In vitro methods for simulating and analyzing the behavior of ophthalmic drugs in the eye

Abstract

The present invention relates to a method and apparatus for simulating and analyzing the behavior of substances in the eye, particularly in the vitreous humor. Furthermore, the present invention refers to a method for providing vitreous humor, and a buffer fluid for use in such a method and apparatus.

Description

[Technical field]

[0001] The present invention relates to a method and apparatus for simulating and analyzing the behavior of substances in the eye, particularly in the vitreous humor. Furthermore, the present invention refers to a method for providing vitreous humor, and a buffer fluid for use in such a method and apparatus. [Background technology]

[0002] Blinding diseases of the posterior segment of the eye, such as age-related macular degeneration or diabetic retinopathy, affect an increasing number of people worldwide as life expectancy increases, and if not treated properly, can lead to severe conditions that lead to complete blindness. Unfortunately, there is a global lack of effective treatments for these diseases, as the eye is a complex organ associated with many biological static, dynamic and metabolic barriers that make drug delivery and development extremely challenging.

[0003] Intravitreal drug delivery has become an efficient and frequently used technique for treating diseases of the posterior segment of the eye. According to this treatment technique, drugs are typically injected directly into the vitreous humor, also called the "vitreous body," of the eye, either in humans or any other vertebrate animals. Some innovative drug delivery strategies, such as implants or microparticles, have also been developed, but they require reliable models to be tested preclinically.

[0004] Because the volume of the vitreous is relatively small (approximately 4 mL) and must remain generally constant, the possible injection volume of intravitreal drugs is usually less than 100 μL. Thus, at the moment of administration, the injected drug is subject to rapid dilution and pH and temperature changes, which can destabilize the API and lead to its aggregation.

[0005] Furthermore, most of the stabilizers in drug formulations have very low molecular weights and are therefore eliminated very quickly compared to large protein drugs, so that the drugs are quickly exposed to the stress conditions of the vitreous humor without stabilizers and may interact with all the various components of the vitreous humor.

[0006] Generally, administering drugs to the posterior segment of the eye is difficult because this organ is inherently complex, and there are many static, dynamic and metabolic barriers, which consistently limit the diffusion and convection of active pharmaceutical ingredients in ocular tissue, thereby affecting their bioavailability.By directly injecting drugs into vitreous humor, some of these biological barriers can be selectively overcome.Therefore, intravitreal drug delivery methods usually allow a high proportion of the administered dose of unchanged drug to reach the intended posterior segment of the eye, thereby contributing to the high bioavailability of ophthalmic drugs.

[0007] Despite circumventing many of the ocular biological barriers mentioned above, drugs applied intravitreally must still traverse remaining barriers and clearance pathways.

[0008] The first barrier is the vitreous humor itself, which significantly affects and limits the diffusion and convection of drugs. The vitreous humor is a complex biological fluid composed mainly of water with additional positively charged collagen and negatively charged proteoglycans. In addition to these, many compounds are present in low concentrations. The interaction between collagen and proteoglycans creates a gel-like rigid structure. As a result of this composition, small molecule drugs usually diffuse more easily in this rigid matrix than macromolecules. Furthermore, positively charged molecules usually have a significantly reduced diffusion rate due to their interaction (and usually aggregation) with the negatively charged proteoglycans. The described vitreous humor composition may change, for example, with age, pathological conditions, etc., and with it, its diffusion characteristics may change.

[0009] The second barrier is the diffusion of drugs to other parts of the eye, which causes the clearance of drugs from the vitreous humor before they reach their intended posterior segment. These dynamic barriers are constituted, for example, by the forward flow pathway, where the vitreous flows and exchanges material with the aqueous humor, during which the drug is removed by the aqueous humor outflow. In addition, small lipophilic drug molecules can also be eliminated through the retina-choroid-scleral pathway by diffusing through the retinal pigment epithelium and being eliminated through the circulation of the blood-retinal barrier. Drugs that are substrates for efflux pumps can also be eliminated by active transport through the retina-choroid-scleral pathway. In addition, diffusion and convection can be induced by saccadic eye movements, which can govern the distribution direction of the drug.

[0010] All of the above combine to produce complex interactions between vitreous humor components and applied drugs, which have a decisive effect on the physicochemical stability, bioavailability and therefore therapeutic efficacy of drugs.The selection of effective drug formulations and doses is also important to ensure that drugs remain stable as long as possible and achieve the intended therapeutic effect.

[0011] Therefore, evaluation of interactions between intravitreally applied substances and the vitreous humor (i.e., the physiological environment of the eye) that may affect drug bioavailability is important for the development of effective ocular disease treatments.

[0012] In order to analyze the behavior of a substance, various eye models, i.e. in vitro models, are known to simulate the interaction of the substance with the physiological environment of the eye. Most of these models are static models and typically cannot adequately model the above-mentioned dynamic diffusion and convection phenomena after intravitreal injection. For example, such a static model is known from the article "Prediction of intraocular antibody drug stability using ex-vivo ocular model" by S. Patel et al., published in European Journal of Pharmaceutics and Biopharmaceutics, 2017 Mar;112:177-186.

[0013] Furthermore, a dynamic model is known from the article "Evaluation of protein drug stability with vitreous humor in a novel ex-vivo intraocular model" by S. Patel et al., published in the European Journal of Pharmaceutics and Biopharmaceutics, 2015 Sep;95(Pt B):407-417. The dynamic model includes three compartments separated by a diffusion-controlling membrane. Specifically, the vitreous humor compartment is surrounded by a gel matrix compartment arranged together in a flow-through compartment that serves as a buffer reservoir to ensure the maintenance of the desired pH level of the vitreous humor received in the vitreous humor compartment. Summary of the Invention

[0014] It is an object of the present invention to provide an improved method for simulating and analysing the behaviour of substances in the eye, which allows an improved simulation of the diffusion and convection procedures occurring in the physiological environment of the eye.It is further an object of the present invention to define a method for providing a sample fluid similar to the vitreous humor for use in said method.It is further an object of the present invention to provide a buffer fluid and a device for simulating the behaviour of substances in the eye for use in said method.

[0015] These objects are solved by the subject matter of the independent claims.

[0016] The present disclosure will be more readily understood by reference to the following detailed description when considered in conjunction with the accompanying drawings, in which: [Brief description of the drawings]

[0017] [Figure 1] FIG. 1 is a schematic diagram of an apparatus for testing the behavior of substances in the simulated physiological environment of the eye, in which various sample cells are used in parallel. [Diagram 2] FIG. 2 is a schematic cross-sectional view of a flow cell unit used in the apparatus shown in FIG. 1, the flow cell unit housing a sample cell. [Diagram 3] FIG. 3 shows a flow diagram illustrating a method for simulating and analyzing the behavior of substances in the eye, in which the apparatus shown in FIGS. 1 and 2 is used. [Figure 4] 4 shows a flow diagram illustrating the individual steps of the method shown in FIG. 3. [Diagram 5] 4 shows a flow diagram illustrating the individual steps of the method shown in FIG. 3. [Figure 6] 1 shows a graph showing the change in pH value of a sample over time. [Figure 7] FIG. 1 shows a graph depicting the change in total protein concentration of samples over time. [Figure 8] 1 shows a table showing the change in color and turbidity of the samples over time. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Provided herein is an improved method for simulating and analyzing the behavior of a substance in the eye. The method for simulating and analyzing the behavior of a substance in the eye includes: a) providing at least one sample cell having an interior cavity therein and a wall defining the cavity, the cavity being referred to as a sample chamber, the wall being at least partially a semi-permeable membrane; b) filling said sample chamber with a sample comprising at least one substance constituting a simulated physiological environment of the eye and a sample fluid; c) inducing a flow of a buffer fluid, in particular a continuous flow of a buffer fluid, on the outer surface of the semipermeable membrane for a predetermined period of time; d) analyzing the sample.

[0019] Generally, the sample cell is intended to provide structural conditions that allow for mimicking or simulating physiological conditions within the eye. To do so, the sample cell has a cavity therein, i.e., a sample chamber for receiving a sample to be analyzed, and a wall that defines the cavity, the wall being at least partially a semi-permeable membrane.

[0020] The semi-permeable membrane has two surfaces: an inner surface facing the sample cell, which forms at least part of the inner surface of the wall defining the sample chamber and thereby comes into contact with the sample, and an outer surface in contact with the buffer fluid.

[0021] In the method of the present invention, the structural characteristics of the sample cell can be set according to the physiological conditions of the eye in order to adequately simulate the physiological conditions of the eye.

[0022] Thus, the volume of the sample chamber of the sample cell may be provided according to or substantially corresponding to the volume of the eye whose physiological environment is to be simulated. "Substantially corresponding" in this context means including the volume of the eye to be simulated and differing therefrom by, for example, up to 20% or 30% or 40% or 50%. For reference, the volume of the human eye is about 4 mL. The volume of the sample chamber may therefore be in the range of 3 mL to 6 mL, or in the range of 4 mL to 6 mL, or in the range of 4 mL to 5 mL. In particular, the volume of the sample chamber may be 5 mL or about 5 mL. Generally speaking, the volume of the sample chamber may be larger, in particular slightly larger, than the volume of the eye whose physiological environment is to be simulated.

[0023] The sample chamber of the sample cell may be provided by a dialysis tube or cassette, such as, for example, a Float-A-Lyzer® dialysis device. In this configuration, a semipermeable membrane forms at least a part of the wall that defines the sample chamber, with the inner surface of the semipermeable membrane and optionally the wall defining the sample chamber, and the outer surface of the semipermeable membrane and optionally the wall defining the sample cell. Generally, semipermeable membranes are used to control diffusion phenomena. Thus, the term "semipermeable membrane" refers to a diffusion-controlled membrane, and therefore can also be considered a molecular weight size selective membrane. The semipermeable membrane may be a dialysis membrane. A dialysis membrane is a semipermeable film, for example a sheet of regenerated cellulose or cellulose ester, that contains pores of various sizes. Generally, molecules larger than the pores cannot pass through the semipermeable membrane, while molecules smaller than the pores can pass freely. The separation properties determined by the pore size range of the semipermeable membrane are referred to as the molecular weight cut off (MWCO) of the membrane.

[0024] Alternatively or additionally, the structural and functional configuration of the semipermeable membrane of the sample cell may be provided according to the physiological conditions of the eye to be simulated. In particular, the molecular weight cut-off (MWCO) of the semipermeable membrane may be provided according to or substantially corresponding to the Retinal Exclusion Limit (REL) of the eye to be simulated. "Substantially corresponding" in this context means to include the REL as well as MWCO values ​​that differ from the REL by, for example, up to about 30% or 40% or 50%. The REL is generally known as the maximum size of a molecule that can freely diffuse across the retina of the eye. For a healthy human eye, the REL is approximately 50 kDa (10 3 The REL is defined as being in the range of 50 kDa to 100 kDa, preferably 70 kDa. However, this range can vary significantly, especially depending on the state of the eye (changes due to age, death, etc.). Furthermore, the REL is highly dependent on the structure of the diffusing molecule, for example the linear or globular structure of the molecule. Thus, the MWCO of the semipermeable membrane of the sample cell may be in the range of 50 kDa to 100 kDa, especially 70 kDa or about 70 kDa.

[0025] According to step b) of the method, the sample chamber is filled with a sample comprising at least one substance and a sample fluid constituting a simulated physiological environment of the eye. In the context of the present disclosure, the term "substance" is intended to refer to any substance of interest for its behavior in the eye, in particular in the vitreous humor. The term "substance" thus refers to any active agent designed or intended to be applied to or in a patient's eye for the therapeutic treatment of an ocular disease, in particular a posterior ocular disease, or to alleviate symptoms associated therewith. The substance may be a small molecule or a large molecule, such as a peptide or a protein. The term "substance" may also refer to a diagnostic agent. The term "substance" may also refer to an excipient used to formulate an active or diagnostic agent. The term "substance" may also refer to a formulation of said active or diagnostic agent(s). In general, the term "substance" is not limited to the above and may refer to any substance that may come into contact with the eye or vitreous humor or that may be the subject of testing for its behavior in the physiological environment of the eye.

[0026] Preferably, the "substance" is an active agent for treating and treating ocular diseases, particularly diseases of the posterior segment of the eye. The substance may be an active agent for topical, systemic, intravitreal, intrathecal, subcutaneous, subconjunctival, retrobulbar, or intracameral administration. More preferably, the active agent is an intravitreal active agent intended to be injected into the vitreous humor of the eye, particularly into the vitreous humor of humans and other vertebrates. Such active agents may be used to treat various ocular diseases, such as age-related macular degeneration, diabetic retinopathy, and infections inside the eye, such as endophthalmitis. For example, the active agent may be or include a monoclonal antibody, particularly a monoclonal antibody in a sterile formulation.

[0027] The term "simulated physiological environment of the eye" or "simulated physiological ocular environment" as used herein refers to a fluid that simulates the natural fluids or conditions of the eye, in particular of humans and other vertebrates. The simulated physiological environment may therefore be or include a fluid extracted from the eye of a human or other vertebrate, in particular vitreous humor. For example, the simulated physiological ocular environment may include or be provided based on porcine vitreous humor. Alternatively or additionally, the simulated physiological ocular environment may be an artificial fluid that simulates such natural fluids or conditions of the eye.

[0028] In the method of the invention, the "sample fluid" is or simulates the physiological environment of the vitreous humor of the eye. In other words, the sample fluid may be a fluid having structural and / or functional characteristics that are equal to or correspond to the structural and / or functional characteristics of the natural fluid or state of the eye. The sample fluid may be a fluid extracted from the vitreous humor of a human or other vertebrate, i.e. an extracted vitreous humor, or a mixture of fluids, i.e. provided by mixing or combining fluids. For example, the sample fluid may comprise a first fluid extracted from the vitreous humor of a human or other vertebrate, i.e. an extracted vitreous humor, and a second fluid constituting a buffer solution. The buffer solution may be identical to the buffer fluid defined herein below. However, it is preferred that the sample fluid is a fluid extracted from the vitreous humor of a human or other vertebrate, i.e. an extracted vitreous humor.

[0029] In particular, step b) of the method can comprise a first sub-step of filling the sample chamber with a sample fluid and a second sub-step of supplying or injecting the at least one substance into the sample chamber. These sub-steps can be performed in any order. However, it is preferred that the first sub-step of filling the sample chamber with a sample fluid is performed before the second sub-step of supplying or injecting the at least one substance into the sample chamber. Alternatively, the at least one substance and the sample fluid can be mixed to provide the sample before filling the sample chamber with the sample.

[0030] The substep of providing or injecting at least one substance into the sample chamber may be performed such that a typical volume of the at least one substance injected into the sample chamber may range from 1 to 200 microliters, e.g., from 2 to 100 microliters, or from 10 to 100 microliters. The amount of substance typically corresponds to the amount of substance that is applied to the eye for therapeutic or diagnostic purposes.

[0031] As mentioned above, the sample fluid constituting the simulated physiological environment may be extracted vitreous humor or may be a mixture of a first fluid, which may be extracted vitreous humor, and a second fluid, which is a buffer solution. Thus, the substep of filling the sample chamber with the sample fluid may include filling the first fluid, e.g. extracted vitreous humor, and optionally a second fluid, e.g. a buffer solution, into the sample chamber. This may be performed sequentially in any order or simultaneously. Alternatively, the first and second fluids, i.e. extracted vitreous humor and buffer solution, may be mixed before the step of filling the sample chamber with the sample fluid.

[0032] The buffer solution may be any suitable buffer solution known to those skilled in the art. However, it is preferred that the buffer solution used to prepare the buffer solution of the sample fluid is the same as the "buffer fluid" as defined herein, i.e. the same as the buffer fluid introduced onto the outer surface of the semipermeable membrane that at least partially defines the sample chamber that contains the sample to be analyzed.

[0033] According to a further development, the method may further comprise a step of providing a sample fluid. The sample fluid may be provided in the form of a gel matrix. In particular, this step may comprise sub-steps, in particular a sub-step of extracting vitreous fluid from at least one, preferably two or more isolated vertebrate eyes, in particular an isolated porcine eye. The extraction may be carried out with a syringe, for example a needleless syringe. In particular, the syringe may be inserted into at least one eye. The syringe may then be used to aspirate vitreous fluid from the eye. To allow proper insertion of the syringe, the eye may be opened by a slit, in particular spaced apart from the iris of the eye, for example at least about 3 mm or 1 cm away from the iris. From each eye (for example a porcine eye), a volume of up to 2 mL or 3 mL of vitreous fluid may be extracted. By limiting the amount of extracted vitreous fluid to 2 mL or 3 mL per eye, an excessive contamination of the extracted vitreous fluid, for example by tissue cells, may be effectively prevented. The vitreous humor thus extracted may then be pooled and / or cooled, for example with ice or in a refrigerator, particularly at a temperature of about 5°C.

[0034] In a further optional substep, the extracted vitreous humor may be subjected to a centrifugation step, in which the extracted vitreous humor is centrifuged, in particular by using a centrifuge, to separate the clear fraction of the extracted vitreous humor, in particular from the cellular debris, after which the clear fraction can be extracted and isolated, for example by aspirating with a pipette.

[0035] In an alternative or additional optional substep, the extracted vitreous humor may be filtered, in particular by applying pressure filtration, for example using a pressure filtration unit equipped with a polyethersulfone filter having a pore size of about 0.10 to 0.30 μm, preferably about 0.15 to 0.25 μm, more preferably about 0.20 to 0.24 μm, in particular 0.22 μm.

[0036] In an alternative or additional optional substep, the extracted vitreous humor may then be frozen, in particular at a temperature of about -80°C, before being further processed or used in the method.

[0037] In an alternative or additional optional substep, the extracted vitreous humor may be combined with a buffer solution to provide a sample fluid, which may be the same or different from the buffer fluid, but is preferably the same.

[0038] According to step c) of the method described above, a flow of buffer fluid, also referred to herein as "buffer fluid flow", is directed onto the outer surface of the semi-permeable membrane which at least partially defines a sample chamber containing the sample to be analyzed.

[0039] The buffer fluid directed across the surface of the semi-permeable membrane may be the same or different, and preferably is the same, as the buffer solution used as a component of the sample fluid in the sample chamber.

[0040] Preferably, the buffer fluid flow is a continuous flow. During step c), the buffer fluid flow is continuously directed onto the outer surface of the semipermeable membrane for a predetermined period, also referred to herein as the "test time". The test time may refer to a period of 1 hour or more, but may also be less than 1 hour. In particular, the test time may range from 4 hours to a substantial multiple of 24 hours, for example, the test time may be a period of 4 hours or 24 hours or 72 hours or 96 hours or 168 hours or 336 hours or 504 hours.

[0041] Furthermore, the buffer fluid flow can have a constant flow rate. As mentioned above, by inducing a buffer fluid flow on the outer surface of the semipermeable membrane, the outer surface of the sample cell can be continuously provided with fresh buffer fluid, thereby providing dynamic conditions around the sample cell. Preferably, the buffer fluid flow is provided such that a laminar flow of the buffer fluid is induced on the semipermeable membrane. In this way, a uniform and optimal diffusion through the semipermeable membrane can be achieved, which makes it possible to mimic the natural environment of the eye, in particular the dynamic and metabolic barriers that exist therein.

[0042] According to one configuration, the flow rate of the buffer fluid stream may be a constant flow rate, further, the flow rate may be in the range of 5 mL / min to 12 mL / min, specifically in the range of 6 mL / min to 10 mL / min, more specifically about 8 mL / min.

[0043] More preferably, the buffer fluid is provided such that when it is introduced across the outer surface of the semipermeable membrane, it sets or influences a desired condition of the sample contained in the sample chamber, in particular a predetermined pH condition of the sample received in the sample chamber. In certain embodiments, it further sets or influences at least one of a predetermined temperature condition and a predetermined osmolality condition of the sample received in the sample chamber. Alternatively or additionally, when it is introduced across the outer surface of the semipermeable membrane, it can receive or absorb substances that diffuse across the semipermeable membrane from the sample chamber into the buffer fluid, in particular precipitates or decomposition products from the sample contained in the sample chamber.

[0044] Thus, in the context of the present disclosure, the term "buffer fluid" refers to a buffer solution having a desired pH value and / or capable of maintaining the pH value of a sample received in a sample chamber, for example, at a pH between 5.5 and 8.5, specifically between about pH 7.0 and about pH 7.6, and more specifically at about pH 7.4.

[0045] In particular, the buffer fluid may include salts. Preferably, the buffer fluid is an aqueous buffer fluid. The buffer fluid may be, for example, phosphate buffered saline, bicarbonate buffer, Ringer's bicarbonate buffer, Ringer's lactate buffer, simulated body fluids, other isotonic fluids, cell culture media, and any other physiologically representative buffer. Lactated Ringer's buffer, also known as lactated Ringer's solution (RL), sodium lactate solution, and Hartmann's solution, is a mixture of sodium chloride, sodium lactate, potassium chloride, and calcium chloride in water.

[0046] The buffer fluid may also be used as a buffer solution for the preparation of a sample fluid which may be provided in the form of a gel matrix.

[0047] According to one configuration, the buffer fluid is an artificial substitute for vitreous humor, such as human vitreous humor. The buffer fluid can include at least one cationic species selected from the group of sodium, potassium, calcium, and magnesium cations, at least one anionic species selected from the group of chloride, bicarbonate, phosphate, lactate, and optionally a preservative, such as azide. The buffer fluid can include one, two, three, or all four of the respective cationic and anionic species.

[0048] The buffer fluid may further include glucose and / or glutathione disulfide.

[0049] Specifically, the buffer fluid may include sodium, potassium, calcium, magnesium, chloride, bicarbonate, phosphate, lactate, glucose, and / or glutathione disulfide, and optionally a preservative such as an azide.

[0050] Exemplary salts for preparation of buffer fluids include sodium chloride, potassium chloride, dibasic sodium phosphate, sodium bicarbonate, calcium chloride dihydrate, magnesium chloride hexahydrate, sodium lactate, and sodium azide. A buffer fluid may include one, two, three, four, five, six, seven, or all of these salts.

[0051] In one embodiment, the buffer fluid comprises: (A) a buffer comprising sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride hexahydrate, sodium hydrogen phosphate, and sodium bicarbonate; (B) a buffer comprising sodium chloride, potassium chloride, calcium chloride dihydrate, and sodium lactate; and (C) sodium azide.

[0052] In one embodiment, the buffer fluid comprises: (A) a buffer comprising sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride hexahydrate, sodium hydrogen phosphate, sodium bicarbonate, and glucose; (B) a buffer comprising sodium chloride, potassium chloride, calcium chloride dihydrate, and sodium lactate; and (C) sodium azide.

[0053] In one embodiment, the buffer fluid comprises (A) a buffer comprising sodium chloride, potassium chloride, calcium chloride dihydrate, magnesium chloride hexahydrate, dibasic sodium phosphate, sodium bicarbonate, glucose, and glutathione disulfide, (B) a buffer comprising sodium chloride, potassium chloride, calcium chloride dihydrate, and sodium lactate, and (C) sodium azide.

[0054] An example of the above (A) is commercially available BSS PLUS® Sterile Intraocular Irrigating Solution (e.g., manufactured by Alcon). An example of the above (B) is commercially available Ringer's lactate solution (e.g., manufactured by B. Braun).

[0055] In a preferred embodiment, the buffer fluid is About 50 mmol / L to about 250 mmol / L of sodium, About 1.0 mmol / L to about 25 mmol / L of potassium, Approximately 0.2 mmol / L to approximately 5.0 mmol / L of calcium, Approximately 0.1 mmol / L to approximately 4.0 mmol / L of magnesium, About 25 mmol / L to about 200 mmol / L of chloride, About 5 mmol / L to about 100 mmol / L of bicarbonate, About 0.1 mmol / L to about 10 mmol / L of phosphate, About 0.8 mmol / L to about 20 mmol / L of lactate, About 0.8 mmol / L to about 20 mmol / L of glucose, Approximately 0.1 mmol / L to approximately 10 mmol / L of glutathione disulfide, Optionally, a preservative is included, for example, about 0.5 mmol / L to about 10 mmol / L of azide.

[0056] Specifically, the buffer fluid is Approximately 100 mmol / L to approximately 200 mmol / L of sodium, Approximately 2.0 mmol / L to approximately 10 mmol / L of potassium, Approximately 0.4 mmol / L to approximately 2.5 mmol / L of calcium, Approximately 0.2 mmol / L to approximately 2.0 mmol / L of magnesium, About 75 mmol / L to about 175 mmol / L of chloride, About 10 mmol / L to about 50 mmol / L of bicarbonate, About 0.1 mmol / L to about 5.0 mmol / L of phosphate; About 1.5 mmol / L to about 15 mmol / L of lactate, About 1.5 mmol / L to about 15 mmol / L of glucose, About 0.1 mmol / L to about 5.0 mmol / L of glutathione disulfide, Optionally, a preservative may be included, for example, about 1.0 mmol / L to about 5.0 mmol / L of azide.

[0057] More specifically, the buffer fluid is Approximately 125 mmol / L to approximately 175 mmol / L of sodium; Approximately 3.0 mmol / L to approximately 7.0 mmol / L of potassium, Approximately 0.6 mmol / L to approximately 1.5 mmol / L of calcium, Approximately 0.5 mmol / L to approximately 1.3 mmol / L of magnesium, About 100 mmol / L to about 150 mmol / L of chloride, About 10 mmol / L to about 40 mmol / L of bicarbonate, About 0.1 mmol / L to about 4.0 mmol / L of phosphate; About 1.5 mmol / L to about 8.0 mmol / L of lactate, Glucose of about 2.0 mmol / L to about 8.0 mmol / L, About 0.1 mmol / L to about 4.0 mmol / L of glutathione disulfide, Optionally, a preservative may be included, for example, about 1.5 mmol / L to about 4.5 mmol / L of azide.

[0058] Even more specifically, the buffer fluid is Approximately 140 mmol / L to approximately 160 mmol / L of sodium, Approximately 4.5 mmol / L to approximately 6.0 mmol / L of potassium, Approximately 0.9 mmol / L to approximately 1.3 mmol / L of calcium, Approximately 0.6 mmol / L to approximately 1.0 mmol / L of magnesium, About 120 mmol / L to about 130 mmol / L of chloride, About 17 mmol / L to about 25 mmol / L of bicarbonate; About 0.2 mmol / L to about 3.0 mmol / L of phosphate; About 3.0 mmol / L to about 5.0 mmol / L of lactate, About 2.0 mmol / L to about 5.0 mmol / L of glucose; Approximately 0.2 mmol / L to approximately 3.0 mmol / L of glutathione disulfide, Optionally, a preservative may be included, for example, about 2.5 mmol / L to about 3.5 mmol / L of azide.

[0059] In a particularly preferred embodiment, the buffer fluid comprises Approximately 155.6 mmol / L ± 10% sodium, Approximately 5.1mmol / L ±10% potassium, Approximately 1.1mmol / L ±10% calcium, Approximately 0.9 mmol / L ± 10% magnesium, Approximately 127.3 mmol / L ± 10% chloride, Approximately 21.2 mmol / L ± 10% bicarbonate and Approximately 2.5 mmol / L ± 10% phosphate, Approximately 4.3 mmol / L ± 10% lactate, Approximately 4.2 mmol / L ± 10% glucose, Approximately 2.5 mmol / L ± 10% glutathione disulfide, Optionally, a preservative is included, for example, about 3.0 mmol / L ± 10% azide.

[0060] In a more particularly preferred embodiment, the buffer fluid is Approximately 155.6 mmol / L ± 5% sodium, Approximately 5.1 mmol / L ± 5% potassium, Approximately 1.1mmol / L ±5% calcium, Approximately 0.9 mmol / L ± 5% magnesium, Approximately 127.3 mmol / L ± 5% chloride, Approximately 21.2 mmol / L ± 5% bicarbonate and Approximately 2.5 mmol / L ± 5% phosphate; Approximately 4.3 mmol / L ± 5% lactate, Approximately 4.2 mmol / L ± 5% glucose, Approximately 2.5 mmol / L ± 5% glutathione disulfide, Optionally, a preservative is included, for example, about 3.0 mmol / L ± 5% azide.

[0061] In a most particularly preferred embodiment, the buffer fluid comprises Approximately 155.6 mmol / L of sodium; About 5.1mmol / L of potassium, Approximately 1.1 mmol / L of calcium, Approximately 0.9 mmol / L of magnesium, About 127.3 mmol / L chloride, Bicarbonate at approximately 21.2 mmol / L; Approximately 2.5 mmol / L of phosphate; Approximately 4.3 mmol / L lactate; Glucose at approximately 4.2 mmol / L; Approximately 2.5 mmol / L of glutathione disulfide; Optionally, a preservative is included, for example, about 3.0 mmol / L azide.

[0062] The osmolality of the freshly prepared buffer fluid is preferably about 280 mOsm / kg HO to about 330 mOsm / kg HO, more preferably about 300 mOsm / kg HO to about 315 mOsm / kg HO, and most preferably about 305 mOsm / kg HO to about 310 mOsm / kg HO.

[0063] According to the methods of the present invention, the buffer fluid is conditioned by subjecting the buffer fluid to an atmosphere of a predetermined CO2 concentration for a predetermined period of time prior to and / or while directing the buffer fluid stream over the exterior surface of the semipermeable membrane. In certain embodiments, conditioning further comprises subjecting the buffer fluid to a predetermined temperature for a predetermined period of time prior to and / or while directing the buffer fluid stream over the exterior surface of the semipermeable membrane.

[0064] The acclimation step may be performed such that the buffer fluid is subjected to an atmosphere of predetermined conditions (e.g., an atmosphere of a predetermined CO2 concentration and, optionally, a predetermined temperature). The acclimation may be performed for a predetermined period of time. To do so, the buffer fluid may be contained in an incubator configured to generate an atmosphere of the predetermined conditions. By doing so, the properties of the buffer fluid may be set to desired conditions and / or maintained stable. For example, the acclimation step may prevent the buffer fluid from being subjected to unintended pH shifts.

[0065] The acclimatization may be performed once or continuously, for example once before the buffer fluid is used or continuously during the use of the buffer fluid. For example, the buffer fluid may be subjected to continuous acclimatization while being induced in a flow, in particular in a continuous flow, on the outer surface of the semipermeable membrane to an atmosphere of a given condition, for example an atmosphere of a given CO2 concentration and optionally a given temperature.

[0066] For example, the buffer fluid may be subjected to an atmosphere having a temperature of about 37° C. and a CO2 concentration of about 7%. To do so, the buffer fluid may be housed in an incubator configured to generate an atmosphere of a predetermined condition for a predetermined period of time. As mentioned above, the acclimation may be carried out for a predetermined period of time, which may also be referred to herein as an "acclimation time". The acclimation time may be set to allow for proper acclimation of the buffer fluid. The acclimation time may be 1 hour or more. Specifically, the acclimation time may be at least 2 hours or 6 hours or 8 hours, for example, the acclimation may be carried out overnight, i.e., for at least 6 hours.

[0067] Advantageously, the buffer fluid is acclimatized in a CO2 incubator before and during use at a temperature of about 35°C to 40°C and a CO2 concentration of about 5% to 10%, preferably at a temperature of about 37°C and a CO2 concentration of about 7%.

[0068] In certain embodiments, the buffer fluid is similar to the human vitreous humor, meaning that the properties of the buffer fluid are similar or essentially the same as those of the human vitreous humor, including the type and concentration of anionic and cationic species, and preferably further including pH, osmolality, and viscosity.

[0069] The inventors have observed that buffer fluids, being highly nutritional, are likely to be subject to microbial contamination during long-term stability studies despite sterile filtration. This can be prevented by the addition of preservatives. Azide salts, such as sodium azide, have been found to effectively inhibit microbial growth in buffer solutions.

[0070] The buffer fluids of the present invention are advantageous in that they are standardized and easy to prepare and handle, while closely reflecting the conditions of the vitreous humor, particularly the human vitreous humor.

[0071] In the context of the present invention, it has surprisingly been found that the dynamic conditions in the sample chamber and at the outer surface of the sample chamber can be guaranteed by inducing a flow of a buffer fluid (preferably conditioned as outlined above), in particular a continuous laminar flow of such a buffer fluid, on the outer surface of the semipermeable membrane that at least partially defines the sample chamber. The laminar flow of the buffer fluid corresponds to a stable diffusion process of at least one substance in the sample cell and the semipermeable membrane interface. This allows to better mimic the natural environment of the eye, in particular the dynamic and metabolic barriers prevailing therein. As a result, the suggested method provides an improved in vitro model that allows to more realistically analyze the behavior of substances in the eye, without having to revert to invasive procedures.

[0072] Generally, the proposed methods refer to in vitro methods: they can be carried out using components of organisms isolated from their normal biological environment.

[0073] The proposed method allows to assess the behavior of substances in a simulated physiological environment of the eye, in particular of humans and other vertebrates. Thus, the method may be used, for example, to allow to simulate, determine or predict how a substance is affected over time by the biological environment in the eye, in particular in the vitreous humor. Potential effects on the intended therapeutic effect may also be determined on this basis.

[0074] To that end, the method may be applied to analyze and assess the stability of a substance in a simulated physiological ocular environment, without being limited thereto. Such analysis may refer, for example, to the concentration-dependent precipitation of a substance. Furthermore, the method may be used to determine the interaction of a substance, such as a macromolecule and / or excipient, in a particular fluid environment, such as the vitreous humor, with, for example, proteins in the vitreous humor, the stability of a substance upon dilution in a particular fluid, such as the vitreous humor, and after loss of stabilizing excipients, such as surfactants, sugars, buffer species, and tonicity agents, as well as long-term stability in a simulated physiological ocular environment, particularly in the vitreous humor.

[0075] According to step d), the method further comprises analyzing the sample. To do so, the sample may be removed from the sample chamber before performing the step of analyzing the sample. In one embodiment, the method may further comprise a step of separating said at least one substance from the sample fluid, for example separating a proteinaceous active agent from the sample fluid, before performing the step of analyzing.

[0076] In particular, the step of analyzing the sample may include determining the pH value of the sample, in particular at a given temperature of the sample, for example 37° C. This may be performed by a pH meter.

[0077] Alternatively or additionally, the total protein concentration of the sample may be measured, particularly by spectrophotometry, for example at about 280 nm.

[0078] Alternatively or additionally, the osmolality of a sample may be determined, for example, by freezing point depression methods.

[0079] Alternatively or additionally, the step of analyzing the sample may include measuring or determining the physicochemical stability of a substance in the sample, such as an active agent contained therein. In particular, this step may be carried out to analyze the degradation, denaturation and aggregation of at least one substance. To do so, the step of analyzing the physicochemical stability of the sample may include visual inspection, for example, visual inspection under a black and white box or low magnification, to determine the presence or concentration of visible particles in the sample.

[0080] Alternatively or additionally, the presence and density of sub-visible particles may be determined, for example, by optical obscuration or by flow imaging microscopy techniques (such as microflow imaging or FlowCA).

[0081] Alternatively or additionally, the color of the sample may be determined, for example, by visual comparison with a color standard, or by colorimetry, etc. Alternatively or additionally, the turbidity of the sample may be determined, for example, by plate-based or cuvette-based spectrophotometry, or by performing nephelometry, etc. Alternatively or additionally, the aggregation and oligomerization of at least one substance in the sample may be determined, for example, by gel permeation chromatography, or by size exclusion chromatography, in particular by UV-visible spectroscopy, for example at about 280 nm, or by fluorescence spectroscopy.

[0082] Alternatively or additionally, the step of analyzing the physicochemical stability of the sample may comprise determining the total substance concentration, e.g. the total protein concentration, by methods known in the art, such as, for example, spectrophotometry at about 280 nm, or by surface plasmon resonance (SPR), or by any other method known for this purpose by the skilled artisan.

[0083] Furthermore, the step of analyzing the sample may be carried out to measure or determine the chemical purity and / or chemical stability of the sample. In this way, in particular the chemical modification and degradation of at least one substance may be determined. In particular, the chemical purity may be determined by any method known in the art, for example by size exclusion chromatography, in particular by UV-Vis spectroscopy, for example at about 280 nm.

[0084] Alternatively or additionally, the charge distribution may be determined, for example, by anion exchange chromatography or by cation exchange chromatography, particularly with UV-Visible spectroscopy detection at 280 nm.

[0085] Alternatively or additionally, subunit analysis can be performed, for example, by capillary electrophoresis under denaturing and non-denaturing conditions. Other suitable methods, including those outlined above, will be known to those of skill in the art.

[0086] In a further development, in order to analyze the behavior of the substance, the method may further comprise a step of analyzing the buffer fluid, in particular after it has been guided over the outer surface of the semipermeable membrane. This step may be carried out while the outer surface of the semipermeable membrane is subjected to a flow of buffer fluid and / or during the subsequent test time. In other words, the step of analyzing the buffer fluid may be carried out in parallel with the step of analyzing the sample and / or in parallel with the step of directing a flow of buffer fluid over the outer surface of the semipermeable membrane. This step may be applicable when the device is equipped with a closed or an open buffer fluid circuit.

[0087] In particular, the step of analyzing the buffer fluid may comprise determining the pH value of the buffer fluid, in particular at its predetermined temperature, e.g. 37° C. This may be performed, for example, by a pH meter. Alternatively or additionally, the total protein concentration in the buffer fluid may be measured, in particular, by spectrophotometry, e.g. at about 280 nm, or by other suitable methods generally known for this purpose. Alternatively or additionally, the osmolality of the buffer fluid may be determined, for example, by freezing point depression, or by measuring the conductivity, or by other generally known analytical methods.

[0088] Alternatively or additionally, the step of analyzing the buffer fluid may include measuring or determining the degradation, denaturation and aggregation products of at least one substance (e.g., a protein drug such as a mAb) in the buffer fluid. To do so, the step of analyzing the physicochemical stability of the buffer fluid may include visual inspection to determine the presence or density of visible particles therein. Alternatively or additionally, the presence and density of sub-visible particles may be determined, for example, by light obscuration. Alternatively or additionally, the color of the buffer fluid may be determined, for example, by colorimetry. Alternatively or additionally, the turbidity of the buffer fluid may be determined, for example, by performing nephelometry. Alternatively or additionally, the aggregation and oligomerization products of substances in the buffer fluid may be determined, for example, by size exclusion chromatography, in particular by UV-Vis spectroscopy, for example at about 280 nm. Alternatively or additionally, the denaturation products of substances may be determined, for example, by micro-differential scanning calorimetry. Alternatively or additionally, the step of analysing the physicochemical stability of the substance may comprise determining the total protein concentration, in particular by spectrophotometry, for example at about 280 nm.

[0089] All of the above analytical methods are generally known, and other methods known to those skilled in the art for the above analytical purposes may also be used.

[0090] Acclimatization According to a further development, the method may further comprise a step of conditioning the sample fluid before adding the at least one substance. In particular, the sample fluid may be conditioned before being filled into the sample chamber, but it is equally preferred that the sample fluid is conditioned after being filled into the sample cell. The conditioned step may be performed such that the sample cell containing the sample fluid is subjected to an atmosphere of predetermined conditions, such as an atmosphere of a predetermined CO2 concentration. The conditioned step may further comprise subjecting the sample cell containing the sample fluid to a predetermined temperature. The subjecting may be performed for a predetermined period of time. For example, the sample fluid, preferably the extracted vitreous humor, may be subjected to an atmosphere having a CO2 concentration of about 7% and optionally a temperature of about 37°C. To do so, the sample cell containing the sample fluid, preferably the extracted vitreous humor, may be accommodated in an incubator configured to generate an atmosphere of predetermined conditions for a predetermined period of time. As mentioned above, the conditioned may be performed for a predetermined period of time, which may also be referred to herein as an "conditioned time". The conditioned time may be set to allow proper conditioned of the sample fluid. The acclimation time may be 1 hour or more. Specifically, the acclimation time may be at least 2 hours, or 6 hours, or 8 hours, for example, acclimation may be carried out overnight, i.e., for at least 6 hours.

[0091] Alternatively or additionally, conditioning of the sample fluid, preferably the extracted vitreous humor, may be conveniently achieved by using a buffer fluid conditioned as outlined above as the buffer solution used to form the sample fluid.

[0092] Alternatively or additionally, the sample fluid, preferably the extracted vitreous humor, may be conditioned after being loaded into the sample chamber by directing a flow of conditioned buffer fluid over the outer surface of the semi-permeable membrane during the test, and optionally also for a predetermined period of time prior to the test.

[0093] In the context of the present invention, it has been found that the step of conditioning the sample fluid, in particular the extracted vitreous humor, has the effect that the pH value of the sample fluid, for example the extracted vitreous humor, in particular the porcine vitreous humor, remains constant or is more stable during the test. In other words, the pH level of the conditioned sample fluid, in particular the conditioned extracted vitreous humor, may be more stable when performing the method compared to a non-conditioned sample fluid.

[0094] The sub-step of providing or injecting a substance into the sample chamber may be performed after the step of conditioning the sample fluid as outlined above.

[0095] sign According to a further development, the method may comprise a step of labeling the at least one substance, for example with a fluorescent dye. If the at least one substance is a pharmaceutical formulation of an active agent and a pharma- ceutical acceptable excipient, it is sufficient if the active agent is labeled, for example with a fluorescent dye. By doing so, the step of analyzing the sample can be carried out more effectively, since the determination of the purity of the at least one substance in the sample can be more accurately assessed, for example by using fluorescence detection, and can be more accurately differentiated from proteins in the sample, for example porcine vitreous humor proteins.

[0096] The method may include the further step of acclimating the material, optionally after labeling the material (e.g., with a fluorescent dye), before being provided to the sample chamber. To do so, the material may be subjected to predetermined conditions, i.e., a predetermined CO2 atmosphere, optionally at a predetermined temperature, e.g., about 7% CO2, and optionally at about 37°C, which may be performed for a predetermined period of time, i.e., the acclimation time as described herein, and all embodiments thereof. Acclimating the material may be performed before providing the material to the sample chamber.

[0097] Parallel sample cells In a further development, the method disclosed herein may be carried out such that two or more sample cells are provided and filled with a sample as described above. Each of the sample cells may be subjected to a flow of buffer fluid as described above in parallel or simultaneously with any other sample cell. The step of directing a flow of buffer fluid across a semi-permeable membrane at least partially defining the sample chamber of the different sample cells may be carried out such that each sample cell is subjected to a flow of buffer fluid for an individual period, i.e. an individual test time. For example, when providing a first and a second sample cell filled with a sample, the first sample cell may be subjected to a flow of buffer fluid for a first period and the second sample cell may be subjected to a second period different from the first period.

[0098] Furthermore, the step of analyzing the sample may be performed such that different samples, when analyzed, are placed in their associated sample cells for different individual periods of time, in parallel or simultaneously with each other. For example, when filling a first sample in a first sample chamber and a second sample in a second sample chamber, the first sample is analyzed after the first sample cell is placed in the first sample chamber exposed to a flow of buffer fluid for a first period of time. Thus, the second sample is analyzed after the second sample cell is placed in the second sample chamber exposed to a flow of buffer fluid for a second period of time. This configuration allows for analysis at different times, while at the same time preventing the analysis of the same sample and sample cell several times, which would disturb the osmotic equilibrium in the sample cell. In other words, in this way, the method allows for the testing and analysis of multiple different samples simultaneously, which is performed in that each sample cell is subjected to a flow of buffer fluid for an individual period of time (Tj) that may differ between sample cells.

[0099] Sample fluids, methods for providing sample fluids and uses - Patents.com Provided herein is a sample fluid prepared by extracting vitreous humor from a vertebrate eye, centrifuging the extracted vitreous humor to separate a clear fraction of the extracted vitreous humor, filtering the extracted vitreous humor, and optionally acclimating the extracted vitreous humor by subjecting the extracted vitreous fluid to an atmosphere of a predetermined CO2 concentration for a predetermined period of time. In one embodiment, the extracted vitreous humor may be further subjected to a predetermined temperature for a predetermined period of time.

[0100] Further, a method for providing a sample fluid for use in the above-mentioned method is provided. In particular, the method for providing a sample fluid includes the steps of extracting vitreous humor from a vertebrate eye, centrifuging the extracted vitreous humor to separate a clear fraction of the extracted vitreous humor, filtering the extracted vitreous humor, and optionally acclimating the extracted vitreous humor by subjecting the extracted vitreous humor to an atmosphere with a predetermined CO2 concentration for a predetermined period of time. In one embodiment, the extracted vitreous humor may be subjected to a predetermined temperature for a predetermined period of time. Since the method for providing a sample fluid is intended to be used in the above-mentioned method, the technical features described in relation to the method for simulating and analyzing the behavior of an ophthalmic substance in an eye may also be related to and applied to the method for providing a sample fluid or the sample fluid, and vice versa.

[0101] Further provided is a sample fluid obtainable by the method for providing a sample fluid described above.

[0102] Also provided herein is the use of a sample fluid as described herein in a method for simulating and analyzing the behavior of a substance in the eye as described herein above.

[0103] Buffer fluids and their uses Still further, a buffer fluid is provided, the buffer fluid being configured as described herein above and optionally also conditioned as described above. Further provided is the use of a buffer fluid in the methods described herein, the buffer fluid being configured as described herein above and optionally conditioned as described above. In one embodiment, the buffer fluid comprises sodium, potassium, calcium, magnesium, chloride, bicarbonate, phosphate, lactate, glucose, and optionally a preservative such as azide.

[0104] Since a buffer fluid is intended to be used in the above-mentioned methods, the technical features described in relation to the method for simulating and analysing the behaviour of an ophthalmic substance in the eye also relate to and may apply to the method for providing a sample fluid, and vice versa.

[0105] Device As is evident from FIG. 2, the sample cell may be placed, in particular removably mounted, in a buffer cell having an internal cavity referred to as a flow chamber. A buffer fluid, i.e. a flow of buffer fluid, flows through the flow chamber. According to one configuration, the buffer cell has a flow chamber, a buffer fluid inlet configured to supply the buffer fluid to the flow chamber, i.e. to direct the flow of buffer fluid into the flow chamber of the buffer cell, and a buffer fluid outlet configured to drain the buffer fluid after being directed through the flow chamber, i.e. to direct the flow of buffer fluid out of the flow chamber of the buffer cell. The flow chamber thereby fluidically connects the buffer fluid inlet to the buffer fluid outlet.

[0106] Specifically, the sample cell may be disposed within the flow chamber of the buffer cell. According to this configuration, the sample cell may be disposed wholly or partially within the flow chamber. At least the semi-permeable membrane of the sample cell may be disposed within the flow chamber. With such a configuration, the buffer fluid is then directed into the flow chamber, flows through the flow chamber while being guided on the outer surface of the semi-permeable membrane, and is then discharged from the flow chamber, thereby generating a continuous flow over the semi-permeable membrane.

[0107] The buffer cell, and in particular the structural arrangement of the buffer fluid inlet, buffer fluid outlet and flow chamber, may be designed such that a continuous flow of buffer fluid directed through the buffer cell flows along the outer surface of the semipermeable membrane of the sample cell, thereby continuously replacing the buffer fluid present in the buffer cell. According to one configuration, the flow rate of the buffer fluid through the buffer cell may be a constant flow rate. The flow rate may be in the range of 5 mL / min to 12 mL / min, particularly in the range of 6 mL / min to 10 mL / min, more particularly about 8 mL / min.

[0108] In a further development, the buffer cell, i.e. the inlet and the outlet of the buffer cell, may be connected to a buffer fluid circuit, which may be a closed buffer fluid circuit.

[0109] According to this configuration, the buffer fluid discharged from the buffer cell may be treated, for example filtered and conditioned, as described above, before being redirected through the buffer cell. To do so, the closed buffer fluid circuit may be provided with a filter, for example a fiber filter, which may be arranged downstream of the buffer cell. In the context of the present disclosure, the terms "downstream" and "upstream" refer, respectively, to the flow direction of the buffer fluid flow and to a position before or after the sample cell. The filter may be configured to filter the buffer fluid after being guided through the buffer cell. By doing so, clogging of aggregated degradation products received by the buffer fluid as it flows through the buffer cell may be prevented. The filter may in particular be a glass fiber filter with a pore size of 1 μm. Alternatively or additionally, the closed buffer circuit may comprise a buffer reservoir in which the buffer fluid discharged from the buffer cell may be supplied and stored before being redirected to the buffer cell, in particular after being filtered.

[0110] Alternatively, the buffer cell may be connected to an open buffer fluid circuit. In this configuration, the buffer fluid discharged from the buffer cell is collected and stored in a discharge container rather than being redirected back into the buffer cell. Fresh buffer fluid may be supplied to the buffer cell from a buffer reservoir.

[0111] In one configuration, the sample cell 26 with the sample chamber 28 and the buffer cell 24 with the flow chamber 32, the buffer fluid inlet 36, and the buffer fluid outlet 38 are arranged such that the sample chamber 28 and the flow chamber 32 are in direct contact with each other and are separated from each other by the semipermeable membrane 30. In this configuration, the buffer fluid inlet 36 is configured to supply buffer fluid to the flow chamber 32, i.e., direct the flow of buffer fluid 34 into the flow chamber 32 of the buffer cell 24, and the buffer fluid outlet 38 is configured to discharge the buffer fluid 34 after being directed through the flow chamber 32, i.e., direct the flow of buffer fluid 34 out of the flow chamber 32 of the buffer cell 24. The buffer fluid thereby communicably connects the buffer fluid inlet to the buffer fluid outlet. The buffer fluid is delivered into the flow chamber, flows through the flow chamber while being directed over the outer surface of the semipermeable membrane, and is then discharged from the flow chamber, thereby generating a flow (e.g., continuous flow) over the semipermeable membrane.

[0112] Since the device 10 is intended to be used in the above-mentioned method for simulating and analysing the behaviour of ophthalmic substances in the eye, the technical features described in relation to said method, in particular the technical features relating to the flow of buffer fluids, the sample cell, the sample chamber, the buffer cell and the flow chamber, or the buffer fluid, also relate and may apply to the device, and vice versa.

[0113] Use of the device Further provided is the use of the device in the above-mentioned method for simulating and analyzing the behavior of an ophthalmic substance in an eye. Specifically, the device is configured to stimulate the behavior of an ophthalmic substance in an eye, the device comprising at least one sample cell having a sample chamber defined by a semipermeable membrane, the sample cell being configured to receive a sample containing the ophthalmic substance and a sample fluid constituting a simulated physiological environment of the eye, and a measuring device for measuring the amount of the sample in the sample chamber for a predetermined period of time (T j and a buffer supply system configured to direct a flow of a buffer fluid across the outer surface of the semipermeable membrane.

[0114] Since the apparatus is intended to be used in the above-mentioned methods, the technical features described in relation to the method for simulating and analysing the behaviour of ophthalmic substances in the eye may also be relevant and applicable to the method for providing a sample fluid, and vice versa.

[0115] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The invention will now be described in more detail with reference to the accompanying drawings, in which similar elements are indicated by the same reference numerals and repeated description thereof may be omitted to avoid redundancy, in which:

[0116] FIG. 1 shows an apparatus 10 for simulating the behavior of a substance in the eye. The apparatus comprises a base 12 that accommodates a number of flow cell units 14. The illustrated apparatus 10 comprises seven flow cell units 14a-14g, but the apparatus is not limited to this number and may therefore comprise more or less than seven flow cell units 14. For better visualization, only three flow cell units 14a, 14b and 14g are shown, the other four flow cell units being indicated by curved lines. Preferably, the different flow cell units 14 are structurally identical.

[0117] The apparatus 10 further comprises a buffer supply system 16 configured to supply a buffer fluid to the plurality of flow cell units 14 .

[0118] 2 shows an enlarged view of one exemplary flow cell unit 14 of the device 10, which is shown separated from other parts of the device 10 for better visualization. In the following, features of the flow cell unit 14 are exemplarily described based on the flow cell unit 14 shown in FIG. 2, which may correspondingly be applied to any one of the different flow cell units 14 of the device 10.

[0119] The flow cell unit 14 comprises a cell mount 18 designed to removably mount the flow cell unit 14 to the base 12 of the device 10. In particular, the cell mount 18 is configured to be mounted securely, i.e. by a threaded connection, to the top of the base 12 and remain there during normal operation of the device 10. The flow cell unit 14 further comprises a casing 20 having a hollow cylindrical shape. At its lower end, the casing 20 is integrally connected to the cell mount 18 as one component.

[0120] The upper part of the cell mount 18 is provided with a centrally located recess into which a flat gasket 22 is inserted. The buffer cell 24 is inserted centrally into the casing 20 such that the bottom end face of the buffer cell 24 rests on the gasket 22 in the recess of the cell mount 18, as can be gathered from FIG. 2. The buffer cell 24 is thereby directly connected to the gasket 22 as one component. The contact surface between the upper end of the buffer cell 24 and the upper end of the casing 20 may be provided with at least one further gasket (not shown) to ensure a watertight connection therebetween. Also, the connection surface between the bottom of the casing 20 and the upper part of the cell mount 18 may be provided with a gasket (not shown) to ensure a sealed connection.

[0121] In the center of the buffer cell 24, the sample cell 26 is inserted, such that the sample chamber 28 and the semi-permeable membrane 30 that defines the sample chamber 28 are completely located in the buffer cell 24. In an alternative configuration, the sample cell 26 may also be completely located in the buffer cell 24, for example provided in the form of a floating cell. The sample cell 26 is configured to receive a sample whose behavior is to be tested during operation of the device 10. To do so, the sample cell 26 is preferably submerged in the buffer cell 24. The connection between the sample cell 26 and the buffer cell 24 is a sealed connection that can be realized, for example, by a corresponding thread or by a further gasket (not shown). In particular, the sample cell 26 may be provided in the form of or include a dialysis tube or cassette, such as a Float-A-Lyzer® dialysis device. A buffer fluid outlet 38 may be located on the screw cap.

[0122] The flow cell unit 14 comprises three different chambers or compartments designated in flow.

[0123] The sample cell 26 is provided with a sample chamber 28 that is defined at least in part by a semi-permeable membrane 30. In the illustrated configuration, the sample chamber 28 has a cylindrical shape, with at least the cylindrical surface and the bottom surface of the cylinder being provided by the semi-permeable membrane 30. Specifically, the sample chamber 28, in all of its embodiments, has a volume as described herein.

[0124] The buffer cell 24 is provided with a flow chamber 32, and the sample cell 26 is disposed within the flow chamber 32 such that the semipermeable membrane 30 defining the sample chamber 28 is fully received within the flow chamber 32. The flow chamber 32 is defined by the outer wall of the buffer cell 24 and is configured for the flow of a buffer fluid. In other words, the flow chamber 32 constitutes a buffer flow channel through which a buffer fluid flow 34, preferably a laminar flow, is directed as indicated by the arrows in FIG. 2. To generate the buffer fluid flow 34 within the flow chamber 32, the buffer cell 24 comprises a buffer fluid inlet 36 for supplying the buffer fluid into the flow chamber 32, and a buffer fluid outlet 38 for discharging the buffer fluid from the flow chamber 32 after the buffer fluid has been guided through the flow chamber 32. The buffer fluid inlet 36 and the buffer fluid outlet 38 are fluidly connected to the buffer supply system 16 shown in FIG. 1, which is configured to guide the buffer fluid to the buffer fluid inlet 36 and to discharge the buffer fluid from the buffer fluid outlet 38 after the buffer fluid has been guided through the flow chamber 32. As can be inferred from FIG. 2, the cell mount 18 and gasket 22 are provided with an inlet channel through which a buffer fluid inlet 36 is fluidly connected to the buffer supply system 16.

[0125] As shown in FIG. 1, the buffer supply system 16 constitutes a closed buffer fluid circuit. Thus, the buffer supply system 16 comprises a buffer reservoir 40 for storing and conditioning the buffer fluid to be delivered to the buffer cell 24. In particular, the buffer reservoir 40 is arranged in an incubator 42, which is configured to subject the buffer reservoir 40, i.e. the buffer fluid stored therein, to a predetermined atmosphere with a predetermined CO2 concentration and optionally a predetermined temperature, the predetermined CO2 concentration and the predetermined temperature being described herein and in all embodiments thereof. A buffer supply line 44 is provided with a supply manifold that delivers the buffer fluid to the different flow cell units 14 respectively. To do so, a pump unit 46 is arranged in the supply line 44 upstream of the supply manifold and configured to selectively and variably generate a continuous and constant flow through the flow chambers 32 of the different flow cell units 14. Specifically, in one mode of operation, the buffer supply system 16 operates with all of its embodiments such that buffer fluid flows through each flow chamber 32 of the different flow cell units 14 at a flow rate as described herein. Before being sent to the buffer cell units 14, the buffer fluid is filtered in a filter unit 50 (e.g., including a glass fiber filter having a pore size similar to all of the embodiments as described herein) and then redirected to the buffer reservoir 40. Additionally, a buffer outlet line 48 is provided having an outlet manifold that outlets and collects the buffer fluid from the different flow cell units 14, respectively.

[0126] Furthermore, as can be gathered from Fig. 2, a conditioning chamber 52 is provided between the casing 20 and the buffer cell 24, which is configured to guide a conditioning medium, such as conditioned water, having a predetermined temperature, for example of about 37°C, over the outer surface of the buffer cell 24. In this way, the temperature of the buffer cell 24, and therefore the temperature of the sample cell 26, can be set and maintained at a desired temperature. To do so, the casing 20 is provided with a conditioning medium inlet 54 and a conditioning medium outlet 56, which are designed so that the conditioning medium supplied to the conditioning chamber 52 via the conditioning medium inlet 54 is guided around the outer surface of the buffer cell 24 before being discharged via the conditioning medium outlet 56. To supply the conditioning medium to the conditioning chamber 52, the device 10 comprises a conditioning system (not shown) configured to supply the conditioning medium to the conditioning chamber 52 via the conditioning medium inlet 54 and to discharge the conditioning medium from the conditioning chamber 52 via the conditioning medium outlet 56. The conditioning system may further comprise a heat exchanger unit configured to provide heat exchange between the buffer fluid and the conditioning medium, particularly before directing the buffer fluid into the flow chamber 32 .

[0127] In the following, with reference to Figure 3, a method for simulating and analysing the behaviour of substances in the eye is described using the device 10 shown in Figures 1 and 2. In particular, the method is applied for example to test and analyse the behaviour of substances in vitreous humor, in particular human vitreous humor, i.e. in a simulated ocular environment.

[0128] In a first step S1 of the method, an apparatus 10 is provided comprising a plurality of flow cell units 14, each of which comprises one sample cell 26 having a sample chamber 28 at least partially defined by a semi-permeable membrane 30.

[0129] In a next step S2, a buffer fluid is provided which is stored in a buffer reservoir 40. The buffer fluid may be a buffer fluid configured as described herein and in all its embodiments.

[0130] In step S3, a sample fluid is provided, which is intended to simulate the vitreous fluid of a human eye, and is preferably extracted vitreous fluid. This is carried out in several substeps, as shown in FIG. 4. In a first substep S3.1, porcine vitreous fluid is extracted from a number of porcine eyes. Then, in substep S3.2, the extracted vitreous fluid is centrifuged. In particular, the extracted vitreous fluid is placed in a centrifuge tube of a centrifuge and centrifuged, for example, at 3000-11000 rpm for 10-30 minutes, at 3900 rpm for 10-20 minutes, or at 10000 rpm for 20 minutes. By doing so, a clear fraction of the extracted vitreous fluid is separated, for example, from cellular debris and then extracted, for example by aspirating with a pipette. The extracted clear fraction of the vitreous fluid is then filtered in substep S3.3, for example, by a pressurized filtration unit having a polyethersulfone filter, in particular having a pore size as described herein, and with all its embodiments.

[0131] Optionally, the filtered vitreous humor can be frozen for a predetermined time before being further processed. Then, in optional sub-step S3.4, the vitreous humor is mixed with a buffer solution, thereby providing a sample fluid. The buffer solution may be the same as the buffer fluid. In step S4, the sample chamber 28 of each flow cell unit 14 is filled with the sample fluid. To do so, the sample cell 26 of each flow cell unit 14 may be disassembled from its associated flow cell unit 14, and the top of the sample cell 26 is opened. After filling the sample chamber 28 of the sample cell 26 with the sample fluid, the sample cell 26 is closed by placing the top of the sample cell 26, and then each of the sample cells 26 is housed in a respective buffer cell 24.

[0132] Step S5 may be performed after loading the sample into the sample cells 26 and before receiving the sample cells 26 in their respective buffer cells 24. This is performed by placing each sample cell 26 containing sample fluid in an incubator, which may be incubator 42, for a predetermined period of time, i.e. the acclimation time mentioned above, e.g. 6 hours. In this way, the sample fluid is subjected to an atmosphere of a predetermined CO2 concentration and optionally a predetermined temperature for a predetermined period of time.

[0133] In optional step S6, a substance is provided, in particular an intravitreal active agent. This step may be performed simultaneously with steps S4 or S5. In particular, during S6, a substance is provided and may optionally be pre-labeled, for example with a fluorescent dye. The thus labeled substance may then be conditioned by subjecting it to an atmosphere of a predetermined CO2 concentration and optionally a predetermined temperature for a predetermined period of time. To do so, the substance is placed in an incubator, which may be incubator 42, for a predetermined period of time. Additionally or alternatively, conditioning of the substance may be achieved by directing the conditioned buffer fluid 32 onto the outer surface of the semipermeable membrane 30 after step S7, or both.

[0134] Next, in step S7, a substance is injected into each sample chamber 28 containing sample fluid, thus providing the sample cells 26 with the sample to be tested and analysed.

[0135] Next, the sample cell 26, prepared for performing a test simulating the behavior of a sample contained in the sample cell 26, is placed in the associated flow cell unit 14, and the device 10 is placed in a test state ready to perform the test (step S8).

[0136] The behavior of the samples is then tested and analyzed in step S9, described below with reference to Fig. 5. In this step, a total number J of individual flow cell units 14 (each flow cell unit 14 containing one sample cell 26 with one sample) are subjected to simulated test conditions. Thus, the integer J indicates the total number of flow cell units 14, i.e., seven flow cell units 14 in the illustrated configuration, each sample chamber 28 containing a sample of the same composition and condition at the start of the test. However, the integer j refers to one particular one of the samples in the J different flow cell units 14, or sample cells 26, or sample chambers 28, and therefore has a value between 1 and J. The test is performed in such a way that the different sample cells are subjected to different predetermined periods T j , i.e., the test is carried out over different test times, i.e., exposed to simulated conditions. Thus, the parameter T j refers to the test duration of the jth sample cell 26, and this predetermined time differs among different sample cells 26. j may have durations as described herein for all of its embodiments.

[0137] Specifically, T j can be defined as follows: (1)T j =T j -1+D j , 1≦j≦J Here, D j refers to a period greater than 0 that may be constant, i.e., D1 to D J Each of D1 to D2 refers to a period of time that may have the same value or may vary, i.e., D1 to D J may have different values. In the present configuration, for example, D1 is equal to 0, D2 is equal to 4h, D3 is equal to 20h, D4 is equal to 72h, D5 is equal to 72h, D6 is equal to 168h, and D7 is equal to 168h.

[0138] In a first substep S9.1, the variable j is set equal to 1. As long as the variable j does not exceed the number J of sample cells 26 used, as indicated by S9.2, the following substeps are performed. Then, in substep S9.3, the device 10 is operated such that the buffer fluid flows around each sample cell 26, such that a flow 34 of the buffer fluid is induced on the outer surface of the semipermeable membrane 30 of each sample cell 26. In particular, this step is performed such that a laminar flow 34 of the buffer fluid is induced on the outer surface of the semipermeable membrane 30. By doing so, the buffer fluid sets the predetermined pH conditions, the predetermined temperature conditions, and the predetermined osmolality conditions of the samples received in the different sample chambers 28. Furthermore, the buffer fluid flowing over the semipermeable membrane 30 of the sample cells 26 receives any substances (e.g., precipitates or decomposition products from the samples received in the sample chambers) that diffuse from the samples through the semipermeable membrane into the buffer fluid.

[0139] This substep is for a parameter t, which indicates the elapsed time from the start of the flow of the buffer fluid in substep S9.3, to be T, as indicated by S9.4. j It will be implemented unless t reaches T j If so, the method proceeds to substep S9.5, where the jth sample cell 26 is removed from the apparatus 10. During this substep S9.5, the flow of buffer fluid 34 may be interrupted or continued. Then, in substep S9.6, the removed jth sample cell 26 is analyzed, for example, by determining the physicochemical stability of the sample. In other words, the sample received in the jth sample cell 26 is passed through the semipermeable membrane 30 of the jth sample cell 26 for a predetermined period T j The sample received in the sample cell 26 is then subjected to a flow of buffer fluid 34 for a period of time and then analyzed. Analyzing the sample received in the sample cell 26 may be performed as described herein and with all embodiments thereof. At the same time, the variable j is incremented by 1 in substep S9.7 and the method returns to substep S9.2, thereby repeating substeps S9.3-9.7 until all sample cells 26 have been removed and analyzed.

[0140] Thus, in the methods disclosed herein, two or more sample cells 26 are provided, the sample chambers 28 of which are filled with sample, and the step of directing a flow 34 of a buffer fluid over the semi-permeable membranes 30 of the different sample cells 26 results in each sample cell 26 having an individual time period T that varies between the sample cells 26. j The buffer fluid is then subjected to a flow of buffer fluid 34 over the entire surface of the substrate 30 .

[0141] In a further development, the method may further comprise a step of analyzing the buffer fluid, in particular after it has been guided over the outer surface of the semipermeable membrane 30. This step may be performed in parallel with step 9.6 and / or in parallel with step 9.3. The step of analyzing the buffer fluid may be performed as described herein and with all embodiments thereof. EXAMPLES

[0142] In the following, a particular configuration of the method is described by way of example. material: Pig eyes were obtained from a local slaughterhouse in Basel, Switzerland. The drug product (monoclonal antibody (mAb)) was provided by Lonza AG. ·BSS PLUS® Irrigating Solution (catalog number 0008000048) was purchased from Alcon (Fort Worth, USA). BSS PLUS® is a sterile intraocular irrigating solution consisting of two parts. Part I is a sterile 480 mL solution in a 500 mL single dose bag to which the Part II concentrate is added. Each mL of Part I contains 7.44 mg sodium chloride, 0.395 mg potassium chloride, 0.433 mg dibasic sodium phosphate, 2.19 mg sodium bicarbonate, hydrochloric acid and / or sodium hydroxide (to adjust pH) in water for injection. Part II is a sterile concentrate in a 20 mL single dose vial for addition to Part I. Each mL of Part II contains 3.85 mg calcium chloride dihydrate, 5 mg magnesium chloride hexahydrate, 23 mg glucose, 4.6 mg glutathione disulfide (oxidized glutathione) in water for injection. After adding BSS PLUS® Solution Part II to the Part I bag, each mL of reconstituted product contains 7.14 mg sodium chloride, 0.38 mg potassium chloride, 0.154 mg calcium chloride dihydrate, 0.2 mg magnesium chloride hexahydrate, 0.42 mg sodium phosphate dibasic, 2.1 mg sodium bicarbonate, 0.92 mg glucose, 0.184 mg glutathione disulfide (oxidized glutathione), hydrochloric acid and / or sodium hydroxide (to adjust pH) in water for injection. The reconstituted product has a pH of about 7.4. The osmolality is about 305 mOsm. Ri-lac nach Hartmann solution (catalog number FV10452) was purchased from BBraun (Sempach, Switzerland). 1000 mL of solution contained 6.0 g sodium chloride, 0.40 g potassium chloride, 0.27 g calcium chloride dihydrate, 6.24 g sodium lactate as sodium lactate solution (50% w / w, equivalent to 3.12 g sodium lactate), water; (electrolyte concentrations in mmol / l: Na + 131, K + 5.4, ​​Ca 2+ 1.8, Cl -112, lactate ion 28); pH: 5.0-7.0; theoretical osmolality = 277 mOsm / L. ·Float-A-Lyzer G2 dialysis device MWCO 50 kDa 5 mL (catalog number G235058) was purchased from Spectrum Laboratories Inc. (USA). Vivaspin® (6 and 20) centrifugal concentrators equipped with 10 or 30 kDa membrane filters (catalog no. Z614483-25EA) were purchased from Sartorius (Steiheim, Germany). Sodium azide: Sigma-Aldrich (MW 65g / mol) Material no. 71289 All chemicals were of analytical grade.

[0143] Device: The test was carried out using a "USP 4 DFZ II" dissolution tester (Flow-Through Cell USP Apparatus 4 dissolution tester) from the manufacturer ERWEKA, which was configured to receive seven separate flow cell units. The different flow cell units were customized to receive a Float-A-Lyzer® G2 dialysis device from the manufacturer Spectra-Por as a sample cell with a semipermeable membrane having a MWCO of 50 kDa and a sample chamber with a volume of 5 mL to mimic the REL of the human eye (approximately 70 kDa) and its volume (approximately 4 mL).

[0144] Preparation of buffer fluid (simulating vitreous humor): To prevent the use of large amounts of extracted porcine vitreous humor (PVH), an artificial vitreous humor (VH) buffer was made and used with a similar composition as human VH for most of its electrolytes (see Table 1 below). The buffer fluid was obtained by combining two commercially available components: BSS PLUS® Irrigating Solution and Ri-lac nach Hartmann solution to reach the concentrations in Table 1. For this purpose, 1 L of BSS irrigation solution was added to a suitable beaker and 180 mL of Ri-Lac solution was added. Afterwards, 0.235 g of sodium azide was weighed and added to the buffer fluid (to obtain a final concentration of 0.02% w / v or 3.0 mmol / L therein) to prevent microbial growth despite sterile filtration, since the PVH content is very nutritious for microorganisms and may be subject to contamination for long-term stability studies. [Table 1]

[0145] The buffer fluid was constantly conditioned by subjecting it to (and storing it for the entire duration of the stability study) in a CO2 incubator (Fisherbrand®, Thermo Fisher Scientific) at 37°C and 7% CO2 and equilibrating the pH to 7.4.

[0146] Acclimatizing the buffer fluid in a CO incubator at 37° C. and 7% CO to equilibrate the pH to 7.4 prevents the pH from shifting as previously observed during PVH incubations where the buffer was not acclimated in this manner.

[0147] Porcine vitreous humor extraction: Pig eyes were kept in ice and handled with sterile materials under a laminar flow hood. The eyes were fixed and incised with a scalpel approximately 1 cm from the iris (towards the posterior segment) to reach and open the vitreous cavity. Care was taken to extract as much PVH as possible with a needleless syringe (2 mL) without opening the aqueous chamber (aqueous humors are contaminants due to their different composition). Despite a theoretical volume of approximately 4 mL, no more than 2-3 mL was extracted from the vitreous cavity because of too many tissue cell contaminants that tend to be aspirated after this volume.

[0148] Samples were then pooled and kept on ice or in a refrigerator at 5° C. at all times. The PVH was first centrifuged (3900 rpm for 20 min using an Eppendorf 581 OR Refrigerated Centrifuge) to sediment cells or tissue debris. The top fraction was then isolated and further filtered with a pressure filtration unit (Milipore) equipped with a 0.22 pm disposable polyether sulfone (PES) filter.

[0149] Due to the high viscosity of PVH and the large amount of cell debris / tissue contamination from the dissection process, the filtration process required frequent replacement of filters.

[0150] All filtered samples were aliquoted into 6 mL fractions and frozen at -80°C until use.

[0151] Preparation of Float-A-Lyzer® G2 Cassette: A Float-A-Lyzer® G2 cassette (sample cell) was opened, filled, and immersed in a 10% ethanol:water (v:v) solution for 10 minutes. The solution was then removed and the sample chamber was flushed with deionized water.

[0152] One day prior to the start of the stability study run, the PVH sample was thawed and injected (4.5 mL of PVH) into an activated Float-A-Lyzer® cassette (sample cell), which was placed into the buffer fluid contained in a beaker, and the beaker was incubated overnight at 37° C. and 7% CO2.

[0153] Specifically, each sample chamber was filled with 4 mL of extracted PVH at room temperature. To do so, the extracted PVH was filtered with a syringe (e.g., 18G, silicone oil-free) having a syringe filter with a pore size of 0.22 μm before being filled into the sample chamber of the Float-A-Lyzer® cassette. Each sample cell was then placed vertically in a beaker filled with buffer fluid, which was placed in an incubator overnight (i.e., at least 8 hours) to subject the buffer and sample cell to an atmosphere having a temperature of about 37° C. and a CO2 concentration of about 7%. During this acclimatization step, the buffer in the beaker was stirred.

[0154] The contents of each sample chamber were then checked by determining that the pH of the PVH was in the range of pH 7.2-7.4. Furthermore, it was ensured that the protein concentration of the vitreous humor was comparable in the different sample chambers. Usually, if the pH is outside the desired range, the acclimation step may be repeated for a longer duration and / or with improved stirring and gas exchange conditions, e.g., with a higher beaker volume, fresh vitreous humor, and / or a higher stirring rate. If the protein concentration in one sample chamber is significantly lower compared to the other sample chambers, this sample chamber can be exchanged by exchanging the sample cell.

[0155] Stability testing performed: On the starting day (T0), the cassettes (=sample cells) were transferred to a VH-FTC (Flow-Through Cell USP Apparatus 4 dissolution tester) and monoclonal antibody (mAb) drugs were injected into each cell (approximately 3 mg) as described in Table 2.

[0156] For this purpose, 50 μL of monoclonal antibody, pre-filtered with an 18G filter needle, was injected into six of the seven sample chambers containing conditioned PVH (conditioned in buffer fluid as described above). The time point at which the monoclonal antibody was loaded into the sample chamber was marked as T0. Each time point required the use of the entire contents of the cassette, since sampling each cell several times would disturb the osmotic equilibrium in the cassette for the subsequent time points. Therefore, at each sampling, the buffer fluid flow was stopped and the entire contents of the cassette were transferred to a glass vial for analysis. The buffer fluid flow rate applied during the test was 8 mL / min. [Table 2]

[0157] analysis: Constant pH over time The pH values ​​of the different samples were determined using a pH meter at a given temperature of 37° C. The results of the measurements are shown in FIG. 6, which shows the change in pH value of the samples over time (time points are shown in Table 2).

[0158] Specifically, the abscissa of the graph in FIG. 6 represents the test time after which the corresponding sample measurement was performed, and the ordinate represents the pH value.

[0159] As can be inferred from Figure 6, in the suggested configuration of the device as described above, the pH value of the sample was maintained within the desired range throughout the test time, i.e., within 0.5 around the initial value. These pH variations are quite small compared to the pH shifts (moving from more than 1 pH unit) observed by others (e.g., Patel et al., Eur J Pharm Biopharm. 2015;95(Pt B):407-417). This favorable result is believed to be due to the applied CO2 acclimation, which appears to maintain the pH appropriately in this configuration.

[0160] This indicates that the natural environment of the eye was adequately simulated under the applied conditions.

[0161] Total protein concentration Additionally, the total protein concentration of the different samples was determined spectrophotometrically, and the results are shown in Figure 7, which shows a plot of the change in protein concentration of the samples over time.

[0162] Specifically, the abscissa of the figure refers to the test time after the corresponding sample measurement was performed, and the ordinate of the figure refers to the total drug protein concentration (mg / mL). As can be inferred from Figure 7, the protein amount showed a gradual decrease in protein content, with a small degree in the first few days and a more pronounced decrease after 2 weeks.

[0163] All intact drugs with associated aggregates and PVH proteins larger than 50 kDa should remain in the cassette unless they are degraded to smaller precursors. This was observed in Figure 7, when the amount of protein (from both PVH and drug) decreased over time but remained close to the initial mass (at least over the first week, still very high even after 2 weeks). This means that although some products were washed out (inevitably degradation products, since the initially present LMWS of PVH is removed during the equilibration step), the majority of the drug and PVH remained in the cassette throughout the period. Thus, the drug can still interact with the PVH protein throughout this period, which should allow this model to effectively maintain the stress conditions of the PVH environment with associated physicochemical changes over several weeks.

[0164] This demonstrates that the method retains the drug within the dialysis cassette while removing degradation products, thus allowing for proper determination of the behavior of protein drugs within a simulated ocular environment.

[0165] Assessment of physicochemical stability changes of mAb drugs over time: Colorimetric and turbidimetric measurements The expected in vivo modifications of the mAb drug (due to physicochemical instability of the mAb) were captured in vitro by this method by analyzing the aggregation and precipitation behavior by colorimetric and turbidimetric assessment.

[0166] The color of the different samples was determined by colorimetric analysis. The color scale is based on Method 1 of the European Pharmacopoeia Color Standard 2.2.2, which consists of 37 coded reference color standards. "BY7" stands for "Brown / Yellow standard n°7", and "<" and ">" stand for "higher" or "lower intensity", since the closest reference standard with the highest intensity is always compared.

[0167] The turbidity of the samples was determined by performing a nephelometric method. The turbidity was measured in Nephelometric Turbidity Units (NTU).

[0168] The results of these analyses are shown in Figure 8, which shows a table showing the color and turbidity changes over time. The first row of the table refers to the turbidity measurements. The second row is the colorimetric measurements after different test durations T.

[0169] In the table shown in FIG. 8, the last column refers to the reference sample, which is a sample of vitreous humor without monoclonal antibody and which was subjected to buffer fluid flow for 3 weeks (T6).

[0170] This demonstrates that the method is sensitive enough to study the physicochemical behavior of intravitreal drugs in vivo over time in vitro.

[0171] Labeling of mAbs The mab drugs are labeled with a fluorescent dye (Alexa Fluor™ 488, Cat. No. A20000, Invitrogen™) at a molar mab:dye ratio of 1:4 according to the manufacturer of Alexa Fluor 488. Before incubating the drugs, unbound dye was removed by centrifugation (Princeton PRO SPIN, Cat. No. CS-800, Nippon Genetics Europe GmbH). The labeled drugs were injected into the PVH in the VH-FTC and analyzed using SEC HPLC with fluorescence detection (excitation at 495 nm / emission at 519 nm). This allowed an accurate assessment of the purity of the drug and allowed it to be distinguished from the PVH protein.

[0172] Stability testing during intravitreal drug development is extremely challenging because the vitreous humor is a complex biological fluid within a complex organ with many static, dynamic and metabolic barriers, and in vivo, ex vivo and in vitro models known in the art cannot adequately represent this system or allow for good assessment of drug stability changes. The above results demonstrate that, unlike previous in vitro models, the present in vitro method successfully mimics and analyzes the behavior of intravitreal drugs in extracted VH under simulated in vivo conditions.

[0173] These exemplary embodiments and items are merely examples of multiple possibilities. Therefore, the embodiments shown herein should not be understood to form limitations on these features and configurations. Any possible combination and configuration of the described features can be selected according to the scope of the present invention.

[0174] List of reference numbers 10 equipment 12 Base 14 Flow cell unit 16 Buffer Supply System 18 Cell mount 20 Casing 22 Gasket 24 Buffer Cell 26 Sample Cell 28 Sample Chamber 30 Semi-permeable membrane 32 Flow Chamber 34 Buffer fluid flow 36 Buffer fluid inlet 38 Buffer fluid outlet 40 Buffer reservoir 42 Incubator 44 Buffer Supply Line 46 Pump Unit 48 Buffer solution discharge line 50 Filter Unit 52 Air conditioning chamber 54 Tempering medium inlet 56 Tempering medium outlet

[0175] Problem to be solved by the invention The present invention further includes the following items.

[0176] 1. A method for simulating and analyzing the behavior of a substance in the eye, comprising: a) providing at least one sample cell (26) having a sample chamber (28) at least partially defined by a semipermeable membrane (30); b) filling the sample chamber (28) with a sample comprising at least one substance constituting a simulated physiological environment of the eye and a sample fluid; c) A given period (T j ), directing a flow of a buffer fluid (34) over the exterior surface of the semipermeable membrane (30); d) analyzing the sample.

[0177] 2. The method according to item 1, wherein prior to carrying out step c), the buffer fluid is acclimatized by subjecting the buffer fluid to an atmosphere of a predetermined CO2 concentration for a predetermined period of time.

[0178] 3. The method according to item 1 or 2, wherein the flow of buffer fluid in step c) is a laminar flow, preferably a continuous laminar flow, and / or the buffer fluid flow has a flow rate in the range of 6 mL / min to 10 mL / min, in particular about 8 mL / min.

[0179] 4. The method of any one of the preceding items, wherein the substance is an active agent, preferably an active agent for topical, systemic, intravitreal, intrathecal, subcutaneous, subconjunctival, retrobulbar, or intracameral administration, and the sample fluid is or simulates vitreous humor.

[0180] 5. The method further includes providing a sample fluid, the step comprising: - extracting vitreous humor from at least one isolated vertebrate eye; - centrifuging the extracted vitreous humor to separate a clear fraction of the extracted vitreous humor; - filtering the extracted vitreous humor; Optionally, mixing the extracted vitreous humor with a buffer solution to provide a sample fluid.

[0181] 6. The method according to any one of the preceding items, wherein the sample chamber (28) 5 has a volume in the range of 4 mL to 6 mL, in particular about 5 mL, and / or the semi-permeable membrane (30) has a molecular weight cut-off in the range of 50 kDa to 100 kDa, in particular about 70 kDa.

[0182] 7. The method of any one of the preceding claims, further comprising the step of labelling the substance with a fluorescent dye.

[0183] 8. As the buffer fluid is directed over the semipermeable membrane (30) of the sample cell (26), - setting a predetermined pH condition for the sample received in the sample chamber (28); 5. The method according to any one of the preceding items, comprising receiving substances diffusing from the sample chamber (28) across the semipermeable membrane into the buffer fluid, in particular precipitates or decomposition products from the sample received in the sample chamber (28).

[0184] 9. The method of any one of the preceding items, wherein the buffer fluid comprises at least one cationic species selected from the group of sodium, potassium, calcium, magnesium, at least one anionic species selected from the group of chloride, bicarbonate, phosphate, lactate, glucose, and optionally a preservative such as azide.

[0185] 10. The method of any one of the preceding items, wherein the step of analyzing the sample includes determining the physicochemical stability of at least one substance.

[0186] 11. The method of any one of the preceding items, wherein in step a) two or more sample cells are provided and the predetermined time periods (Tj) in step c) of inducing a flow of buffer fluid (34) over the outer surface of the semipermeable membrane (30) of each sample cell (26) are different for the different sample cells (26).

[0187] 12. A sample fluid prepared by extracting vitreous humor from a vertebrate eye, centrifuging the extracted vitreous fluid to separate a clear fraction of the extracted vitreous fluid, filtering the extracted vitreous fluid, and optionally acclimating the extracted vitreous fluid by subjecting the extracted vitreous fluid to an atmosphere having a predetermined CO2 concentration for a predetermined period of time.

[0188] 13. A method for providing a sample fluid for use in the method according to any one of items 1 to 11, comprising: - extracting vitreous humor from a vertebrate eye; - centrifuging the extracted vitreous humor to separate a clear fraction of the extracted vitreous humor; - filtering the extracted vitreous humor; and optionally - acclimating the extracted vitreous humor by subjecting the extracted vitreous humor to an atmosphere having a predetermined CO2 concentration for a predetermined period of time.

[0189] 14. Use of a sample fluid according to item 12 or a sample fluid prepared by the method according to item 13 in the method according to item 1.

[0190] 15. Use of a buffer fluid in the method according to any one of items 1 to 11, wherein the buffer fluid comprises sodium, potassium, calcium, magnesium, chloride, bicarbonate, phosphate, lactate, glucose, and optionally a preservative such as azide.

[0191] 16. Use of the device (10) in the method according to any one of items 1 to 11, the device being configured to stimulate the behavior of a substance in the eye, at least one sample cell (26) having a sample chamber (28) defined at least in part by a semipermeable membrane (30), the sample cell being configured to receive a sample containing substances constituting a simulated physiological environment of the eye and a sample fluid; - A given period (T j and a buffer supply system (16) configured to direct a flow of a buffer fluid (34) across the outer surface of the semipermeable membrane (30).

[0192] 17. A buffer solution is About 50 mmol / L to about 250 mmol / L of sodium, About 1.0 mmol / L to about 25 mmol / L of potassium, Approximately 0.2 mmol / L to approximately 5.0 mmol / L of calcium, Approximately 0.1 mmol / L to approximately 4.0 mmol / L of magnesium, About 25 mmol / L to about 200 mmol / L of chloride, About 5 mmol / L to about 100 mmol / L of bicarbonate, About 0.1 mmol / L to about 10 mmol / L of phosphate, About 0.8 mmol / L to about 20 mmol / L of lactate, About 0.8 mmol / L to about 20 mmol / L of glucose, Approximately 0.1 mmol / L to approximately 10 mmol / L of glutathione disulfide, 6. The method of claim 5, optionally comprising a preservative, for example, about 0.5 mmol / L to about 10 mmol / L of azide.

Claims

1. 1. A method for simulating and analyzing the behavior of a substance in the eye, comprising: a) providing at least one sample cell (26) having a sample chamber (28) at least partially defined by a semi-permeable membrane (30); b) filling said sample chamber (28) with a sample comprising at least one substance constituting a simulated physiological environment of the eye and a sample fluid; c) A predetermined period (T ｊ ), directing a flow of a buffer fluid (34) over the exterior surface of the semipermeable membrane (30); d) analyzing the sample.

2. Before carrying out step c), the buffer fluid is diluted with a predetermined amount of CO ２ The method of claim 1 , wherein the acclimation is performed by subjecting the material to an atmosphere of a concentration of 0.5% for a predetermined period of time.

3. 3. The method according to claim 1 or 2, wherein the flow of buffer fluid in step c) is a laminar flow, preferably a continuous laminar flow, and / or the buffer fluid flow has a flow rate in the range of 6 mL / min to 10 mL / min, in particular about 8 mL / min.

4. 10. The method of any one of the preceding claims, wherein the substance is an active agent, preferably an active agent for topical, systemic, intravitreal, intrathecal, subcutaneous, subconjunctival, retrobulbar or intracameral administration, and the sample fluid is or simulates vitreous humor.

5. Further comprising the step of providing the sample fluid, the step comprising: - extracting vitreous humor from at least one isolated vertebrate eye; - centrifuging the extracted vitreous humor to separate a clear fraction of the extracted vitreous humor; - filtering the extracted vitreous humor; Optionally, mixing the extracted vitreous humor with a buffer solution to provide the sample fluid.

6. 10. The method according to any one of the preceding claims, wherein the sample chamber (28) has a volume in the range of 4 mL to 6 mL, in particular about 5 mL, and / or the semi-permeable membrane (30) has a molecular weight cut-off in the range of 50 kDa to 100 kDa, in particular about 70 kDa.

7. 10. A method according to any one of the preceding claims, further comprising the step of labelling said substance with a fluorescent dye.

8. As the buffer fluid is directed over the semi-permeable membrane (30) of the sample cell (26), - setting a predetermined pH condition for the sample received in the sample chamber (28); The method according to any one of the preceding claims, wherein the buffer fluid is adapted to receive substances diffusing from the sample chamber (28) across the semi-permeable membrane into the buffer fluid, in particular precipitates or decomposition products from the sample received in the sample chamber (28).

9. 10. The method of any one of the preceding claims, wherein the buffer fluid comprises at least one cationic species selected from the group of sodium, potassium, calcium, magnesium, at least one anionic species selected from the group of chloride, bicarbonate, phosphate, lactate, glucose, and optionally a preservative such as azide.

10. 10. The method according to any one of the preceding claims, wherein the step of analysing the sample comprises determining the physico-chemical stability of the at least one substance.

11. 10. The method according to any one of the preceding claims, wherein in step a) two or more sample cells are provided and the predetermined time periods (Tj) in step c) of inducing a flow of buffer fluid (34) on the outer surface of the semipermeable membrane (30) of each sample cell (26) are different for different sample cells (26).

12. A sample fluid comprising: extracting vitreous humor from a vertebrate eye; centrifuging the extracted vitreous humor to separate a clear fraction of the extracted vitreous humor; filtering the extracted vitreous humor; and optionally subjecting the extracted vitreous fluid to a predetermined CO ２ and acclimating the extracted vitreous humor by subjecting it to an atmosphere of a concentration of 0.1% or more for a predetermined period of time.

13. A method for providing a sample fluid for use in the method according to any one of claims 1 to 11, comprising the steps of: - extracting vitreous humor from a vertebrate eye; - centrifuging the extracted vitreous humor to separate a clear fraction of the extracted vitreous humor; - filtering the extracted vitreous humor, and optionally - The extracted vitreous humor is treated with a predetermined CO ２ and acclimating the extracted vitreous humor by subjecting it to an atmosphere of a concentration of 0.1% to 1.0% for a predetermined period of time.

14. 14. Use of a sample fluid according to claim 12 or of a sample fluid prepared by the method according to claim 13 in the method according to claim 1.

15. 12. Use of a buffer fluid in the method according to any one of claims 1 to 11, said buffer fluid comprising sodium, potassium, calcium, magnesium, chloride, bicarbonate, phosphate, lactate, glucose and optionally a preservative such as azide.

16. Use of a device (10) in a method according to any one of claims 1 to 11, said device being configured to stimulate the behaviour of a substance in the eye, at least one sample cell (26) having a sample chamber (28) at least partially defined by a semipermeable membrane (30), said sample cell being configured to receive a sample containing substances constituting a simulated physiological environment of the eye and a sample fluid; - a given period (T ｊ ), and a buffer supply system (16) configured to induce a flow of a buffer fluid (34) over the outer surface of the semipermeable membrane (30).

17. The buffer solution is About 50 mmol / L to about 250 mmol / L of sodium; about 1.0 mmol / L to about 25 mmol / L of potassium; about 0.2 mmol / L to about 5.0 mmol / L calcium; about 0.1 mmol / L to about 4.0 mmol / L magnesium; about 25 mmol / L to about 200 mmol / L chloride; about 5 mmol / L to about 100 mmol / L of bicarbonate; about 0.1 mmol / L to about 10 mmol / L of phosphate; about 0.8 mmol / L to about 20 mmol / L of lactate; about 0.8 mmol / L to about 20 mmol / L glucose; about 0.1 mmol / L to about 10 mmol / L of glutathione disulfide; 6. The method of claim 5, optionally including a preservative, for example, about 0.5 mmol / L to about 10 mmol / L of azide.

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