more detail in W02010/060748 and

WO2011/135067.

Further specific antibody mimetics with VEGF antagonist activity are the 40 kD pegylated anticalin PRS-050 and the monobody angiocept (CT-322).

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The afore-mentioned non-antibody VEGF antagonist may be modified to further improve their pharmacokinetic properties or bioavailability. For example, a non-antibody VEGF antagonist may be chemically modified (e.g., pegylated) to extend its in vivo half-life. Alternatively or in addition, it may be modified by glycosylation or the addition of further glycosylation sites not 5 present in the protein sequence of the natural protein from which the VEGF antagonist was derived.

Variants of the above-specified VEGF antagonists that have improved characteristics for the desired application may be produced by the addition or deletion of amino acids. Ordinarily, these amino acid sequence variants will have an amino acid sequence having at least 60% amino acid 0 sequence identity with the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95%, including for example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%. Identity or homology with respect to this sequence is defined herein as the percentage of amino acid 5 residues in the candidate sequence that are identical with SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.

Sequence identity can be determined by standard methods that are commonly used to compare the similarity in position of the amino acids of two polypeptides. Using a computer program such as BLAST or FASTA, two polypeptides are aligned for optimal matching of their respective amino acids (either along the full length of one or both sequences or along a predetermined portion of one or both sequences). The programs provide a default opening penalty and a default gap penalty, and a scoring matrix such as PAM 250 [a standard scoring matrix; see

Dayhoff et al., in Atlas of Protein Sequence and Structure, voi. 5, supp. 3 (1978)] can be used in conjunction with the computer program. For example, the percent identity can then be calculated as: the total number of identical matches multiplied by 100 and then divided by the sum of the length of the longer sequence within the matched span and the number of gaps introduced into the longer sequences in order to align the two sequences.

Preferably, the non-antibody VEGF antagonist of the invention binds to VEGF via one or more protein domain(s) that are not derived from the antigen-binding domain of an antibody. The non-1055157

2018201120 15 Feb 2018 antibody VEGF antagonist of the invention are preferably proteinaceous, but may include modifications that are non-proteinaceous (e.g., pegylation, glycosylation).

Therapy

The syringe of the invention may be used to treat an ocular disease, including but not limited to choroidal neovascularisation, age-related macular degeneration (both wet and dry forms), macular edema secondary to retinal vein occlusion (RVO) including both branch RVO (bRVO) and central RVO (eRVO), choroidal neovascularisation secondary to pathologic myopia (PM), diabetic macular edema (DME), diabetic retinopathy, and proliferative retinopathy.

Thus the invention provides a method of treating a patient suffering from of an ocular disease selected from choroidal neovascularisation, wet age-related macular degeneration, macular edema secondary to retinal vein occlusion (RVO) including both branch RVO (bRVO) and central RVO (eRVO), choroidal neovascularisation secondary to pathologic myopia (PM), diabetic macular edema (DME), diabetic retinopathy, and proliferative retinopathy, comprising the step of administering an ophthalmic solution to the patient using a pre-filled syringe of the invention. This method preferably further comprises art initial priming step in which the physician depresses the plunger of the pre-filled syringe to align the pre-determined part of the stopper with the priming mark.

In one embodiment, the invention provides a method of treating an ocular disease selected from choroidal neovascularisation, wet age-related macular degeneration, macular edema secondary to .0 retinal vein occlusion (RVO) including both branch RVO (bRVO) and central RVO (cRVO). choroidal neovascularisation secondary to pathologic myopia (PM), diabetic macular edema (DME), diabetic retinopathy, and proliferative retinopathy, comprising administering a nonantibody VEGF antagonist with a pre-filled syringe of the invention, wherein the patient has previously received treatment with an antibody VEGF antagonist.

Kits

Also provided are kits comprising the pre-filled syringes of the invention. In one embodiment, such a kit comprises a pre-filled syringe of the invention in a blister pack. The blister pack may itself be sterile on the inside. In one embodiment, syringes according to the invention may be placed inside such blister packs prior to undergoing sterilisation, for example terminal sterilisation.

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Such a kit may further comprise a needle for administration of the VEGF antagonist. If the VEGF antagonist is to be administered intravitreally, it is typical to use a 30-gauge x /2 inch needle, though 31-gauge and 32-gauge needles may be used. For intravitreal administration, 33-gauge or 34-gauge needles could alternatively be used. Such kits may further comprise 5 instructions for use. In one embodiment, the invention provides a carton containing a pre-fdled syringe according to the invention contained within a blister pack, a needle and optionally instructions for administration.

Sterilisation

As noted above, a terminal sterilisation process may be used to sterilise the syringe and such a 0 process may use a known process such as an ethylene oxide (EtO) or a hydrogen peroxide (H2O2) sterilisation process. Needles to be used with the syringe may be sterilised by the same method, as may kits according to the invention.

The package is exposed to the sterilising gas until the outside of the syringe is sterile. Following such a process, the outer surface of the syringe may remain sterile (whilst in its blister pack) for up to 6 months, 9 months, 12 months, 15 months, 18 months, 24 months or longer. Thus, in one embodiment, a syringe according to the invention (whilst in its blister pack) may have a shelf life of up to 6 months, 9 months, 12 months, 15 months, 18 months, 24 months or longer, in one embodiment, less than one syringe in a million has detectable microbial presence on the outside of the syringe after 18 months of storage. In one embodiment, the pre-fdled syringe has been

Ϊ0 sterilised using EtO with a Sterility Assurance Level of at least 1 O’⁶. In one embodiment, the prefdled syringe has been sterilised using hydrogen peroxide with a Sterility Assurance Level of at least 10⁶. Of course, it is a requirement that significant amounts of the sterilising gas should not enter the variable volume chamber of the syringe. The term “significant amounts’⁷ as used herein refers to an amount of gas that would cause unacceptable modification of the ophthalmic solution within the variable volume chamber. In one embodiment, the sterilisation process causes <10% (preferably <5%, <3%, <I%) alkylation of the VEGF antagonist, in one embodiment, the pre-filied syringe has been sterilised using EtO, but the outer surface of the syringe has <lppm, preferably <0.2ppm EtO residue. In one embodiment, the pre-filled syringe has been sterilised using hydrogen peroxide, but the outer surface ofthe syringe has <lppm, preferably <0.2ppm hydrogen peroxide residue. In another embodiment, the pre-filled syringe has been sterilised using EtO, and the total EtO residue found on the outside of the syringe and inside of the blister pack is <0.1mg. In another embodiment, the pre-filled syringe has been sterilised using hydrogen

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2018201120 15 Feb 2018 peroxide, and the total hydrogen peroxide residue found on the outside of the syringe and inside of the blister pack is <0.1 mg.

General

The term “comprising” means “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X + Y.

The term “about” in relation to a numerical value x means, for example, x+10%.

References to a percentage sequence identity between two amino acid sequences means that, when aligned, that percentage of amino acids are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software .0 programs known in the art, for example those described in section 7.7.18 of Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987) Supplement 30. A preferred alignment is determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62, The SmithWaterman homology search algorithm is disclosed in Smith & Waterman (1981) Adv. Appl.

Math. 2: 482-489

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a side view of a syringe

Figure 2 shows a cross section of a top down view of a syringe

Figure 3 shows a view of a plunger

Figure 4 shows a cross section though a plunger

Figure 5 shows a stopper

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MODES FOR CARRYING OUT THE INVENTION

The invention will now be further described, by way of example only, with reference to the drawings.

Figure 1 shows a view from a side of a syringe 1 comprising a body 2, plunger 4, backstop 6 and a 5 sealing device 8.

Figure 2 shows a cross section through the syringe 1 of Figure 1 from above. The syringe 1 is suitable for use in an ophthalmic injection. The syringe 1 comprises a body 2, a stopper 10 and a plunger 4. The syringe 1 extends along a first axis A. The body 2 comprises an outlet 12 at an outlet end 14 and the stopper 10 is arranged within the body 2 such that a front surface 16 of the 0 stopper 10 and the body 2 define a variable volume chamber 18. The variable volume chamber 18 contains an injectable medicament 20 comprising an ophthalmic solution comprising a VEGF antagonist such as ranibizumab. The injectable fluid 20 can be expelled though the outlet 12 by movement of the stopper 10 towards the outlet end 14 thereby reducing the volume of the variable volume chamber 18. The plunger 4 comprises a plunger contact surface 22 at a first end 24 and a 5 rod 26 extending between the plunger contact surface 22 and a rear portion 25. The plunger contact surface 22 Is arranged to contact the stopper 10, such that the plunger 4 can be used to move the stopper 10 towards the outlet end 14 of the body 2. Such movement reduces the volume of the variable volume chamber 18 and causes fluid therein to be expelled though the outlet.

The backstop 6 is attached to the body 2 by coupling to a terminal flange 28 of the body 2. The 20 backstop 6 includes sandwich portion 30 which is adapted to substantially sandwich at least some of the terminal flange 28 of the body 2. The backstop 6 is adapted to be coupled to the body 2 from the side by leaving one side of the backstop 6 open so that the backstop 6 can be fitted to the syringe

2.

The body 2 defines a substantially cylindrical bore 36 which has a bore radius. The rod 26 25 comprises a rod shoulder 32 directed away from the outlet end 14. The rod shoulder 32 extends from to a rod shoulder radius from the first axis A which is such that it is slightly less than the bore radius so that the shoulder fits within the bore 36. The backstop 6 includes a backstop shoulder 34 directed towards the outlet end 14. The shoulders 32, 34 are configured to cooperate to substantially prevent movement of the rod 26 away from the outlet end 14 when the backstop shoulder 34 and rod shoulder 32 are in contact. The backstop shoulder 34 extends from outside the bore radius to a radius less than the rod shoulder radius so that the rod shoulder 32 cannot pass the

-1455Ί57

2018201120 15 Feb 2018 backstop shoulder 34 by moving along the first axis A. In this case the rod shoulder 32 is substantially disc, or ring, shaped and the backstop shoulder 34 includes an arc around a rear end 38 of the body 2.

The backstop 6 also includes two finger projections 40 which extend in opposite directions away 5 from the body 2 substantially perpendicular to the first axis A to facilitate manual handling of the syringe I during use.

In this example the syringe comprises a 0.5ml body 2 filled with between about 0.1 and 0.3 ml of an injectable medicament 20 comprising a lOmg/ml injectable solution comprising ranibizumab. The syringe body 2 has an internal diameter of about between about 4.5mm and 4.8mm, a length of 0 between about 45mm and 50mm.

The plunger 4 and stopper 10 will be described in more detail with reference to later figures.

Figure 3 shows a perspective view of the plunger 4 of Figure 1 showing the plunger contact surface 22 at the first end 24 of the plunger 4. The rod 26 extends from the first end 24 to the rear portion

25. The rear portion 25 includes a disc shaped flange 42 to facilitate user handling of the device.

The flange 42 provides a larger surface area for contact by the user than a bare end of the rod 26,

Figure 4 shows a cross section though a syringe body 2 and rod 26. The rod 26 includes four longitudinal ribs 44 and the angle between the ribs is 90°.

Figure 5 shows a detailed view of a stopper 10 showing a conical shaped front surface 16 and three circumferential ribs 52,54,56 around a substantially cylindrical body 5.8. The axial gap between the first rib 52 and the last rib 56 is about 3mm. The rear surface 60 of the stopper 10 includes a substantially central recess 62. The central recess 62 includes an initial bore 64 having a first diameter. The initial bore 64 leading from the rear surface 60 into the stopper 10 to an inner recess 66 having a second diameter, the second diameter being larger than the first diameter.

Stopper movement forces

0.5ml syringes siliconised with <100pg silicone oil, filled with Lucentis, comprising one of two different stopper designs were tested for maximal and average break out and slide force. Prior to testing, 30G x 0.5” needles were attached to the syringes. The testing was carried out at a stopper speed of 190mm/min over a travel length of 10.9mm. Stopper design 2 had a 45% increase in the distance between the front circumferential rib and rear circumferential rib.

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		Stopper design 1	Stopper design 2
		Batch A	Batch B	Batch C	Batch D	Batch E
Break loose force of syringes	Average of ! 0 syringes	2.2N	2.3N	1.9N	2.IN	2.5N
Max individual value	2.5N	2.5N	2.3N	2.6N	2.7N
Sliding force	Average of 10 syringes	3.IN	3.2N	3.1N	4.1N	4.6N
Max individual value	3.5N	3.5N	3.6N	4.7N	4.8N

For both stopper designs, average and maximum break out force remained below 3N. For both stopper designs, average and maximum sliding force remained below 5N.

It will be understood that the invention has been described by way of example only and 5 modifications may be made whilst remaining within the scope and spirit of the invention.

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Claims (36)

1. A pre-filled syringe, the syringe comprising a glass body, a stopper and a plunger, the body comprising an outlet at an outlet end and the stopper being arranged within the body such that a front surface of the stopper and the body define a variable volume chamber from which a fluid

5 can be expelled though the outlet, the plunger comprising a plunger contact surface at a first end and a rod extending between the plunger contact surface and a rear portion, the plunger contact surface arranged to contact the stopper, such that the plunger can be used to force the stopper towards the outlet end of the body, reducing the volume of the variable volume chamber, characterised in that the fluid is an ophthalmic solution which comprises a VEGF-antagonist

0 wherein:

(a) the syringe has a nominal maximum fill volume of between about 0.5ml and about 1ml, (b) the syringe is filled a dosage volume of between about 0.03ml and about 0.05ml of said VEGF antagonist solution, (c) the syringe barrel comprises less than about 500pg silicone oil, and

5 (d) the VEGF antagonist solution comprises no more than 2 particles >50pm in diameter per ml.

2. A pre-filled syringe according to claim 1, wherein the syringe is filled with between about 0.15ml and about 0,175ml of a VEGF antagonist solution.

3. A pre-filled syringe according to claim 1 or claim 2, wherein the syringe is filled with about 0.165ml of said VEGF antagonist solution.

20

4. A pre-filled syringe according to any previous claim, wherein the syringe is filled with dosage volume of about 0.05ml of a VEGF antagonist solution.

5. A pre-filled syringe according to any previous claim, in which the dosage volume is determined by the volume of the variable volume chamber when a predetermined part of the stopper is aligned with a priming mark on the syringe.

25

6. A pre-filled syringe according to any previous claim, wherein the syringe barrel has an internal coating of silicone oil that has an average thickness of about 450nm or less, preferably 400nm or less, preferably 350nm or less, preferably 300nm or less, preferably 200nm or less, preferably lOOnm or less, preferably 50nm or less, preferably 20nm or less.

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7. A pre-filled syringe according to any previous claim, wherein the syringe barrel has an internal coating of less than about 500p.g silicone oil, preferably less than about lOOpg silicone oil, preferably less than about 50pg silicone oil, preferably less than about 25pg silicone oil, preferably less than about 10pg silicone oil.

5

8, A pre-filled syringe according to any previous claim, wherein the syringe barrel has an internal coating of more than about lpg, more than about 3pg, more than about 5pg, more than about 7pg or more than about 10pg silicone oil.

9. A pre-filled syringe according to any previous claim, wherein the syringe barrel has an internal coating of about lpg-about 500pg, about 3pg-about 200pg, about 5pg-about lOOpg or

0 about lOpg-about 50pg silicone oil.

10. A pre-filled syringe according to any previous claim, wherein the silicone oil is DC365 emulsion.

11. A pre-filled syringe according to any one of claims 1-5, wherein the syringe is silicone oil free.

5

12. A pre-ftlled syringe according to any previous claim, wherein the VEGF antagonist solution further comprises one or more of (i) no more than 5 particles >25 pm in diameter per ml, and (ii) no more than 50 particles > 10pm in diameter per ml.

13. A pre-filled syringe according to any previous claim, wherein the VEGF antagonist solution meets USP789.

20

14. A pre-filled syringe according to any previous claim, wherein tlie VEGF antagonist is an anti-VEGF antibody.

15, A pre-filled syringe according to claim 14, wherein the anti-VEGF antibody is ranibizumab.

16. A pre-filled syringe according to claim 15, wherein the ranibizumab is at a concentration of 1 Omg/ml.

25

17. A pre-filled syringe according to any one of claims 1-13 wherein the VEGF antagonist is a non-antibody VEGF antagonist.

18. A pre-filled syringe according to claim 17, wherein the non-antibody VEGF antagonist is aflibercept or conbercept.

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19. A pre-filled syringe according to claim 18, wherein the non-antibody VEGF antagonist is aflibercept at a concentration of 40mg/ml.

20. A pre-filled syringe according to any previous claim, wherein the syringe has a stopper break loose force of less than about 1 IN.

5

21. A pre-filled syringe according to claim 20, wherein the syringe has a stopper break loose force of less than about 5N.

22. A pre-filled syringe according to any previous claim, wherein the syringe has a stopper slide force ofless than about 11N.

23. A pre-filled syringe according to claim 22, wherein the syringe has a stopper slide force of 0 less than about 5N.

24. A pre-filled syringe according to any of claims 20-23, wherein the stopper break loose force or stopper slide force is measured using a filled syringe, at a stopper travelling speed of 190mm/min, with a 30Gx 0.5 inch needle attached to the syringe.

25. A blister pack comprising a pre-filled syringe according to any previous claim, wherein the 5 syringe has been sterilised using H2O2 or EtO.

26. A blister pack comprising a pre-filled syringe according to claim 25, wherein the outer surface of the syringe has <lppm EtO or H2O2 residue.

27. A blister pack comprising a pre-filled syringe according to claim 25, w-herein the syringe has been sterilised using EtO or FLO2 and the total EtO or FLOj residue found on the outside of the

20 syringe and inside of the blister pack is <0,1 mg.

28. A blister pack comprising a pre-filled syringe according to any one of claims 25-27, wherein <5% of the VEGF antagonist is alkylated.

29. A blister pack comprising a pre-filled syringe according to any of claims 25-28, wherein the syringe has been sterilised using EtO or H2O2 with a Sterility Assurance Level of at least 10'⁶.

25

30. A blister pack according to any of claims 25-29, wherein the pre-filled syringe has a shelf life of up to 6 months, 9 months, 12 months, 15 months, 18 months, 24 months or longer.

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31. A kit comprising: (i) a pre-filled syringe according to any one of claims 1-24, or a blister pack comprising a pre-filled syringe according to any one of claims 25-30, (ii) a needle, and optionally (iii) instructions for administration.

32. A kit according to claim 31, wherein the needle is a 30-gauge χ ‘Λ inch needle.

5

33. A pre-Filled syringe according to any one of claims 1-24 for use in therapy.

34. A pre-filled syringe according to any one o f claims 1-24 for use in the treatment of an ocular disease selected from choroidal neovascularisation, wet age-related macular degeneration, macular edema secondary to retinal vein occlusion (RVO) including both branch RVO (bRVO) and central RVO (cRVO), choroidal neovascularisation secondary to pathologic myopia (PM),

0 diabetic macular edema (DME). diabetic retinopathy, and proliferative retinopathy,

35. A method of treating a patient suffering from of an ocular disease selected from choroidal neovascularisation, wet age-related macular degeneration, macular edema secondary to retinal vein occlusion (RVO) including both branch RVO (bRVO) and central RVO (cRVO), choroidal neovascularisation secondary to pathologic myopia (PM), diabetic macular edema (DME),

5 diabetic retinopathy, and proliferative retinopathy, comprising the step of administering an ophthalmic solution to the patient using a pre-filled syringe according to any one of claims 1-24.

36. The method of claim 35, further comprising an initial priming step in which the physician depresses the plunger of the pre-filled syringe to align the pre-determined part of the stopper with the priming mark.

20 37. A method according to claim 35 or 36, wherein the VEGF antagonist administered is a nonantibody VEGF antagonist and wherein the patient has previously received treatment with an antibody VEGF antagonist.

-202018201120 15 Feb 2018

Figi

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SEQUENCE LI STI NG <110> Novartis AG <120> SYRINGE <130> PAT055157-WO <150> EP 12174860.2 <151> 2012-07-03 <150> EP 12189649.2 <1 51 > 201 2- 1 0- 23 <1 50> DE 20201 201 1 01 6. 0 <1 51 > 201 2- 1 1 - 1 6 <1 50> AU 201 21 01 677 <1 51 > 201 2- 1 1 - 1 6 <1 50> AU 201 21 01 678 <1 51 > 201 2- 1 1 - 1 6 <1 50> DE 20201 201 1 260. 0 <1 51 > 201 2- 1 1 - 23 <1 50> DE 20201 201 1 259. 7 <1 51 > 201 2- 1 1 - 23 <150> EP 12195360. 8 <1 51 > 201 2- 1 2- 03 <150> DE 202013000688. 9 <151> 2013-01-23 <150> AU 2013100071 <151> 2013-01-23 <150> AU 2013100070 <151> 2013-01-23 <1 60> 3 <170> PatentIn version 3.5 <21 0> 1 <211> 431 <21 2> PRT <213> Artificial Sequence <220>

<223> afli ber cept <400> 1

Ser 1 As p Thr Gly Arg 5 Pr o Phe Val Gl u Met 10 Tyr Ser Gl u Ile Pr o 15 Gl u Ile Ile Hi s Mfet 20 Thr Gl u Gl y Ar g Gl u 25 Le u Val Ile Pr o Cys 30 Ar g Val Thr Ser Pr o 35 As n Ile Thr Val Thr 40 Le u Lys Lys Phe Pr o 45 Le u As p Thr Le u Ile Pr o As p Gl y Lys Ar g Ile Ile Tr p As p Ser Ar g Lys Gl y Phe

50 55 60

Pa ge 1

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Ile 65 Ile Ser As n Al a Thr 70 Tyr Lys Gl u Ile Gl y 75 Le u Le u Thr Cys Gl u 80 Al a Thr Val As n Gl y 85 Hi s Le u Tyr Lys Thr 90 As n Tyr Le u Thr Hi s 95 Ar g Gl n Thr As n Thr 100 Ile Ile As p Val Val 105 Le u Ser Pr o Ser Hi s 110 Gl y Ile Gl u Le u Ser 115 Val Gl y Gl u Lys Le u 120 Val Le u As n Cys Thr 125 Al a Ar g Thr Gl u Le u 130 As n Val Gl y Ile As p 135 Phe As n Tr p Gl u Tyr 140 Pr o Ser Ser Lys Hi s 145 Gl n Hi s Lys Lys Le u 150 Val As n Ar g As p Le u 155 Lys Thr Gl n Ser Gl y 160 Ser Gl u Mfet Lys Lys 165 Phe Le u Ser Thr Le u 170 Thr Ile As p Gl y Val 175 Thr Arg Ser Asp Gl n 180 Gl y Le u Tyr Thr Cys 185 Al a Al a Ser Ser Gl y 190 Le u Met Thr Lys Lys 195 As n Ser Thr Phe Val 200 Ar g Val Hi s Gl u Lys 205 As p Lys Thr Hi s Thr 21 0 Cys Pr o Pr o Cys Pr o 21 5 Al a Pr o Gl u Le u Le u 220 Gl y Gl y Pr o Ser Val 225 Phe Le u Phe Pr o Pr o 230 Lys Pr o Lys As p Thr 235 Le u Met Ile Ser Ar g 240 Thr Pr o Gl u Val Thr 245 Cys Val Val Val As p 250 Val Ser Hi s Gl u As p 255 Pr o Gl u Val Lys Phe 260 As n Tr p Tyr Val As p 265 Gl y Val Gl u Val Hi s 270 As n Al a Lys Thr Lys 275 Pr o Ar g Gl u Gl u Gl n 280 Tyr As n Ser Thr Tyr 285 Ar g Val Val Ser Val 290 Le u Thr Val Le u Hi s 295 Gl n As p Tr p Le u As n 300 Gl y Lys Gl u Tyr Lys 305 Cys Lys Val Ser As n 310 Lys Al a Le u Pr o Al a 315 Pr o Ile Gl u Lys Thr 320 Ile Ser Lys Al a Lys 325 Gl y Gl n Pr o Ar g Gl u 330 Pr o Gl n Val Tyr Thr 335 Le u Pr o Pr o Ser Ar g As p Gl u Le u Thr Lys As n Gl n Page 2 Val Ser Le u Thr Cys

2018201120 15 Feb 2018

340 345 350 Leu Val Lys Gl y Phe Tyr Pr o Ser As p Ile Al a Val Gl u Tr p Gl u Ser 355 360 365 Asn Gly Gl n Pr o Gl u As n As n Tyr Lys Thr Thr Pr o Pr o Val Le u As p 370 375 380 Ser Asp Gl y Ser Phe Phe Le u Tyr Ser Lys Le u Thr Val As p Lys Ser 385 390 395 400 Arg Trp Gl n Gl n Gl y As n Val Phe Ser Cys Ser Val Mfet Hi s Gl u Al a 405 410 41 5 Le u Hi s As n Hi s Tyr Thr Gl n Lys Ser Le u Ser Le u Ser Pr o Gl y 420 425 430

<210> 2 <211> 552 <212> PRT <213> Artificial Sequence <220> <223> c onbe r c e pt <400> 2 Mtt Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Al a Le u Le u Se r 1 5 10 15

Cys Le u Le u Le u 20 Thr Gl y Ser Ser Ser 25 Gl y Gl y Ar g Pr o Phe 30 Val Gl u Met Tyr Ser 35 Gl u Ile Pr o Gl u Ile 40 Ile Hi s Met Thr Gl u 45 Gl y Ar g Gl u Le u Val 50 Ile Pr o Cys Ar g Val 55 Thr Ser Pr o As n Ile 60 Thr Val Thr Le u Lys 65 Lys Phe Pr o Le u As p 70 Thr Le u Ile Pr o As p 75 Gl y Lys Ar g Ile Ile 80 Tr p As p Ser Ar g Lys 85 Gl y Phe Ile Ile Ser 90 As n Al a Thr Tyr Lys 95 Gl u Ile Gl y Le u Le u 100 Thr Cys Gl u Al a Thr 105 Val As n Gl y Hi s Le u 110 Tyr Lys Thr As n Tyr 115 Le u Thr Hi s Ar g Gl n 120 Thr As n Thr Ile Ile 1 25 As p Val Val Le u Ser 130 Pr o Ser Hi s Gl y Ile 135 Gl u Le u Ser Val Gl y 140 Gl u Lys Le u Val Le u As n Cys Thr Al a Ar g Thr Gl u Le u As n Val Gl y Ile As p Phe As n

Page 3

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145 150 155 160

Trp Gl u Tyr Pr o Ser 165 Ser Lys Hi s Gl n Hi s 170 Lys Lys Le u Val As n 175 Ar g Asp Le u Lys Thr 180 Gl n Ser Gl y Ser Gl u 185 Met Lys Lys Phe Le u 190 Ser Thr Leu Thr Ile 195 As p Gl y Val Thr Ar g 200 Ser As p Gl n Gl y Le u 205 Tyr Thr Cys Al a Al a 21 0 Ser Ser Gl y Le u Met 21 5 Thr Lys Lys As n Ser 220 Thr Phe Val Ar g Val 225 Hi s Gl u Lys Pr o Phe 230 Val Al a Phe Gl y Ser 235 Gl y Met Gl u Ser Le u 240 Val Gl u Al a Thr Val 245 Gl y Gl u Ar g Val Ar g 250 Le u Pr o Al a Lys Tyr 255 Le u Gly Tyr Pr o Pr o 260 Pr o Gl u Ile Lys Tr p 265 Tyr Lys As n Gl y Ile 270 Pr o Le u Gl u Ser As n 275 Hi s Thr Ile Lys Al a 280 Gl y Hi s Val Le u Thr 285 Ile Met Gl u Val Ser 290 Gl u Ar g As p Thr Gl y 295 As n Tyr Thr Val Ile 300 Le u Thr As n Pr o Ile 305 Ser Lys Gl u Lys Gl n 310 Ser Hi s Val Val Ser 315 Le u Val Val Tyr Val 320 Pr o Pr o Gl y Pr o Gl y 325 As p Lys Thr Hi s Thr 330 Cys Pr o Le u Cys Pr o 335 Al a Pr o Gl u Le u Le u 340 Gl y Gl y Pr o Ser Val 345 Phe Le u Phe Pr o Pr o 350 Lys Pr o Lys As p Thr 355 Le u Mfet Ile Ser Ar g 360 Thr Pr o Gl u Val Thr 365 Cys Val Val Val As p 370 Val Ser Hi s Gl u As p 375 Pr o Gl u Val Lys Phe 380 As n Tr p Tyr Val As p 385 Gl y Val Gl u Val Hi s 390 As n Al a Lys Thr Lys 395 Pr o Ar g Gl u Gl u Gl n 400 Tyr Asn Ser Thr Tyr 405 Ar g Val Val Ser Val 41 0 Le u Thr Val Le u Hi s 41 5 Gl n As p Tr p Le u As n 420 Gl y Lys Gl u Tyr Lys 425 Cys Lys Val Ser As n 430 Lys Al a

Page 4

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Leu Pr o Al a 435 Pr o Ile Gl u Lys Thr 440 Ile Ser Lys Al a Lys 445 Gl y Gl n Pr o Arg Gl u Pr o Gl n Va l Tyr Thr Le u Pr o Pr o Ser Ar g As p Gl u Le u Thr 450 455 460 Lys As n Gl n Val Ser Le u Thr Cys Le u Val Lys Gl y Phe Tyr Pr o Ser 465 470 475 480 Asp Ile Al a Val Gl u Tr p Gl u Ser As n Gl y Gl n Pr o Gl u As n As n Tyr 485 490 495 Lys Al a Thr Pr o Pr o Val Le u As p Ser As p Gl y Ser Phe Phe Le u Tyr 500 505 510 Ser Lys Le u Thr Va l As p Lys Ser Ar g Tr p Gl n Gl n Gl y As n Val Phe 515 520 525 Ser Cys Ser Val Me t Hi s Gl u Al a Le u Hi s As n Hi s Tyr Thr Gl n Lys 530 535 540 Ser Le u Ser Le u Ser Pr o Gl y Lys 545 550 <210> 3 <211> 126 <212> PRT <213> Ar t i f i ci a l Se que nc e <220> <223> DARPi n MP01 1 2 <400> 3 Gly Ser As p Le u Gl y Lys Lys Le u Le u Gl u Al a Al a Ar g Al a Gl y Gl n 1 5 10 15 As p As p Gl u Val Ar g Ile Le u Mfet Al a As n Gl y Al a As p Val As n Thr 20 25 30 Al a As p Ser Thr Gl y Tr p Thr Pr o Le u Hi s Le u Al a Val Pr o Tr p Gl y 35 40 45 Hi s Le u Gl u Ile Va l Gl u Val Le u Le u Lys Tyr Gl y Al a As p Val As n 50 55 60 Al a Lys As p Phe Gl n Gl y Tr p Thr Pr o Le u Hi s Le u Al a Al a Al a Ile 65 70 75 80 Gl y Hi s Gl n Gl u Ile Val Gl u Val Le u Le u Lys As n Gl y Al a As p Val 85 90 95 Asn Al a Gl n As p Lys Phe Gl y Lys Thr Al a Phe As p Ile Ser Ile As p 100 105 110

Page 5

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Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala 115 120 125

Page 6