Ophthalmic composition of bevacizumab

Abstract

To provide an ophthalmic composition of bevacizumab.SOLUTION: Provided is an ophthalmic composition of bevacizumab; a. herein, the limit of Bacterial Endotoxins Test (BET) is within the range of 0.001 to 0.4EU / mg, preferably within the range of 0.001 to 0.2Eu / mg, and more preferably within the range of 0.001 to 0.16Eu / mg; b. herein, a particulate substance is as follows; 1 to 50 particles≥diameter 10 μm per iv.ml, 0 to 5 particles≥diameter 25 μm per v.ml, and 0 to 2 particles≥diameter 50 μm per vi.ml; and c. herein, the composition has an aggregate of 0.1 to 5%, more preferably 0.1 to 4%, and most preferably 0.1 to 3.5% for 2 to 3 years, preferably for 2 to 2.5 years, and most preferably for 2 years.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Field of the invention: The present invention relates to the fields of biotechnology and drug delivery. In particular, the present invention relates to ophthalmic compositions of bevacizumab and devices containing said compositions. [Background technology]

[0002] Background of the invention: Bevacizumab is a recombinant humanized monoclonal antibody containing human framework regions and murine complementarity-determining regions that binds to and inhibits the biological activity of human vascular endothelial growth factor (VEGF). Bevacizumab has been approved for the treatment of various cancers, including metastatic colorectal cancer (mCRC), non-squamous non-small cell lung cancer (NSCLC), metastatic breast cancer (MBC), metastatic renal cell carcinoma (mRCC), glioblastoma, and advanced cervical cancer. In these indications, bevacizumab has demonstrated efficacy in terms of patient safety and survival. Additionally, it is also used off-label to treat wet age-related macular degeneration (wAMD), choroidal neovascularization (CNV), diabetic macular edema (DME), retinal vein occlusion (RVO), and neovascular glaucoma (NVG). Alternatively, another monoclonal antibody, ranibizumab, a USFDA-approved drug for AMD, CNV, DME, etc., is also on the market. Although there appears to be no difference in efficacy and safety between bevacizumab and ranibizumab as evidenced by several studies, there is a huge cost advantage when bevacizumab is used instead of ranibizumab.

[0003] Bevacizumab is approved for cancer and is available in 100 mg or 400 mg vials, intended to provide a full dose to individuals based on their body weight. When using bevacizumab for wAMD, ophthalmologists typically inject significantly smaller amounts, as little as 1.25 mg per dose, intravitreally from small vials, possibly repeatedly (multidose use). Any drug intended for intravitreal administration needs to be prepared and maintained in a sterile environment. There is a risk of contamination when injections are prepared in vials by withdrawing the solution reconstituted in a device prior to administration. Contaminated drug puts patients at risk for several additional infections, such as endophthalmitis and alpha-streptococcal infections. In some parts of the world, the physician himself withdraws the required medication from the original vial, and the same vial is used for many consecutive patients, which has proven to be a bad practice in the context of patient safety, since the increased number of punctures from the same vial increases the chances of contamination and infection to the patient. Furthermore, there can be inaccuracies in the dosage due to human error by the administering medical or paramedical personnel.

[0004] In some countries, compounding pharmacies repackage multiple syringes from the original vial. The need to repackage medication into smaller doses from available vials increases the likelihood of contamination transmission, especially since it is difficult to maintain sterility during such procedures, and there is no way to check whether such procedures are being followed at compounding pharmacies. The FDA has documented and issued warning notices in several cases regarding the use of aseptic technique and dosage control at these compounding pharmacies, but such practices remain rampant. In addition, some compounding pharmacies have recalled repackaged bevacizumab syringes due to sterility concerns, and the U.S. FDA is currently warning medical and paramedical teams against the use of repackaged injections of bevacizumab for intraocular use. Additionally, bevacizumab is manufactured for intravenous administration, and therefore, standards for particle size distribution are crucial. If particles are not controlled, they can build up, causing irritation and / or inflammation of sensitive organs such as the eye. It has been reported for such repackaged injectable ophthalmic solutions that particle size distributions fall outside the limits imposed by the United States Pharmacopoeia and are generally not standardized and are not uniform across different pharmacies.

[0005] In addition to safety issues, the drug itself may have varying efficacies related to product aliquoting, handling, and distribution. Deterioration may be due to issues with the special packaging used, and reduced stability of repackaged bevacizumab may be related to the duration of storage. In addition to the potential safety implications of these changes, drug efficacy may also be affected. The shelf-life stability of repackaged bevacizumab is also a major concern, and guidelines have been issued by the DCGI of India, which recommend that repackaged bevacizumab syringes be used within 14 days under certain conditions to prevent degradation and deterioration. However, there is no effective mechanism for checking the shelf-life after reconstitution and / or time limits for degradation or deterioration. Further reconstitution of the packaged product for administration is a complex and cumbersome procedure and does not necessarily result in a homogeneous solution. Furthermore, as the global supply chain has become more complex, this has proven increasingly difficult, with reports of counterfeit Avastin being traced internationally.

[0006] Although the specific problems associated with dispensing bevacizumab into small units for intravitreal use and counterfeit bevacizumab may be reduced by the implementation of strict regulatory guidelines, prior art issues such as bacterial endotoxin testing (BET) and particulate matter testing (PMT) limitations limit the specific technical solutions and manufacturing controls required to eliminate the prior art issues and provide a product acceptable for intravitreal injection into the eye. There are increasing reports of adverse events associated with intravitreal injection of bevacizumab, including the occurrence of intraocular sterile inflammation, infectious endophthalmitis, and elevated intraocular pressure.

[0007] Certain reported adverse events associated with intravitreal bevacizumab administration may be related to poor quality of the drug due to the way it is manufactured or repackaging into plastic syringes. Additionally, silicone droplets from the needles and syringes used for intravitreal injection exacerbate the condition. There are problems with the quality of the prior art products, as follows:

[0008] · Bevacizumab currently only meets intravenous quality standards, rather than the more stringent ocular standards for subvisible particulate matter and endotoxin levels.

[0009] The presence of particles and / or silicone oil that are not visible to the naked eye is significant in the case of intravitreal injection (accumulation in the eye, potentially leading to severe intraocular inflammation).

[0010] No data are presented to consider the quality of the product after repackaging from a microbiological and physicochemical point of view. The compatibility of the solution with the primary packaging is not considered. In most parts of the world, there is no shelf life testing of repackaged bevacizumab and no testing for safety or quality effects.

[0011] Thus, there is a pressing need for ophthalmic compositions for bevacizumab that are safe, non-toxic, and effective for as-needed administration with a long shelf life.

[0012] Object of the invention It is an object of the present invention to provide ophthalmic compositions of bevacizumab with controlled particulate matter, bacterial endotoxins, and aggregation during shelf life, processes for preparing said compositions, and devices that are safe, non-toxic, and effective for as-needed administration. Summary of the Invention

[0013] The present invention discloses a bevacizumab ophthalmic composition, which is administered as a single-use, pre-filled syringe. The ophthalmic composition of the present invention controls bacterial endotoxin test (BET), particulate matter, and aggregate limits during a shelf life of two years. The present invention also provides a process for obtaining an active ingredient, bevacizumab, with negligible or no bacterial endotoxin test (BET) and particulate matter, and the composition of the present invention, when formulated therein, has extremely low levels of aggregates, BET, and particulate matter during its shelf life and is suitable for intravitreal use. The present invention also provides a device, such as a pre-filled syringe or kit, containing the bevacizumab ophthalmic composition. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 depicts a schematic representation of the method for obtaining an ophthalmic composition of the present invention that is controlled in terms of sub-visible particulate matter count, low endotoxin levels during the shelf life of the product. [Figure 2] Figures 2a and 2b depict the sub-visible particle size of the sample of the present invention compared to that of other samples in the prior art. DETAILED DESCRIPTION OF THE INVENTION

[0015] Detailed Description of the Invention Ophthalmic compositions of bevacizumab that are safe, non-toxic, and effective for administration throughout their shelf life are characterized in that the ophthalmic compositions are controlled with respect to sub-visible particulate matter, low endotoxin levels, and aggregates throughout the shelf life of the product. The ophthalmic compositions of the present invention are preferably solutions. The ophthalmic solutions may be presented as individual injectable drug units in pre-filled syringes.

[0016] The ophthalmic compositions of the present invention are controlled for particulate matter, which consists of mobile, undissolved particles derived from any component of the composition, other than gas bubbles, impurities, and any unstable substances, unintentionally present in the solution, where sub-visible particulate matter for intravitreal injection includes: (i) 1 to 50 particles per ml ≥ 10 μm in diameter (ii) 0-5 particles per ml ≥ 25 μm in diameter, and (iii) 0-2 particles per ml ≥ 50 μm in diameter

[0017] The presence of endotoxins in recombinant therapeutics is a major concern due to the diverse and potentially harmful biological activities of these molecules. Maintaining sterility in the processes used in the manufacture of biologics, along with stringent protocols for device preparation, helps ensure that the product has acceptably low levels of endotoxins. It is clear from literature that the posterior segment (the site of intravitreal injection) is more sensitive to endotoxins than the anterior and posterior segments of the eye, and endotoxin residence time in this segment is due more to the higher viscosity and lower fluidity of the vitreous cavity, which results in slower recovery of the vitreous cell response. Even minimal endotoxin levels may result in intraocular inflammation, depending on the patient's sensitivity. Intravitreal injections of bevacizumab are administered monthly, and 7–8 injections per year are required for wAMD. It is imperative to keep endotoxin levels as low as possible to avoid endotoxin accumulation in the eye due to frequent intravitreal injections. Several cases have been reported in which sterile endophthalmitis and increased intraocular pressure developed after intraocular injection of bevacizumab. Although several theories exist regarding the etiology of intraocular inflammation, its cause remains unknown. Endotoxins, usually produced by commercially produced immunoglobulin products, are thought to play a major role in these sterile reactions. In recent years, there have been several reports of TASS-like culture-negative sterile endophthalmitis after IVB injection. This may be due to endotoxins related to degradation products resulting from imperfect storage. With the increasing number of IVB injections, the number of such cases is sure to rise, thus necessitating the need to find intelligent methods for treating such situations. The etiology of sterile endophthalmitis is poorly understood. However, there are numerous possible explanations. For example, the common practice of dispensing individual aliquots from commercially available vials of bevacizumab as a cost-effective method may pose an increased risk of contamination with bacterial endotoxins.Although prior art bevacizumab compositions as administered contain traces of endotoxin at levels considered safe for intravenous use, they can still induce inflammatory reactions within the vitreous. In post-injection endophthalmitis, extraneous drug handling during preparation also plays an important role. Due to several outbreaks of infectious endophthalmitis reported in the United States, concerns about repackaging bevacizumab for intravitreal injection are increasing.

[0018] The ophthalmic compositions of the present invention control the level of endotoxin concentration in the range of 0.001 to 0.4 EU / mg, and preferably in the range of 0.001 to 0.2 EU / mg, and more preferably in the range of 0.001 to 0.16 EU / mg.

[0019] The ophthalmic compositions of the present invention may be aggregate-free up to the formulated shelf life or may have aggregates in the range of 0.1-5%, more preferably 0.1-4%, and most preferably 0.1-3.5% for a period of 2-3 years, preferably 2-2.5 years, and most preferably 2 years.

[0020] Even if the cold chain is intermittently broken for short periods of time, e.g., during transport, the ophthalmic compositions of the present invention are able to maintain bacterial endotoxins, particulate matter limits, and aggregate limits throughout their shelf life. In this context, the products of the present invention have excellent tolerances with respect to stability and efficacy. The ophthalmic compositions of the present invention do not have dose variation between units because they can be pre-filled to doses determined through automation. Furthermore, the dosage of the individual units remains constant over the prescribed shelf life.

[0021] The bevacizumab ophthalmic composition of the present invention comprises: a. Bacterial Endotoxin Test (BET) in the range of 0.001-0.4 Eu / mg, and preferably in the range of 0.001-0.2 Eu / mg, and more preferably in the range of 0.001-0.16 Eu / mg; b. Wherein particulate matter is: 1-50 particles per i.ml ≥ 10 μm diameter ii. 0-5 particles per ml ≥ 25 μm in diameter, and iii. 0-2 particles per ml ≥ 50 μm in diameter; and

[0022] c. Wherein the composition has 0.1-5%, more preferably 0.1-4%, and most preferably 0.1-3.5% aggregates for 2-3 years, preferably 2-2.5 years, and most preferably 2 years.

[0023] In another aspect, a process is provided for preparing a composition that is controlled with respect to subvisible particulate matter count, low endotoxin levels, and is substantially or completely devoid of silicone oil during the life of the product of the invention, comprising the steps of: i. Cultivating cells of bevacizumab by continuous fermentation method of CHO cells; ii. subjecting the cell-free harvest obtained from the CHO cell culture to adsorption-based depth filtration for further clarification; iii. concentrating the sample of step (ii) using single-pass tangential flow filtration to obtain a concentrated harvest;

[0024] iv. subjecting the concentrated harvest of step (iii) to Protein A chromatography to capture bevacizumab to obtain an eluate containing partially purified bevacizumab; v. subjecting the eluate of step (iv) to low pH viral inactivation to obtain a viral inactivated sample;

[0025] vi. For further purification, subjecting the virally inactivated sample of step (v) to further cation exchange chromatography to obtain an eluate containing primarily highly purified bevacizumab;

[0026] vii. subjecting the eluate of step (vi) to anion exchange chromatography to remove trace impurities, such as host cell proteins (HCPs), host cell DNA (HCDs), and endotoxins; viii. subjecting the eluate of step (vii) to virus-reducing filtration and complete removal of viruses; ix. concentrating and diafiltering the sample of step (viii) to obtain bevacizumab; and x. Formulating the sample obtained from step (ix) to achieve a bevacizumab composition of the present invention.

[0027] Weak viral inactivation may be carried out at a pH in the range of 3.0 to 5.0, preferably in the range of 3.0 to 4.0, most preferably in the range of 3.5 to 4.0.

[0028] Cation exchange chromatography of the eluate may be carried out by using a matrix or stationary phase selected from the group comprising SO3-sulfonylisobutyl, SO3-sulfoethyl, sulfopropyl, carboxymethyl, etc., preferably the chromatography matrix or stationary phase used is a sulfonate.

[0029] Anion exchange chromatography of the eluate may be carried out by using a matrix or stationary phase selected from the group comprising diethylaminoethyl, quaternaryamine, polyquaternium, N-benzyl-N-methylethanolamine, etc., preferably the chromatography matrix or stationary phase used is quaternaryamine. Diafiltration may be carried out in the presence of TFF-II diafiltration buffer. The processes described herein result in products and compositions with desirable endotoxin effects. By way of illustration, endotoxin levels may be sequentially reduced as follows: (See Table A) [Table A]

[0030] From the table above, the process of the present invention results in a several-fold reduction in endotoxin content as determined by the gel clot method. Before the harvested broth is subjected to the purification process, the endotoxin content is extremely high. After Protein A affinity purification, the endotoxin content is usually much higher than the acceptable level, below 10 EU / mg. In addition, anion exchange chromatography has been used to separate and remove endotoxin from the active bevacizumab.

[0031] At the end of the anion exchange chromatography step, the endotoxin content in the protein is less than 0.5 Eu / mg. The use of anion exchange chromatography in the process of the present invention allows for control of important quality attributes of the drug product, such as BET, bioburden, aggregates, and, to a narrower extent, PMT.

[0032] Endotoxin removal is one of the most difficult steps in downstream process purification. The bevacizumab endotoxin limit of the present invention is achieved by obtaining an ophthalmic composition having an endotoxin concentration of less than 0.4 Eu / mg, and more preferably less than 0.2 Eu / mg, and more preferably less than 0.16 Eu / mg. In addition, the process must involve: (i) Use of sterile solutions and equipment that minimizes the introduction of microorganisms; (ii) using filtration during purification through a 0.22 micron filter; (iii) working at low temperatures to minimize microbial growth; (iv) adding a bacteriostatic agent to the purified product; (v) high purity raw materials; (vi) an orthogonal purification step; (vii) Strict clean-in-place (CIP) procedures for vessels, columns, and process equipment. The inventive process disclosed herein results in a product with novel and desirable parameters.

[0033] The process must be followed in the order listed herein to achieve a product with the desired properties. This process develops after much experimentation, trial and error. The process of the present invention provides an ophthalmic composition of bevacizumab;

[0034] a. Wherein the limit of the bacterial endotoxin test (BET) is in the range of 0.001 to 0.4 Eu / mg, and preferably in the range of 0.001 to 0.2 Eu / mg, and more preferably in the range of 0.001 to 0.16 Eu / mg. b. Wherein particulate matter is: 1-50 particles per i.ml ≥ 10 μm diameter ii. 0-5 particles per ml ≥ 25 μm in diameter, and iii. 0-2 particles per ml ≥ 50 μm in diameter; and

[0035] c. Wherein the composition has 0.1-5%, more preferably 0.1-4%, and most preferably 0.1-3.5% aggregates for 2-3 years, preferably 2-2.5 years, and most preferably 2 years. In another aspect, the present invention discloses an ophthalmic composition comprising bevacizumab, a buffer, a stabilizer, and a surfactant.

[0036] The concentration of bevacizumab in the composition may be in the range of 24 mg / ml to 26 mg / ml, preferably in the range of 25 mg / ml to 26 mg / ml, more preferably 25 mg / ml.

[0037] The buffer present in the composition may be selected from the group including phosphate, citrate, acetate, histidine, succinate, gluconate, glycine, etc., more preferably a phosphate buffer. The concentration of the buffer may preferably be in the range of 40 mM to 60 mM, more preferably in the range of 50 mM to 60 mM. The pH of the buffer may preferably be in the range of 6.0 to 7.0, more preferably in the range of 6.1 to 6.3.

[0038] The stabilizer may be a sugar. The sugar may be a monosaccharide, disaccharide, trisaccharide, polysaccharide, sugar alcohol, reducing sugar, non-reducing sugar, etc. Examples of sugars herein include glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, dextran, erythritol, glycerol, arabitol, ciritol, sorbitol, mannitol, melibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, glucitol, maltitol, lactitol, isomaltulose, etc. A preferred sugar may be a non-reducing disaccharide such as trehalose.

[0039] The amount of sugar in the composition may range from 40 mg / mL to 70 mg / mL, preferably the range may be from 45 mg / mL to 65 mg / mL, more preferably the range is from 50 mg / mL to 60 mg / mL.

[0040] The surfactant present in the composition refers to a surface active agent, preferably a nonionic surfactant.The example of the surfactant herein includes polysorbate (e.g., polysorbate 20 and polysorbate 80).The preferred surfactant herein is polysorbate 20.

[0041] The amount of surfactant in the composition may be in the range of 0.2 mg / mL to 0.6 mg / mL, preferably in the range of 0.3 mg / mL to 0.5 mg / mL, more preferably in the range of 0.35 mg / mL to 0.45 mg / mL. The compositions of the present invention are homogeneous and stable solutions throughout the shelf life of the product and are therefore synergistic compositions.

[0042] In yet another embodiment, the ophthalmic compositions of the present invention may be administered as a predetermined dose in the form of a vial, cartridge or pen or pre-filled syringe, preferably a pre-filled syringe, preferably a single-use pre-filled syringe, with a fill volume of 140 microliters to 200 microliters and a dose of 50 microliters, and the composition may be substantially or completely devoid of silicone oil.

[0043] The body of the syringe may be composed of a material selected from the group including polymer or glass, or indicia coated on either surface; a thermoplastic material; a polyolefin such as a cyclic olefin polymer, a cyclic olefin copolymer, or polypropylene; a polyester such as polyethylene terephthalate; a polycarbonate; or any combination or copolymer thereof.

[0044] The plunger stopper of the prefilled syringe may be constructed of any suitable material selected from chlorobutyl, bromobutyl, or other haloalkyl rubbers. The plunger may optionally be coated with a material selected from the group including silicone, fluoropolymer, parylene, etc. Such coatings may be utilized to minimize any interactions and / or friction that may occur between different parts of the device or between the device and the drug during storage or transportation. The tip of the plunger stopper may be flat or convex. A plunger ring may be present for tight sealing of the plunger and barrel wall. The stopper may be siliconized or coated with bromo-, chloro-, fluoro-, or related polymers.

[0045] Syringes may optionally be coated with an internal coating or layer selected from the group including tie coatings, barrier coatings, pH protective coatings, or lubricious coatings, preferably lubricious coatings with lubricants selected from the group consisting of silicone oil, fluoropolymers, parylene, and related compounds, in the range of 0.1 mg to 0.4 mg of silicone oil. Weakly siliconized borosilicate glass syringe barrels may be spray-coated with a silicone oil-in-water emulsion, followed by heat setting (i.e., "baked silicone") or crosslinking. Such coatings and / or layering may be utilized to minimize any interactions and / or friction that may occur between different parts of the device or between the device and the drug during storage or transportation. Preferably, prefilled syringes are completely free or devoid of silicone oil.

[0046] Certain dosage forms of the present invention, such as pre-filled syringes, may be filled with an inert gas selected from the group including nitrogen, argon, and helium, preferably nitrogen. Pre-filled pharmaceutical syringes may have a fill volume of 140 microliters to 200 microliters and a nominal maximum with a dose of 50 microliters. The compositions of the present invention may be administered as pre-filled syringes.

[0047] The compositions of the present invention may be filled into pre-filled syringes for intravitreal injection. To reduce the possibility of silicone oil droplets being injected into the eye, the pre-filled syringes of the present invention may include barrels that are substantially or completely free of silicone oil. The pre-filled syringes may include an internal coating of silicone oil, which may have 0.1 mg to 0.4 mg of silicone oil. In the alternative, the pre-filled syringes may be internally coated with a tie coating, a barrier coating, a pH protective coating or layer, and optionally a lubricious coating; preferably, the coatings may be fluoropolymers, parylene, and related compounds. In one embodiment, the pre-filled syringes may be completely free of silicone oil. Weakly siliconized borosilicate glass syringe barrels may be spray-coated with a silicone oil-in-water emulsion, which may then be heat-set (i.e., "baked silicon") or cross-linked. Such coatings and / or layering may be utilized to minimize possible interactions and / or friction between different parts of the device or between the device and the drug during storage or during transport.

[0048] In the present invention, the interaction between the contents and the container is minimized and is crucial to maintain the effectiveness to maintain stability for long-term storage / use, especially for the entire shelf life of 2 years. The applicant has carried out several tests and endeavored to ensure that this problem of the prior art is fully addressed in this regard.

[0049] In yet another embodiment, the ophthalmic compositions of the present invention may be used to treat ocular disorders, including but not limited to, exudative age-related macular degeneration, choroidal neovascularization, retinal angiomatous proliferation, pathologic myopia, angioid streaks, Best's disease, adult vitelloid dystrophy, central serous retinochoroidopathy, punctate inner choroidopathy, multifocal choroiditis, presumed ocular histoplasmosis syndrome, choroidal osteoma, toxoplasmosis, uveitis, pseudotumor cerebri, peripapillary idiopathic retinal neovascularization, proliferative diabetic retinopathy, sickle cell retinopathy, retinopathy of prematurity, Eales' disease, macular edema, diabetic retinopathy, central retinal vein occlusion, branch retinal vein occlusion, pseudophakia, uveitis, occlusive vasculitis, retinitis pigmentosa, neovascular glaucoma, central retinal vein occlusion, branch retinal vein occlusion, and hypertrophic glaucoma. The disease may be selected from the group comprising proliferative diabetic retinopathy, central retinal artery occlusion, ocular ischemia syndrome, radiation induced, radiation optic neuropathy, radiation retinopathy, breast cancer with choroidal metastasis, melanoma-associated neovascularization, retinal aneurysm, vascular proliferative tumor, Coats' disease, juxtapapillary capillary hemangioma, idiopathic macular telangiectasia, polypoidal choroidal vasculopathy, central serous retinochoroidopathy, non-arteritic preischemic optic neuropathy, herpetic corneal neovascularization, cicatricial pemphigoid corneal neovascularization, posterior capsule neovascularization, post-corneal transplant rejection neovascularization, corneal neovascularization-associated dry eye, bleb correction, associated with glaucoma filtration surgery, and preferably exudative age-related macular degeneration.

[0050] In yet another aspect, the present invention provides a kit comprising the ophthalmic composition of the present invention in an administrable format, such as a pre-filled syringe, vial, or cartridge, preferably a pre-filled syringe. The kit may include a pre-filled syringe packaged in a blister pack, which may itself be sterile inside. In one aspect, a syringe according to the invention may be placed inside such a blister pack before undergoing sterilization, e.g., terminal sterilization. The blister pack may be formed of a suitable thermoplastic material, such as glass, polyolefin, cyclic olefin polymer or cyclic olefin copolymer, polypropylene, or polyester, or any combination thereof. The kit may include a needle for administration of the ophthalmic composition of the present invention.

[0051] The needle may be any 29-33 gauge x 1 / 2 inch needle, although 31-gauge, 32-gauge, 33-gauge or 34-gauge needles may alternatively be used. The kit may also contain a product insert, which includes instructions for use.

[0052] The present invention discloses a kit comprising an ophthalmic composition in a pre-filled syringe, including a pre-filled syringe, a needle with a gauge in the range of 29-34 gauge, and instructions in a blister package.

[0053] The composition is stable for at least two years at a temperature of 2-10° C., preferably 2-8° C. The ophthalmic composition of the present invention is stable for at least two years and has low sub-visible particulate matter, BET, and aggregates, as well as maintaining safe ease of use throughout its two-year shelf life. advantage: The ophthalmic compositions of the present invention have the following advantages, but the advantages are not limited to those listed herein below: Compared to other leading syringes dispensed from vials, the product is highly stable. · Includes BET, within the limits set forth herein.

[0054] Contains a predetermined dosage, thereby reducing dosage errors, and ensures disposal of the dosage form after a single administration, avoiding multiple use and thereby preventing multiple use and infections resulting from multiple use. Potentially, it has fewer pre-injection and post-injection effects.

[0055] It has a shelf life of 2 years compared to the prior art products which have a shelf life varying from 14 days up to 6 months (14 days in India, FDA has proposed limiting it to only 5 days after repackaging).

[0056] A composition of the invention having no more than 50 particles per ml ≧10 μm in diameter, no more than 5 particles per ml ≧25 μm in diameter, and no more than 2 particles per ml ≧50 μm in diameter. The ophthalmic grade bevacizumab contained in the pre-filled syringes of the present invention is stable at temperatures between 2 and 8°C for the prescribed shelf life. The compositions of the present invention, unlike prior art repackaged or dispensed compositions, are subject to all prescribed quality checks during release. Minimize hospital visits, reduce drug waste, minimize hospital and industrial waste, and eliminate the risk of microbial contamination. The present invention is illustrated by examples, which are intended for illustrative purposes and should not be construed as limiting.

[0057] example Example 1: Purification process of bevacizumab Cell-free harvest containing bevacizumab monoclonal antibody obtained from a perfusion technology-based bioreactor from CHO cells. The harvest was filtered through a 0.2 μm filter and subjected to adsorption-based depth filtration for harvest clarification.

[0058] The filtered harvest was concentrated, and bevacizumab concentration was achieved by using a single-pass TFF module. The "concentrated harvest" from the TFF-I step was filtered using 0.2 μm filtration and applied to an affinity chromatography column packed with Mab Select Sure LX resin (GE Healthcare). The column was equilibrated with equilibration buffer-1 (EB-1, phosphate buffer: 20 mm, NaCl: 150 mm (pH 7.1 ± 0.2)) by passing 5 column volumes (CV) of EB-1 at a flow rate of 150 cm / h. The sample was loaded onto the column at a linear flow rate of 150 cm / h, followed by 5 CV of EB-1 at a flow rate of approximately 150 cm / h. This was followed by 5 CV of wash buffer-1 (WB-1, sodium acetate: 40 mm, pH 6.0) at a flow rate of 150 cm / h. The bound target protein was eluted by passing approximately 5 CV of Elution Buffer-1 (EUB-1, sodium acetate: 30 mm, pH 3.5) through the tube and collected based on absorbance at 280 nm. The eluate (E-1) was analyzed for protein content, glycans, HCPs, charge variants, aggregates, and endotoxin content. At this step, the endotoxin content of E-1 was less than 10 EU / mg (NMT), and the aggregate content was NMT 5%. In the next step, E-1 was subjected to a low-pH inactivation step, incubating at room temperature with gentle continuous agitation for 60 minutes at pH 3.7. After virus inactivation, the solution was diluted with CEX Equilibration Buffer-2 (EB-2, sodium acetate: 50 mm, NaCl: 80 mm (pH 5.3)) and filtered using a 0.2 μm filter. In the next step, a cation exchange column (Capto S ImpAct, GE Healthcare) was equilibrated with 5 CV of equilibration buffer-3 (EB-3, sodium acetate: 50 mm, NaCl: 80 mm, pH 5.3) at a flow rate of 250 cm / h. The sample was loaded onto the column at a flow rate of 250 cm / h, followed by 3 CV of wash buffer-2 (Wb-2, sodium acetate: 50 mm, NaCl: 80 mm, pH 5.3) at a flow rate of 250 cm / h.The sample was run through a linear gradient of 0-15% B in 1.5 CV, followed by 15-25% B in 20 CV using EB-3 and elution buffer-2 (EUB-2, sodium acetate: 50 mM, NaCl: 500 mM, pH 5.3) at a flow rate of 250 cm / h, and the fractions were collected based on UV280. The eluate (E-2) was analyzed for protein amount, charge variants, aggregates, and endotoxin content. After this step, the endotoxin content of E-2 was NMT5Eu / mg.

[0059] In the next step, the CEX elution fractions based on the analysis of charge variants for E-2 were pooled and diluted using AEX equilibration buffer (EB-4, Tris: 20 mm, pH 8.0) followed by filtration using a 0.2 μm filter.

[0060] In the next step, an anion exchange column (Capto Q, GeHealthcare) was equilibrated with 10 CV of equilibration buffer-4 (EB-4, Tris: 20 mm, pH 8.0) at a flow rate of 250 cm / h. The sample was loaded onto the column at a flow rate of 250 cm / h, followed by 2 CV of wash buffer-3 (WB-3, EB-4, Tris: 20 mm, pH 8.0) at a flow rate of 250 cm / h. The column was regenerated with 5 CV of regeneration buffer (Tris: 20 mm, NaCl: 1 M, pH 8.0), and the flow-through (FT) was collected for further analysis, referred to as "AEX FT." It was analyzed for protein content, glycans, HCPs, charge variants, aggregates, and endotoxin content. At this stage, the endotoxin content is less than 0.5 EU / mg (NMT) and the aggregate content is less than 3%. Further, the "AEX FT" is filtered using a prefilter and a nanofilter connected in series at a differential pressure of 1.5±0.5 bar, and the nanofiltrate is concentrated and diafiltered with diafiltration buffer (Pb: 51 mm, trehalose dihydrate: 20 mg / ml (pH 6.0±0.2)).

[0061] The diafiltered sample was spiked with trehalose dihydrate to a final concentration of 60 mg / ml and with 5% polysorbate 20 to a final concentration of 0.04%, and further diluted to achieve the specified bevacizumab concentration to produce the bevacizumab drug substance (DS). The DS was subjected to comprehensive analytical characterization, including but not limited to, protein content, glycans, HCPs, charge variants, aggregates, endotoxin content, particulate matter, and in vitro and in vivo receptor binding assays. For the DS, the endotoxin content was less than 0.16 EU / mg, the aggregate content was 0.8%, and the subvisible particulate matter limit was within the limits specified by the present invention, as described below. (i) Particles ≧10 μm are 50 particles / ml or less. (ii) Particles ≥ 25 μm are 5 particles / ml or less. (iii) Particles ≥ 50 μm are ≤ 2 particles / ml. The ability to achieve the ranges of the present invention for endotoxin, aggregate reduction, and HCP reduction is provided in Table 1 below.

[0062] [Table 1]

[0063] Example 2: Composition according to the invention Bevacizumab obtained in Example 1. The compositions of the present invention may be formulated using various ingredients as follows, as disclosed in Table 2: [Table 2] Composition 3 (C3) was carried forward for further experiments as follows: Some illustrative examples are given in Table 3.

[0064] [Table 3] Composition C33 of the present invention is further analyzed below, and the results are presented in the Examples below.

[0065] Example 3: Analytical characterization of compositions of the present invention in pre-filled syringes Single-use pre-filled syringes were used to fill the bevacizumab ophthalmic composition, which was present as a solution. Three batches of bevacizumab in PFS were extensively characterized to determine physicochemical properties, biological activity, immunochemical properties, purity, and impurities by appropriate techniques. Product properties met the acceptance criteria as shown in Table 4 below:

[0066] [Table 4]

[0067] Example 4: Stability study of compositions of the present invention in a single-use PFS Bevacizumab (ophthalmic grade) in a single-use PFS was subjected to a long-term stability study at 5°C ± 3°C for 24 months. The PFS was filled with 25 mg / ml bevacizumab, phosphate buffer, polysorbate 20, and trehalose dihydrate (pH 6.2). No significant changes in protein concentration or pH were detected during the complete stability program. Data from the 24-month stability study of the drug product were extensively analyzed for physicochemical properties, biological activity, immunochemical properties, purity, and impurities and showed that the product was stable at 5°C ± 3°C for 24 months (Table 5).

[0068] [Table 5] Product characteristics meet acceptance criteria throughout the entire period indicating stability for 24 months at 5°C ± 3°C. In the prior art, bevacizumab repackaged from a vial (Avastin®) into a syringe has not been found to be stable for longer periods.

[0069] The quality of dispensed bevacizumab repackaged into plastic syringes (polypropylene and polycarbonate) for intravitreal injection has been analyzed for up to 3 or 6 months in some studies. In these studies, changes in immunoglobulin G (IgG) content, the presence of silicone oil microdroplets, an increase in subvisible particles, and protein aggregation were reported. It was also observed that repackaged bevacizumab from different suppliers exhibited variable product quality. In one prior art study, a 15.9% degradation of anti-VEGF activity was observed over a 6-month stability period of dispensed bevacizumab.

[0070] Due to stability-related issues, the USFDA has drafted guidelines proposing to limit the expiration date of prefilled Avastin® PFS to 5 days. In India, the Drug Regulatory Commissioner for India (DCGI) has drafted guidelines for dispensing procedures and stated that the use of these syringes should not exceed 14 days. Therefore, the stability of bevacizumab for extended periods of time with the desired quality in repackaged syringes is a significant issue, leading to ocular irritation. The present invention overcomes these limitations and provides for extended storage of bevacizumab by meeting all acceptance criteria imposed herein during shelf life.

[0071] Compared with prior art documents, the compositions of the present invention possess particle sizes of >10 μm and >25 μm throughout their entire shelf life of 2 years. The ophthalmic compositions of the present invention are stable for at least 2 years at temperatures between 2 and 8° C. and are safe, non-toxic, and effective for administration throughout their shelf life.

[0072] Example 5: Toxicity testing of the compositions of the present invention Single-use prefilled syringes filled with ophthalmic-grade bevacizumab were tested for intravitreal repeat-dose (4-week) toxicity studies with a 4-week recovery period in rabbit eyes. The studies were performed in a GLP facility to evaluate the toxicity of bevacizumab in the eye. PFS were filled with 25 mg / ml bevacizumab, 51 mM PB, 0.04% polysorbate 20, and 60 mg / ml trehalose dihydrate at pH 6.2.

[0073] The product met all parameters of an ophthalmic composition throughout its shelf life. During its use in the treatment of eye diseases, bevacizumab was injected directly into the vitreous humor, which comprises a special compartment of the eyeball. The test product, bevacizumab, or a vehicle (diluent) control sample was administered by intravitreal injection into both eyes of each rabbit every two weeks for four weeks, i.e., on days 1, 15, and 29, during the study period.

[0074] The rabbits were observed for the frequency of mortality and signs of local and systemic toxicity throughout the study, then sacrificed and subjected to a complete necropsy. Additional concurrent recovery groups of three rabbits per sex at the vehicle control and high dose levels were treated similarly, but were observed for an additional 28 days after cessation of the treatment period in case of reversal / delayed toxicity.

[0075] The study provided information on target organs, potential for accumulation, reversibility of toxic effects, and estimates of non-effective levels of exposure, which were used to establish safety standards for human exposure. Three dose levels were selected for toxicity testing. The dose levels used in the study were multiples of the human equivalent dose (HED) of bevacizumab for rabbits, i.e., 1.25 mg, 2.50 mg, or 3.75 mg per eye, which were 1X, 2X, and 3X the absolute therapeutic human dose, respectively, and were injected in a volume of 50 μL.

[0076] Based on the findings of the study, it was concluded that the product did not induce any systemic effects, harmful or otherwise, in rabbits treated at a dose level of 3.75 mg per eye. Following intravitreal injection at dose levels of 1.25 mg, 2.50 mg, or 3.75 mg per eye, ophthalmic-grade bevacizumab was well tolerated in rabbit eyes; and the no-observed-adverse-effect level (NOAEL) of the ophthalmic composition of the present invention in rabbits was found to be greater than 3.75 mg per eye. Thus, in intravitreal preclinical studies, the drug was found to be safe for intravitreal administration.

[0077] Specifically, the present invention provides pharmaceutical compositions with significantly reduced endotoxin and particulate matter levels that are suitable for intravitreal use. The present invention also relates to methods for reducing PMT and endotoxin levels in certain compositions, such as pharmaceutical compositions, that can be used for intraocular delivery.

[0078] Example 6: Comparison of compositions of the present invention with prior art compositions As presented in the previous example, the composition of the present invention prepared is compared with a commercially available composition, which is then administered to a patient. A. Comparison of invisible particulate matter: Subvisible particulate matter was tested by a light scattering particle sizer (LOPC) according to USP 789. Particle-free vials of appropriate volume were used for the test. Samples from 10 PFS, forming a volume of approximately 1 ml, were taken into one vial. The sample was further diluted with particle-free water to obtain a final volume of 25 ml. All vials were pooled in a particle-free container, and the sample contents were gently mixed and inverted 20 times consecutively without introducing air bubbles. The sample vials were allowed to stand for at least 15 minutes prior to analysis to remove air bubbles. Four portions of at least 5 ml each were removed, and the number of particles equal to and larger than 10 μm, 25 μm, and 50 μm were counted. The results are presented without regard to the results obtained in the first part.

[0079] The subvisible particulate matter of repackaged bevacizumab from five UK pharmacies (S1-S5) was compared with the ophthalmic composition of the present invention (Eye (2013) 27, 1090-1097). The results are shown in Figures 2a and 2b. Figures 2a and 2b clearly show that the particle size of the present invention is within the range stated in the present invention, thereby making the likelihood of ocular inflammation extremely low.

[0080] B. Comparison of the storage stability of the compositions of the present invention with that of the prior art. The storage stability of repackaged bevacizumab was compared to that of the ophthalmic composition of the present invention and reconstituted bevacizumab, and the results are presented in Table 6 below. [Table 6] Table 6: Comparison of storage stability of prior art compositions with that of the present invention

[0081] In the present invention, it has been found that the compositions of the present invention are stable for two years, which is quite different from the prior art compositions referenced above and offers economic and transportation advantages.

[0082] C. Comparison of bevacizumab concentrations of the present invention with prior art compositions In the study "Retina 2006;26(5):519-22," degradation of bevacizumab was shown when withdrawn in plastic tuberculin syringes, which are commonly used to dispense the drug in most countries. The drug was stored at 4°C for 6 months. The degradation pattern is shown in Table 7 below:

[0083] [Table 7]

[0084] Over a 6-month period, 15.9% degradation was observed in prior art formulated bevacizumab. In the present invention, no protein degradation was observed during the 2-year stability period, as evidenced by % binding efficiency (ELISA) and % VEGF neutralization capacity assays (see Table 8).

[0085] [Table 8]

[0086] Therefore, the present invention, presented in the compositions disclosed herein, is formulated with bevacizumab obtained by the processes disclosed herein and solely possesses the desired product characteristics, stability, and efficacy in the pre-filled syringes disclosed herein.

[0087] The compositions, processes for preparing the compositions, and devices containing the compositions, such as pre-filled syringes, have been arrived at after considerable human effort and experimentation and have their inherent advantages.

Claims

1. Bevacizumab ophthalmic compositions; a. Wherein the bacterial endotoxin test (BET) limit is in the range of 0.001 to 0.4 EU / mg, and preferably in the range of 0.001 to 0.2 Eu / mg, and more preferably in the range of 0.001 to 0.16 Eu / mg; b. wherein the particulate matter is: iv. 1-50 particles per ml >= 10 μm in diameter v. 0-5 particles per ml ≥ 25 μm in diameter, and vi. 0-2 particles per ml >= 50 μm in diameter; and c. wherein the composition has 0.1-5%, more preferably 0.1-4%, and most preferably 0.1-3.5% aggregates for 2-3 years, preferably 2-2.5 years, and most preferably 2 years.

2. 10. A process for preparing the ophthalmic composition of claim 1, comprising the steps of: i. Cultivating the cells of bevacizumab by continuous fermentation method of CHO cells; ii. subjecting the cell-free harvest obtained from the CHO cell culture to adsorption-based depth filtration for further clarification; iii. Concentrating the sample of step (ii) using single-pass tangential flow filtration to obtain a concentrated harvest; iv. subjecting the concentrated harvest of step (iii) to Protein A chromatography to capture bevacizumab to obtain an eluate containing partially purified bevacizumab; v. subjecting the eluate of step (iv) to low pH viral inactivation to obtain a viral inactivated sample; vi. For further purification, subjecting the virally inactivated sample of step (v) to additional cation exchange chromatography to obtain an eluate containing primarily highly purified bevacizumab; vii. subjecting the eluate of step (vi) to anion exchange chromatography to remove trace impurities, such as host cell proteins (HCPs), host cell DNA (HCD), and endotoxins; viii. subjecting the eluate of step (vii) to virus-reducing filtration and complete removal of viruses; ix. concentrating and diafiltering the sample of step (viii) to obtain bevacizumab; and; x. Formulating the sample obtained from step (ix) to achieve a bevacizumab composition of the present invention.

3. 3. The process of claim 2, wherein the weak viral inactivation is carried out at a pH in the range of 3.0 to 5.0, preferably in the range of 3.0 to 4.0, most preferably in the range of 3.5 to 4.

0.

4. The stationary phase of the cation exchange chromatography is SO ３ -sulfonylisobutyl, SO ３ 3. The process according to claim 2, wherein the anion exchange chromatography stationary phase is selected from the group comprising diethylaminoethyl, quaternaryamine, polyquaternium, N-benzyl-N-methylethanolamine, preferably quaternaryamine, and wherein the diafiltration is carried out in the presence of TFF-II diafiltration buffer.

5. 3. An ophthalmic composition produced by the process of claim 2. a. Wherein the bacterial endotoxin test (BET) limit is in the range of 0.001 to 0.4 Eu / mg, and preferably in the range of 0.001 to 0.2 Eu / mg, and more preferably in the range of 0.001 to 0.16 Eu / mg; b. wherein the particulate matter is: i. 1-50 particles per ml ≥ 10 μm in diameter; ii. 0-5 particles per ml >= 25 μm in diameter, and iii. 0-2 particles per ml >= 50 μm in diameter; c. wherein the composition has 0.1-5%, more preferably 0.1-4%, and most preferably 0.1-3.5% aggregates for 2-3 years, preferably 2-2.5 years, and most preferably 2 years.

6. The ophthalmic composition of claim 1 , wherein the composition comprises bevacizumab, a buffer, a stabilizer, and a surfactant.

7. 7. The ophthalmic composition of claim 6, wherein the concentration of bevacizumab in the composition is in the range of 24 mg / ml to 26 mg / ml, preferably in the range of 25 mg / ml to 26 mg / ml, more preferably 25 mg / ml.

8. 7. The ophthalmic composition of claim 6, wherein the buffer is selected from the group comprising phosphate, citrate, acetate, histidine, succinate, gluconate, glycine, more preferably a phosphate buffer, and has a concentration ranging from 40 mM to 60 mM, more preferably from 50 mM to 60 mM, and a pH ranging from 6.0 to 7.0, more preferably from 6.1 to 6.

3.

9. 7. The ophthalmic composition of claim 6, wherein the stabilizer is a sugar selected from monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, preferably the sugar is glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, dextran, erythritol, glycerol, arabitol, sciritol, sorbitol, mannitol, melibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, glucitol, maltitol, lactitol, isomaltulose, more preferably trehalose, and in the range of 40 mg / ml to 70 mg / ml, preferably the range is 45 mg / ml to 65 mg / ml, more preferably 50 mg / ml to 60 mg / ml.

10. 7. The ophthalmic composition of claim 6, wherein the surfactant is selected from the group comprising non-ionic surfactants, preferably polysorbate, polysorbate 20 or polysorbate 80, more preferably polysorbate 20, and is in the range of 0.2 mg / mL to 0.6 mg / mL, preferably in the range of 0.3 mg / mL to 0.5 mg / mL, more preferably in the range of 0.35 mg / mL to 0.45 mg / mL.

11. 10. The ophthalmic composition of claim 1, administered as a predetermined dose in the form of a vial, cartridge or pen or pre-filled syringe, preferably a pre-filled syringe, preferably a single-use pre-filled syringe, with a fill volume of 140 microL to 200 microL, and a dose of 50 microL.

12. 12. The pre-filled syringe of claim 11, wherein the injection is administered intravitreally.

13. Ocular disorders include exudative age-related macular degeneration, choroidal neovascularization, retinal angiomatous proliferation, pathologic myopia, vascular streaks, Best's disease, adult vitelloid dystrophy, central serous retinochoroidopathy, punctate inner choroidopathy, multifocal choroiditis, presumed ocular histoplasmosis syndrome, choroidal osteoma, toxoplasmosis, uveitis, pseudotumor cerebri, peripapillary idiopathic retinal neovascularization, proliferative diabetic retinopathy, sickle cell retinopathy, retinopathy of prematurity, Eales' disease, macular edema, diabetic retinopathy, central retinal vein occlusion, branch retinal vein occlusion, pseudophakia, uveitis, occlusive vasculitis, retinitis pigmentosa, neovascular glaucoma, central retinal vein occlusion, branch retinal vein occlusion, proliferative diabetic retinopathy, central retinal artery occlusion, and ocular ischemic syndrome.

13. The ophthalmic composition and pre-filled syringe of any one of claims 1 and 5 to 12 for use in an eye disorder selected from the group comprising: induced radiation, radiation optic neuropathy, radiation retinopathy, breast cancer with choroidal metastasis, melanoma-associated neovascularization, retinal aneurysm, vascular proliferative tumor, Coats' disease, juxta-papillary capillary hemangioma, idiopathic macular telangiectasia, polypoidal choroidal vasculopathy, central serous retinochoroidopathy, non-arteritic pre-ischemic optic neuropathy, herpetic corneal neovascularization, cicatricial pemphigoid corneal neovascularization, posterior capsule neovascularization, post-corneal transplant rejection neovascularization, corneal neovascularization-associated dry eye, bleb correction, associated with glaucoma filtration surgery, preferably exudative age-related macular degeneration.

14. 14. A kit comprising the ophthalmic composition in a pre-filled syringe according to any one of claims 1 and 5 to 13, including a pre-filled syringe, a needle with a gauge in the range of 29 to 34 gauge, and instructions for use in a blister package.

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