Products suitable for the treatment of tear film deficiency in dry eye disease and methods for producing such products

Abstract

The present invention relates to a product suitable for the treatment of tear film deficiency in dry eye disease and a method for producing such a product. The present invention also relates to a product for use in the treatment of tear film deficiency in dry eye disease. The product according to the present invention improves wound healing in dry eye disease and other pathologies.

Description

Technical Field

[0001] The present invention relates to a product suitable for the treatment of tear film deficiency and corneal epithelium regeneration in dry eye disease and a method for producing the product. The present invention also relates to a product for use in medicine and a product for use in the treatment of dry eye disease. The product according to the present invention improves wound healing in dry eye disease and other pathological conditions.

Background Art

[0002] The lack of the aqueous phase of the tear film is the main causative factor leading to the main pathological condition of tear film disintegration in dry eye disease. Oxidative stress results from the disruption of the balance between the antioxidant system and the pro-oxidant system, damages the ocular surface, and increases the level of oxidized proteins due to the increased inflammatory activity during this disease, thus playing an important role in dry eye disease. Furthermore, due to the lack of integrity of the corneal epithelium, water flows into the stroma.

[0003] Serum has many uses in diagnosis and is also used in medical therapy. Serum refers to the soluble components of whole blood from which coagulation factors and all cell types have been removed, for example, by the coagulation of blood samples followed by subsequent centrifugation and filtration. Thus, serum contains essentially all proteins that do not react to blood coagulation, in addition to electrolytes, antibodies, antigens, and hormones.

[0004] Tear fluid from healthy subjects contains several antioxidants, such as ascorbic acid, lactoferrin, uric acid, and cysteine, to protect the ocular surface from certain radicals. It is speculated that the components present in human serum can provide an antioxidant effect that can be utilized for the treatment of tear film deficiency in dry eye disease.

[0005] International Publication No. WO 2022 / 029109 describes compositions and biologically active blood fractions for treating damaged epithelial surfaces in relation to eye diseases. International Publication No. WO 2022 / 029109 also describes the corresponding production methods, namely obtaining the fractions via ultracentrifugation and subsequent separation from whole serum or whole plasma. The obtained blood fractions are formulated into eye drops. The described fractions of blood obtained after ultracentrifugation of serum are biologically active and have been found to have a healing effect on damaged epithelial cells. The obtained serum fraction is substantially free of lipids and lipoproteins, which has been found to enhance its fluidity and facilitate bottling and filter sterilization for the removal of pathogens, such as bacteria.

[0006] A healthy cornea has restricted water influx (leakage) into the stroma, which is counteracted by water efflux by bicarbonate, lactate, and NaCl solutes across Descemet's membrane and the endothelial monolayer. Oxidative stress results from the breakdown of the tear film, followed by disruption of the balance between the antioxidant system and the pro-oxidant system, damaging the ocular surface and increasing the level of oxidized proteins due to increased inflammatory activity among these diseases, thus playing an important role in dry eye disease. Furthermore, the lack of integrity of the corneal epithelium allows water to flow into the stroma. The described conditions need to be treated appropriately and efficiently. In particular, to avoid long-term damage and exacerbation of dry eye disease, it is necessary to quickly close epithelial defects and stabilize the tear film.

[0007] However, pharmaceutical products known in the art do not provide sufficient regenerative capacity, namely by healing corneal epithelial defects. Therefore, the therapeutic need for novel, more active products for dry eye disease and related conditions remains unmet.

[0008] The above object is solved by the subject matter of the claims and the subject matter described below in this specification.

Summary of the Invention

[0009] In a first aspect, the present invention is a multi-donor human serum product comprising insulin-like growth factor (IGF) bound in a macromolecular complex having a molecular size of 30 kDa or more, comprising IGF-1 at 20 ng / ml or more, comprising less than 70 mg / dl of monosaccharides, and optionally being an isotonic solution or an isotonic gel, relating to a multi-donor human serum product.

[0010] In a related aspect, the present invention is a human serum product comprising insulin-like growth factor (IGF) bound in a macromolecular complex having a molecular size of 30 kDa or more, comprising IGF-1 at 20 - 100 ng / ml, comprising less than 70 mg / dl of monosaccharides, and optionally being an isotonic solution or an isotonic gel, relating to a human serum product.

[0011] The inventors have discovered that human serum contains insulin-like growth factor (IGF) bound in a macromolecular complex having a molecular size of at least 30 kDa, and that these macromolecular complexes, when incorporated intact into pharmaceutical products, provide improved wound healing. Human serum contains specific epithelial affinity factors such as IGF-1, EGF, etc. in addition to high concentrations of proteins such as albumin and fibronectin. Serum directly supports the proliferation and migration of epithelial cells or indirectly enhances epithelial survival rate by binding to and neutralizing inflammatory cytokines. Growth factors and cytokines are important regulatory factors that stimulate the growth, proliferation, migration, differentiation, adhesion of cells involved in wound healing, as well as ECM deposition and protease regulation.

[0012] In a second aspect, the present invention relates to a human serum product for use in medicine.

[0013] In a related aspect, the present invention relates to a human serum product for use in the treatment of dry eye disease.

[0014] In a third aspect, the present invention is a method for manufacturing a human serum product, comprising - Preparing a human allogeneic blood volume fraction from a plurality of donors; - Selecting volume fractions from at least two donors; - Removing serum from the selected volume fractions to obtain a serum fraction; - Pooling the selected serum fractions to obtain a pooled serum, optionally, wherein the selected serum fractions are all from donors aged 45 years or younger; - Obtaining an intermediate product from the pooled serum, wherein the intermediate product contains IGF-1 at 100 ng / ml or more; - Obtaining a human serum product according to the present disclosure from the intermediate product and relates to a method comprising the steps of.

[0015] The method relies on the step of preparing whole blood volume fractions from a plurality of donors selected in such a way as to ensure a sufficient IGF concentration. The IGF-1 concentration in human blood varies between individuals. There are volume fractions that can have very high IGFs and those that can have very low IGF-1 concentrations. By selecting volume fractions with a sufficient IGF-1 concentration, the desired IGF-1 concentration in the product can be obtained. For example, it has been found that the IGF-1 concentration in human serum decreases as the donor's lifespan increases. Therefore, restricting the donor age of a large number of donors to 45 years or younger contributes to an efficient production method because less blood has to be discarded. The method provides for obtaining an intermediate product containing IGF-1 at 100 ng / ml or more. The resulting product contains insulin-like growth factor (IGF) bound in a macromolecular complex with a molecular size of 3 kDa or more. Advantageously, the method is versatile with respect to the selection of variants, parameters and conditions for obtaining the product according to the invention from the intermediate product, thereby providing a product with improved therapeutic and anti-inflammatory properties, for example for repairing corneal epithelial defects.

[0016] In one aspect regarding the product, the present disclosure provides an intermediate product comprising insulin-like growth factor (IGF) bound in a macromolecular complex with a molecular size of 30 kDa or more, and the intermediate product contains IGF-1 at a concentration of 100 to 500 ng / ml.

Brief Description of the Drawings

[0017]

Figure 1-1

Figure 1-2

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Modes for Carrying Out the Invention

[0018] Product In a first aspect, the present invention relates to a multi-donor human serum product comprising insulin-like growth factor (IGF) bound in a macromolecular complex with a molecular size of 30 kDa or more, comprising IGF-1 at 20 ng / ml or more, comprising less than 70 mg / dl of monosaccharides, and optionally being an isotonic solution or an isotonic gel.

[0019] In a related aspect, the present invention relates to a human serum product comprising insulin-like growth factor (IGF) bound in a macromolecular complex with a molecular size of 30 kDa or more, comprising IGF-1 from 20 to 100 ng / ml, comprising less than 70 mg / dl of monosaccharides, and optionally being an isotonic solution or an isotonic gel.

[0020] When the present disclosure refers to a "human serum product", the present disclosure equally refers to both a "human serum product" and a "multi-donor human serum product".

[0021] Human serum contains several growth factors, such as insulin-like growth factors, in particular insulin-like growth factor 1 (IGF-1) and insulin-like growth factor 2 (IGF-2), as well as the binding proteins IGF-BP3 and ALS, all of which can be experimentally quantified. The inventors have found that human serum contains insulin-like growth factors bound in macromolecular complexes with a molecular size exceeding 30 kDa, and that these macromolecular complexes, when incorporated intact into pharmaceutical products, provide improved wound healing. IGF-1 and IGF-2 have molecular weights of 7.65 kDa and 7.5 kDa, respectively (referring to human proteins). Since the macromolecular complexes contain IGF-1 and / or IGF-2 together with their binding proteins, they reach a molecular size of 30 kDa or more. It has been demonstrated and / or confirmed that the macromolecular complexes remain intact in the absence of proteases and at a substantially neutral pH value, i.e., a pH of about 7. High salt conditions or the addition of organic solvents or protease inhibitors are thought to disrupt the macromolecular complexes.

[0022] In one embodiment, the macromolecular complex has a molecular size of 40 to 50 kDa. In one embodiment, the macromolecular complex has a molecular size of up to 150 kDa.

[0023] The IGF bound in the macromolecular complex also makes the active growth factor always available and improves the storage stability of the product. Monomeric IGF is more prone to degradation, which is thought to be due to the lack of its native form.

[0024] In one embodiment, the human serum product contains less than 70 mg / dl of monosaccharides, less than 40 mg / dl of monosaccharides or less than 30 mg / dl of monosaccharides. Advantageously, the human serum product contains IGF bound in a macromolecular complex that has been found to promote wound healing, in combination with a small amount of low molecular weight components typically found in human serum, namely monosaccharides. For example, for reference, whole blood from a healthy donor contains blood glucose levels of 60 - 140 mg / dl (<5.6 - 7.8 mmol / l). Low molecular weight components are generally accepted to have a molecular weight of less than 900 daltons. The concentration of low molecular weight components in serum is drastically reduced, for example, in the retentate obtained after ultracentrifugation or in the human serum product obtained by diluting human serum.

[0025] In one embodiment, the human serum product contains less than 40 mg / dl of total cholesterol, where total cholesterol is the sum of LDL (low density lipoprotein) and HDL (high density lipoprotein). Advantageously, the human serum product contains IGF bound in a macromolecular complex that has been found to promote wound healing, in combination with a certain amount of additional high molecular weight components typically found in human serum, namely cholesterol. For example, for reference, whole blood from a healthy donor contains about 150 mg / dl of total cholesterol. The high molecular weight components in serum are at least somewhat enriched with respect to their concentration in the retentate obtained after ultracentrifugation. In the human serum product obtained by diluting human serum, the concentrations of these high molecular weight components are each diluted.

[0026] In one embodiment, the human serum product contains IGF bound in a macromolecular complex with a molecular size of 30 kDa or more, the product contains IGF-1 at 20 ng / ml or more, and contains less than 70 mg / dl of monosaccharides, less than 55 mg / dl of monosaccharides, less than 40 mg / dl of monosaccharides, less than 30 mg / dl of monosaccharides, less than 20 mg / dl of monosaccharides or less than 10 mg / dl of monosaccharides.

[0027] In one embodiment, the human serum product contains IGF bound in a macromolecular complex with a molecular size of 30 kDa or more, the product contains IGF-1 at 20 to 100 ng / ml, and contains less than 70 mg / dl of monosaccharides, less than 55 mg / dl of monosaccharides, less than 40 mg / dl of monosaccharides, less than 30 mg / dl of monosaccharides, less than 20 mg / dl of monosaccharides or less than 10 mg / dl of monosaccharides.

[0028] In the context of the present disclosure, total cholesterol and urea are components of human serum, and they neither serve nor contribute to the specific functions of the human serum product. On the condition that whole blood is obtained from a healthy donor, the contents of total cholesterol and urea in the human serum product are not considered harmful.

[0029] In one embodiment, the human serum product contains less than 40 mg / dl of total cholesterol, where total cholesterol is the sum of LDL (low-density lipoprotein) and HDL (high-density lipoprotein), and is less than 35 mg / dl of total cholesterol or less than 30 mg / dl of total cholesterol. In one embodiment, the product contains 5 mg / dl or more of total cholesterol, 10 mg / dl or more of total cholesterol or 15 mg / dl or more of total cholesterol. In one embodiment, the product contains 5 to 40 mg / dl of total cholesterol, 10 to 35 mg / dl of total cholesterol or 15 to 30 mg / dl of total cholesterol.

[0030] In one embodiment, the human serum product contains LDL less than 70 mg / dl and / or HDL less than 30 mg / dl. In one embodiment, the product contains LDL of 2 to 70 mg / dl and / or HDL of 1 to 30 mg / dl. In one embodiment, the product contains LDL of 2 to 70 mg / dl, LDL of 5 to 60 mg / dl, LDL of 10 to 50 mg / dl or LDL of 15 to 40 mg / dl. In one embodiment, the product contains HDL of 1 to 30 mg / dl, HDL of 2 to 25 mg / dl or HDL of 5 to 20 mg / dl.

[0031] In one embodiment, the human serum product contains urea less than 100 mg / dL or urea less than 40 mg / dl or urea less than 10 mg / dL or urea less than 3 mg / dL.

[0032] The human serum product may be derived from human serum, preferably from allogeneic human blood. Thus, the human serum product can be easily manufactured and requires only a limited number and amount of additives. In one embodiment, the human serum product contains components and / or additives, such as vitamins, growth factors, pH adjusters, buffers, surfactants, viscosity modifiers, etc., and / or the product contains NaCl in an amount such that it is isotonic with respect to its intended pharmaceutical use.

[0033] In one embodiment, the human serum product essentially does not contain a stabilizer. In one embodiment, the human serum product essentially does not contain a preservative. It is known to those skilled in the art that a stabilizer or a preservative may need to be intentionally added if necessary. The requirement that the human serum product essentially does not contain a stabilizer and the requirement that the human serum product essentially does not contain a preservative mean that neither a stabilizer nor a preservative is intentionally added to the human serum product. Typical pharmaceutical products may require a preservative, such as benzalkonium chloride (which is irritating to the eyes), etc., but the human serum product according to the present disclosure acts in the absence of such agents. Intermediate products obtained from human allogeneic blood are less likely to be contaminated by microorganisms due to their manufacturing method, and thus can be manufactured, further processed, and stored in the absence of a preservative.

[0034] In one embodiment, the human serum product is an isotonic solution or an isotonic gel. In the context of the present invention, the human serum product has pharmaceutical uses and is used in formulations and / or pharmaceuticals. In particular, for the treatment of eye-related conditions, the tonicity or osmolality of the human serum product must be provided and / or maintained at physiological levels. Thus, those skilled in the art will understand that the reference to an isotonic solution or an isotonic gel means that the effective osmolality of the human serum product is essentially the same as that of the relevant body fluid. Thus, the solute concentration of the human serum product is adjusted to be an isotonic solution or an isotonic gel with respect to the solute concentration inside human cells or human tissues.

[0035] Osmolality refers to the concentration of osmotically active solutes in a solution, while tonicity refers to the corresponding physiological effect in the body and is also referred to as "effective osmolality".

[0036] In one embodiment, the human serum product has a pH value between 5.6 and 10.5, between 7.3 and 9.7, or about 7.4.

[0037] In one embodiment, the human serum product has a tonicity of 127 - 475 mosmol / kg, 222 - 445 mosmol / kg, 250 - 320 mosmol / kg or about 285 mosmol / kg.

[0038] In one embodiment, the human serum product has a pH value between 5.6 and 10.5 and a tonicity of 127 - 475 mosmol / kg. In one embodiment, the human serum product has a pH value between 7.3 and 9.7 and a tonicity of 222 - 445 mosmol / kg. In one embodiment, the human serum product has a pH value of about 7.4 and a tonicity of about 285 mosmol / kg. A pH value of about 7.4 and a tonicity of about 285 mosmol / kg are generally regarded as physiological concentrations and thus provide good physiological tolerance of the eye drops.

[0039] In one embodiment, the human serum product contains IGF-1 at a concentration of 20 ng / ml or more, 30 ng / ml or more, 50 ng / ml or more or 100 ng / ml or more. In one embodiment, the human serum product contains IGF-1 at a concentration of 600 ng / ml or less, 500 ng / ml or less, 400 ng / ml or less or 300 ng / ml or less. In one embodiment, the human serum product contains IGF-1 at a concentration of 20 - 600 ng / ml, 30 - 500 ng / ml, 50 - 400 ng / ml or 100 - 300 ng / ml. The presence of IGF-1 in the human serum product contributes to the wound healing properties of the product.

[0040] Insulin-like growth factor-binding protein 3 (IGF-BP3) is a protein encoded by the hIGFBP3 gene. IGF-BP3 is one of six IGF-binding proteins (IGF-BP1 - IGF-BP6) that bind with high affinity to the insulin-like growth factors IGF-1 and IGF-2. IGFBP-3 functions in the blood circulation, extracellular environment and inside cells. It is considered the major IGF transport protein in the blood stream, which transports the growth factor mainly in a stable complex in the blood stream that contains this binding protein, either IGF-1 or IGF-2, and a third protein called the acid-labile subunit (ALS).

[0041] In one embodiment, the human serum product comprises IGF-BP3 at 1300 ng / ml or more, 2000 ng / ml or more, or 3000 ng / ml or more. In one embodiment, the human serum product comprises IGF-BP3 at 8000 ng / ml or less, 7000 ng / ml or less, or 6000 ng / ml or less. In one embodiment, the product comprises IGF-BP3 at 1300 - 8000 ng / ml, 2000 - 7000 ng / ml, or 3000 - 6000 ng / ml.

[0042] In one embodiment, the human serum product comprises the molar ratio of monomer (i.e., free or unbound) IGF-1 to IGF-1 bound in a macromolecular complex (of a molecular size of 30 kDa or more) of 1 / 1000 - 20 / 1000, 2 / 1000 - 15 / 1000, or 3 / 1000 - 10 / 1000. In one embodiment, the human serum product comprises the molar ratio of monomer (i.e., free or unbound) IGF-1 to IGF-1 bound in a macromolecular complex (of a molecular size of 30 kDa or more) of 20 / 1000 or less, 15 / 1000 or less, or 10 / 1000 or less.

[0043] In one embodiment, the human serum product comprises IGF bound in a macromolecular complex of a molecular size of 30 kDa or more, and the product comprises IGF-1 at 20 - 600 ng / ml and IGF-1 bound to IGF-BP3 at a concentration of IGF-BP3 of 1300 - 8000 ng / ml, 2000 - 7000 ng / ml, or 3000 - 6000 ng / ml.

[0044] In one embodiment, the human serum product contains IGF bound in macromolecular complexes with a molecular size of 30 kDa or more, and the product contains IGF-1 bound to IGF-BP3 at concentrations of 20 - 600 ng / ml, 30 - 500 ng / ml, 50 - 400 ng / ml, or 100 - 300 ng / ml of IGF-1 and 1300 - 8000 ng / ml of IGF-BP3. The concentration of IGF-1 refers to the total concentration of IGF-1 protein in the human serum product, i.e., the sum of the concentrations in bound and unbound (free) forms.

[0045] In one embodiment, the human serum product has a wound closure ability of 50% or more in 35 hours, normalized to the area of the injury, in an in vitro wound closure assay. This means that 50% or more of the initial wound area is closed after 35 hours.

[0046] In one embodiment, the human serum product has a wound closure ability of 50% or more in 21 hours, normalized to the area of the injury, in an in vitro wound closure assay. This means that 50% or more of the initial wound area is closed after 21 hours.

[0047] The wound closure ability is based on an in vitro wound closure assay that monitors human corneal epithelial cells (HCE-T cells) over time, and the readout for the reduction (%) of the wound area is based on HCE-T cells with distinct micro-injuries (scratch wounds). A detailed description is provided in the experimental section of this application.

[0048] The in vitro wound closure assay is useful, for example, in mimicking dry eye disease and other pathologies, and monitors wound closure with respect to negative and positive controls, where the negative control is based on serum-free KGM (keratinocyte growth medium - 2), and the positive control is based on KGM supplemented with bovine pituitary extract (i.e., 10 μg / ml).

[0049] In one embodiment, the in vitro wound closure assay includes: (a) preparing an in vitro cell culture containing HCE-T cells; (b) creating a micro-injury, such as a scratch, on the (mono)layer of HCE-T cells; (c) adding a human serum product according to the present invention; (d) monitoring the in vitro cell culture, for example, by an optical microscope; and (e) quantifying time-dependent wound closure as a wound closure ability by calculating the difference in the percentage of the wound area at various time points.

[0050] The quantification of time-dependent wound closure can be performed or assisted by, for example, image analysis software.

[0051] In one embodiment, the human serum product has a wound closure ability of 50% or more, 55% or more, 60% or more, or 65% or more at 35 hours, normalized to the area of the injury, in the in vitro wound closure assay. In one embodiment, the human serum product has a wound closure ability of 90% or less, 85% or less, 80% or less, or 75% or less at 35 hours, normalized to the area of the injury, in the in vitro wound closure assay. In one embodiment, the human serum product has a wound closure ability of 50% - 90%, 55% - 85%, 60% - 80%, or 65% - 75% at 35 hours, normalized to the area of the injury, in the in vitro wound closure assay.

[0052] In one embodiment, the human serum product has a wound closure ability of 50% or more, 55% or more, 60% or more, or 65% or more at 21 hours, normalized to the area of the injury, in the in vitro wound closure assay. In one embodiment, the human serum product has a wound closure ability of 90% or less, 85% or less, 80% or less, or 75% or less at 21 hours, normalized to the area of the injury, in the in vitro wound closure assay. In one embodiment, the human serum product has a wound closure ability of 50% - 90%, 55% - 85%, 60% - 80%, or 6 5% - 75% at 21 hours, normalized to the area of the injury, in the in vitro wound closure assay.

[0053] In one embodiment, the human serum product has anti-inflammatory capacity in an inflammation assay, and the anti-inflammatory capacity is normalized against the normalized expression of LPS-induced TNF-α (as a reference value) and is 50% or more, and / or the human serum product contains C-reactive protein (CRP) less than 0.2 mg / dl.

[0054] The anti-inflammatory capacity in the inflammation assay is - preparing macrophages from at least 5 donors; - combining the isolated macrophages; - stimulating the combined macrophages with LPS (lipopolysaccharide) to induce the secretion of TNF-α (this serves as a reference value for inflammation); - assaying the human serum product in triplicate when stimulating the combined macrophages with LPS (this serves as the value for anti-inflammatory capacity); - assaying prednisolone, preferably prednisolone acetate (1.0% by weight), when stimulating the combined macrophages with LPS (this serves as the value for 100% anti-inflammatory capacity, i.e., the positive control); - assaying the medium when stimulating the combined macrophages with LPS (this serves as the value for 0% anti-inflammatory capacity, i.e., the negative control); and - quantifying the anti-inflammatory capacity of the human serum product in % by normalizing against the positive and negative controls as determined by.

[0055] In one embodiment, the human serum product contains CRP less than 0.2 mg / dl, less than 0.06 mg / dl, or less than 0.02 mg / dl. In one embodiment, the human serum product contains CRP of 0.0002 mg / dl or more, 0.0006 mg / dl or more, or 0.002 mg / dl or more. In one embodiment, the product contains CRP of 0.0002 - 0.2 mg / dl, 0.0006 - 0.06 mg / dl, or 0.002 - 0.02 mg / dl.

[0056] CRP can be detected in blood and serum in response to inflammation. In particular, after inflammation, CRP levels rise in human (or mammalian) blood. Advantageously, the product contains low amounts of CRP that avoid and / or reduce the induction of the inflammatory response.

[0057] The human serum composition may be derived from human serum, preferably from human allogeneic blood. There are several ways to determine that a serum product is obtained from more than one donor, i.e., that it is a multi-donor serum product. In the present disclosure, reference to a "pooled" serum product means that it is derived from more than one donor. Multi-donor human serum products are characterized by one or more of the following criteria: - The presence of antigens indicating that the human serum composition is derived from more than one donor when classified by the International Society of Blood Transfusion (ISBT); - The presence of soluble HLA class I molecules indicating that the human serum composition is derived from more than one donor; and - The presence of glycolipids indicating that the human serum composition is derived from more than one donor.

[0058] In one embodiment, the presence of HLA class I molecules indicating that the human serum product is derived from more than one donor is - preparing the human serum product; - purifying soluble HLA class I molecules such as sHLA-A, sHLA-B, sHLA-C from the human serum composition by affinity chromatography using, for example, polyclonal anti-HLA-A, anti-HLA-B, and anti-HLA-C antibodies to obtain an HLA eluate; - purifying the HLA eluate by, for example, gel permeation chromatography to obtain a purified eluate; - detecting soluble HLA class I molecules by Western blotting using anti-HLA-A, anti-HLA-B, and anti-HLA-C antibodies; - performing trypsin digestion on the soluble HLA class I molecules; - Optionally, extracting trypsin-digested soluble HLA class I molecules; - Analyzing the trypsin-digested soluble HLA class I molecules by mass spectrometry (MS) based on a method comprising.

[0059] Methods for protein isolation, purification and extraction are known to those skilled in the art. It is also known to those skilled in the art that Western blotting requires, for example by SDS-PAGE, pre-separating proteins or peptides according to their sizes. In one embodiment, the extract of trypsin-digested peptides was dried by speed vacuum and resuspended in a buffer suitable for MS analysis.

[0060] Advantageously, in multi-donor human serum products, the quantitative amounts of all serum components are made uniform, and thus donor-to-donor variability in serum components is balanced. Potentially harmful serum components, such as donor antibodies, which can induce a patient reaction, are diluted, thereby minimizing any potential risks and / or adverse reactions on the patient side. Thereby, a more standardized product can be obtained or provided than a similar product based on a human serum composition derived from only one donor. From the point of view of product manufacture, the use of human serum from two or more donors also allows for scaling up of the method, and as a result, larger amounts of product can be manufactured. Advantageously, therefore, working from multi-donor serum enables the manufacture and storage of serum-based eye drops as a finished drug.

[0061] It is also advantageous that serum obtained from healthy donors can be screened and / or optimized with respect to serum components, for example in relation to tolerance and efficacy, for example screening and / or optimizing with respect to establishing a well-defined concentration range of growth factors.

[0062] Several observable factors, such as the presence of various antigens indicating origin from donors with different blood types, enable the evaluation and discrimination of whether a human serum composition is derived from two or more donors. For example, based on antigen analysis, the International Society of Blood Transfusion currently recognizes 43 different blood group systems. Additionally, the presence of glycolipids may indicate that a human serum composition is derived from two or more donors.

[0063] In one embodiment, the human serum product comprises one or more growth factors selected from the list of EGF, IGF-1, IGF-BP3, IGF-2, PDGF-BB, alpha2-macroglobulin, KGF, and PEDF. Advantageously, the growth factors can stimulate cell proliferation, particularly wound healing. The growth factors may be added to the human serum product as recombinant proteins. However, these growth factors may also be essentially present in the human serum composition.

[0064] In one embodiment, the human serum product comprises one or more, particularly all, of the following growth factors in the following concentration ranges: - EGF at 50 - 500 pg / ml or 20 - 200 pg / ml, optionally quantified according to the EGF-Quantikine Elisa Assay (catalog number DEG00) from Bio-Techne Ltd.; - IGF-1 at 50 - 250 ng / ml or 20 - 100 ng / ml, optionally quantified according to the Diasorin Liaison IGF-1 chemiluminescent assay; - IGF-BP3 at 650 - 4000 ng / ml or 260 - 1600 ng / ml, optionally quantified according to a ligand-binding immunoassay; - ALS at 450 - 6000 ng / ml or 180 - 2400 ng / ml, optionally quantified according to the IGFBP-ALS R&D Elisa (my bioresearch, catalog number MBS450295); - Optionally quantified according to Elisa IMD Potsdam, with alpha 2-macroglobulin ranging from 0.37 to 1.49 g / l or alpha 2-macroglobulin ranging from 0.15 to 0.6 g / l; - Optionally quantified according to Bio-Techne Ltd.'s Human Quanitikine Elisa (Catalog No. DBB00), with PDGF-BB ranging from 471 to 3683 pg / ml or PDGF-BB ranging from 188.4 to 1473.2 pg / ml; - Optionally quantified according to R&D Systems, Inc.'s Quantikine® ELISA (Catalog No. DKG00), with KGF ranging from 2 to 100 pg / ml; - Optionally quantified according to the IMD Berlin TGF-beta-1 IVD assay, with TGF-β1 ranging from 1 to 30 ng / ml or TGF-β1 ranging from 0.4 to 12 ng / ml; - Optionally quantified according to IMD Berlin, with VEGF ranging from 30 to 222.5 pg / ml or VEGF ranging from 12 to 89 pg / ml; - Optionally quantified according to the ELISA kit of Bioassay Technology Laboratory R&D, with PEDF ranging from 60 to 5000 ng / ml or PEDF ranging from 24 to 2000 ng / ml; and - Optionally quantified according to MVZ Labor Volkmann Karlsruhe, with albumin ranging from 1750 to 2600 mg / dL or albumin ranging from 700 to 1040 mg / dL.

[0065] In one embodiment, the human serum product optionally contains EGF ranging from 50 to 500 pg / ml or EGF ranging from 100 to 250 pg / ml or EGF ranging from 20 to 200 pg / ml or EGF ranging from 40 to 150 pg / ml, quantified according to Bio-Techne Ltd.'s EGF-Quantikine Elisa Assay (Catalog No. DEG00).

[0066] In one embodiment, the human serum product is optionally quantified according to the Diasorin Liaison IGF-1 chemiluminescent assay and contains IGF-1 of 50-250 ng / ml or IGF-1 of 70-200 ng / ml or IGF-1 of 20-100 ng / ml or IGF-1 of 30-80 ng / ml.

[0067] In one embodiment, the human serum product is optionally quantified according to a ligand-binding immunoassay and contains IGF-BP3 of 650-4000 ng / ml, IGF-BP3 of 1000-3000 ng / ml, IGF-BP3 of 260-1600 ng / ml or IGF-BP3 of 500-1200 ng / ml.

[0068] In one embodiment, the human serum product is optionally quantified according to IGFBP-ALS R&D Elisa (my bioresearch, catalog number MBS450295) and contains ALS of 450-6000 ng / ml, ALS of 1000-4000 ng / ml, ALS of 180-2400 ng / ml or ALS of 500-1500 ng / ml.

[0069] In one embodiment, the human serum product is optionally quantified according to Elisa IMD Potsdam and contains alpha2-macroglobulin of 0.37-1.49 g / l, alpha2-macroglobulin of 0.55-1.20 g / l, alpha2-macroglobulin of 0.15-0.6 g / l or alpha2-macroglobulin of 0.2-0.5 g / l.

[0070] In one embodiment, the human serum product is optionally quantified according to the Human Quanitikine Elisa of Bio-Techne Ltd. (catalog number DBB00) and contains PDGF-BB of 471-3683 pg / ml, PDGF-BB of 1000-3000 pg / ml, PDGF-BB of 188.4-1473.2 pg / ml or PDGF-BB of 400-1200 pg / ml.

[0071] In one embodiment, the human serum product is optionally quantified according to R&D Systems, Inc.'s Quantikine® ELISA (Catalog No. DKG00) and contains 2 - 100 pg / ml of KGF, 5 - 70 pg / ml of KGF, or 10 - 50 pg / ml of KGF.

[0072] In one embodiment, the human serum product is optionally quantified according to the IMD Berlin TGF-beta-1 IVD assay and contains 1 - 30 ng / ml of TGF-β1, 3 - 20 ng / ml of TGF-β1, 0.4 - 12 ng / ml of TGF-β1, or 1.0 - 8 ng / ml of TGF-β1.

[0073] In one embodiment, the human serum product is optionally quantified according to IMD Berlin and contains 30 - 222.5 pg / ml of VEGF, 50 - 150 pg / ml of VEGF, 12 - 89 pg / ml of VEGF, or 20 - 60 pg / ml of VEGF.

[0074] In one embodiment, the human serum product is optionally quantified according to the ELISA kit of Bioassay Technology Laboratory R&D and contains 60 - 5000 ng / ml of PEDF, 300 - 3000 ng / ml of PEDF, 24 - 2000 ng / ml of PEDF, or 120 - 1200 ng / ml of PEDF.

[0075] In one embodiment, the human serum product is optionally quantified according to MVZ Labor Volkmann Karlsruhe and contains 1750 - 2600 mg / dl of albumin or 700 - 1040 mg / dl of albumin.

[0076] Those skilled in the art recognize that enzyme-linked immunosorbent assays are the result of many other (sometimes unappreciated) factors that contribute to the intended design choices and quantitative results. Therefore, it is essential to create a calibration curve to normalize, i.e., correct, the readout values.

[0077] Conveniently, albumin prevents proteins, such as growth factors, from precipitating in aqueous solutions and also acts as a radical scavenger with antioxidant properties during storage. Albumin also prevents hydrophobic interactions between individual serum components and hydrophobic interactions with the surface of pharmaceutical containers.

[0078] In one embodiment, the multi-donor human serum product contains insulin-like growth factor (IGF) bound in macromolecular complexes with a molecular size of 30 kDa or more, the product contains IGF-1 at 20 ng / ml or more, the product contains less than 70 mg / dl of monosaccharides, optionally, the product is an isotonic solution or an isotonic gel, the product has a wound closure ability of 50% or more in 35 hours, normalized to the area of injury, in an in vitro wound closure assay, and the product contains one or more, particularly all, of the following growth factors in the following concentration ranges: - EGF at 50 - 500 pg / ml, optionally quantified according to the EGF-Quantikine Elisa Assay (catalog number DEG00) of Bio-Techne Ltd.; - IGF-1 at 50 - 250 ng / ml, optionally quantified according to the Diasorin Liaison IGF-1 chemiluminescent assay; - IGF-BP3 at 650 - 4000 ng / ml, optionally quantified according to a ligand-binding immunoassay; - ALS at 450 - 6000 ng / ml, optionally quantified according to the IGFBP-ALS R&D Elisa (my bioresearch, catalog number MBS450295); - Alpha2-macroglobulin at 0.37 - 1.49 g / l, optionally quantified according to the Elisa IMD Potsdam; - PDGF-BB at 471 - 3683 pg / ml, optionally quantified according to the Human Quanitikine Elisa (catalog number DBB00) of Bio-Techne Ltd.; - Optionally, quantified according to R&D Systems, Inc.'s Quantikine® ELISA (Catalog No. DKG00), 2 - 100 pg / ml of KGF; - Optionally, quantified according to the IMD Berlin TGF-beta-1 IVD assay, 1 - 30 ng / ml of TGF-β1; - Optionally, quantified according to IMD Berlin, 30 - 222.5 pg / ml of VEGF; - Optionally, quantified according to the ELISA kit of Bioassay Technology Laboratory R&D, 60 - 5000 ng / ml of PEDF; and - Optionally, quantified according to MVZ Labor Volkmann Karlsruhe, 1750 - 2600 mg / dl of albumin.

[0079] In one embodiment, the multi-donor human serum product comprises insulin-like growth factor (IGF) bound in a macromolecular complex with a molecular size of 30 kDa or more, the product comprises IGF-1 at 20 ng / ml or more, the product comprises less than 70 mg / dl of monosaccharides, optionally, the product is an isotonic solution or an isotonic gel, the product has a wound closure ability of 50% or more in 35 hours, normalized to the area of the injury, in an in vitro wound closure assay, and the product comprises one or more, particularly all, of the following growth factors in the following concentration ranges: - Optionally, quantified according to Bio-Techne Ltd.'s EGF-Quantikine Elisa Assay (Catalog No. DEG00), 20 - 200 pg / ml of EGF; - Optionally, quantified according to the Diasorin Liaison IGF-1 chemiluminescent assay, 20 - 100 ng / ml of IGF-1; - Optionally, quantified according to a ligand-binding immunoassay, 260 - 1600 ng / ml of IGF-BP3; - Optionally, quantify according to IGFBP-ALS R&D Elisa (my bioresearch, catalog number MBS450295) to obtain ALS in the range of 180 - 2400 ng / ml; - Optionally, quantify according to Elisa IMD Potsdam to obtain alpha2-macroglobulin in the range of 0.15 - 0.6 g / l; - Optionally, quantify according to Human Quanitikine Elisa (catalog number DBB00) of Bio-Techne Ltd. to obtain PDGF-BB in the range of 188.4 - 1473.2 pg / ml; - Optionally, quantify according to Quantikine® ELISA (catalog number DKG00) of R&D Systems, Inc. to obtain KGF in the range of 2 - 100 pg / ml; - Optionally, quantify according to IMD Berlin TGF-beta-1 IVD assay to obtain TGF-β1 in the range of 0.4 - 12 ng / ml; - Optionally, quantify according to IMD Berlin to obtain VEGF in the range of 12 - 89 pg / ml; - Optionally, quantify according to the ELISA kit of Bioassay Technology Laboratory R&D to obtain PEDF in the range of 24 - 2000 ng / ml; and - Optionally, quantify according to MVZ Labor Volkmann Karlsruhe to obtain albumin in the range of 700 - 1040 mg / dl.

[0080] In one embodiment, the human serum product contains insulin-like growth factor (IGF) bound in a macromolecular complex with a molecular size of 30 kDa or more. The product contains 20 - 100 ng / ml of IGF-1, the product contains less than 70 mg / dl of monosaccharides, and optionally, the product is an isotonic solution or an isotonic gel. The product has a wound closure ability of 50% or more in 35 hours, normalized to the area of the injury, in an in vitro wound closure assay. The product contains one or more, particularly all, of the following growth factors in the following concentration ranges.

[0081] Medical Use In a second aspect, the present invention relates to a product for use in medicine.

[0082] By virtue of its composition, the human serum product according to the invention stimulates cell proliferation and exhibits epithelial defect healing properties. Thus, the human serum product can be used for medical and pharmaceutical purposes, for example after inflammation and / or injury. Similarly, the human serum product can be used after surgery to assist and / or promote recovery.

[0083] In a related aspect, the present invention relates to a human serum product for use in the treatment of dry eye disease.

[0084] In one embodiment, the human serum product according to the present disclosure is for use in the treatment of macular degeneration, epithelial corneal injury, conditions resulting from or after corneal transplantation, conditions related to or resulting from impaired wound healing, or conditions related to or resulting from laser treatment of the eye.

[0085] The inventors have shown that the human serum product has superior performance compared to current compositions and provides improved epithelial defect healing ability (Figures 2A - 4). The assays described have been shown to be useful models of dry eye disease and related pathologies (Hahne M, Reichl S, Simulation of corneal epithelial injuries by mechanical and corrosive damage: Influence of fetal bovine serum and dexpanthenol on epithelial regeneration in a cell culture model, Ophthalmologe 107, 2010, 529 - 532, and 534 - 536). Thus, the product provides an improved therapy for dry eye disease and related pathologies.

[0086] In one embodiment of a human serum product for use in medicine or a human serum product for use in the treatment of dry eye disease, the human serum product is derived from allogeneic blood.

[0087] In one embodiment of a human serum product for use in the treatment of macular degeneration, epithelial corneal damage, conditions resulting from or following corneal transplantation, conditions related to or resulting from impaired wound healing, or conditions related to or resulting from laser treatment of the eye, the human serum product is derived from allogeneic blood.

[0088] Allogeneic blood can be obtained by preparing and / or pooling blood from a number of donors. Such allogeneic donations are advantageous for the manufacture of pharmaceuticals, i.e., pharmaceutical products, because they balance compositional variations between blood donors and enable scale-up of production. Furthermore, blood can be selected from a number of donors to obtain the desired concentrations shown herein. For example, the blood and / or serum of a single donor can be examined for IGF content and, if the IGF level is present at a minimum, it can be used for pooling.

[0089] In one aspect, the present invention relates to an ophthalmic composition, such as an eye drop or a gel, containing the product. Eye drops and gels correspond to typical formulations useful, for example, in the treatment of dry eye disease or related or similar conditions.

[0090] Method for manufacturing the product In one aspect, the present invention is - a step of preparing a human allogeneic blood volume fraction from a number of donors, - a step of selecting volume fractions from at least two donors, - a step of removing serum from the selected volume fractions to obtain a serum fraction, - A step of pooling the selected serum portions to obtain a pooled serum, optionally, where the selected serum portions are all from donors aged 45 years or younger, and - A step of obtaining an intermediate product from the pooled serum, where the intermediate product contains IGF-1 at 100 ng / ml or more, and - A step of obtaining a human serum product according to the present disclosure from the intermediate product and relates to a method for manufacturing a human serum product.

[0091] This method relies on preparing whole blood from a number of human donors, and the whole blood is selected and pooled in such a way as to ensure a sufficient IGF concentration such that the intermediate product contains IGF-1 at 100 ng / ml or more. It has been demonstrated that the IGF-1 concentration in human allogeneic blood varies significantly and decreases as the donor's lifespan extends (Figure 1A). Therefore, restricting the donor age of a number of donors to 45 years or younger contributes to ensuring an efficient process as it reduces the volume portion that has to be discarded. The selection step ensures a sufficient IGF-1 concentration in the human serum product.

[0092] In one embodiment of the method, the intermediate product is serum, and the step of obtaining the intermediate product includes the step of preparing serum from human allogeneic blood.

[0093] In one embodiment of the method, the intermediate product is the retentate of an ultrafiltration process, and the step of obtaining the intermediate product includes the step of preparing serum from human allogeneic blood, the step of ultrafiltering the serum to obtain a filtrate and a retentate, and the step of selecting the retentate.

[0094] In one embodiment of the method, the step of obtaining the human serum product includes the step of diluting the intermediate product.

[0095] In one embodiment, the dilution of the intermediate product comprises dilution of the intermediate product, such as serum (derived from allogeneic human blood), to 10 - 30% by volume or 15 - 25% by volume or about 20% by volume in an essentially isotonic solution, such as an NaCl solution.

[0096] In one embodiment, the product can be obtained, for example, by adding a recombinant protein such as a recombinant growth factor.

[0097] In one embodiment of the method, the ultrafiltration process is carried out using a molecular cut-off membrane of at least 20 kDa or at least 30 kDa or at least 50 kDa or at least 100 kDa. It has been found that the retentate contains insulin-like growth factor (IGF) bound in macromolecular complexes with a molecular size of 30 kDa or more. Advantageously, the method provides for the acquisition of a product with improved therapeutic and anti-inflammatory properties, for example, for wound healing.

[0098] In one embodiment of the method, the obtained product has anti-inflammatory ability in an inflammation assay, and the anti-inflammatory ability is normalized to be 50% or more relative to the normalized expression of LPS-induced TNF-α (as a reference value), and / or the product contains less than 0.2 mg / dl of C-reactive protein (CRP).

[0099] In one embodiment, the step of obtaining serum from allogeneic human blood includes the step of enabling coagulation, also referred to as clotting, and the step of removing blood clots and any cells present in the blood. Optionally, the step of removing blood clots and any cells present in the blood is performed by centrifugation. The resulting pale yellow liquid supernatant is the serum.

[0100] In an alternative embodiment, the step of obtaining serum from human allogeneic blood comprises the steps of preparing a volume portion of human allogeneic blood from a number of donors, selecting volume portions from at least two donors, obtaining a serum portion from the selected volume portions, and pooling the selected serum portions, optionally wherein the selected volume portions are all from donors aged 45 years or less. In this alternative embodiment, serum rather than blood is pooled. Other than this change, the remaining method steps may be the same as those of the method according to other embodiments of the present disclosure.

[0101] In one embodiment, the step of obtaining the product comprises adding a solution to the intermediate product, the solution optionally comprising components and / or additives such as vitamins, growth factors, pH adjusters, buffers, surfactants, viscosity modifiers, etc., and / or the solution comprising NaCl in an amount such that the product is isotonic with respect to its intended pharmaceutical use.

[0102] The method according to the present disclosure is advantageous as the resulting product does not require the addition of a stabilizer as part of its manufacture. Typical pharmaceutical products may require preservatives such as benzalkonium chloride, which is irritating to the eye, but the method according to the present disclosure operates in the absence of such agents. The intermediate product obtained from human allogeneic blood is less prone to microbial contamination due to its method of manufacture and can thus be manufactured, further processed, and stored in the absence of preservatives.

[0103] In one embodiment - preparing a volume portion of human allogeneic blood from a number of donors, - selecting volume portions from at least two donors, - removing serum from the selected volume portions to obtain a serum portion, - pooling the selected serum portions to obtain a pooled serum, optionally wherein the selected serum portions are all from donors aged 45 years or less, - A step of obtaining an intermediate product from pooled serum, wherein the intermediate product contains IGF-1 at 100 ng / ml or more, and - A step of obtaining a human serum product according to the present disclosure from the intermediate product in the form of an alginate dressing, and A method for producing a product is provided, which includes the above steps.

[0104] Advantageously, the alginate dressing allows for the sustained release of active serum components and / or recombinant growth factors that can be added to the alginate dressing. The alginate dressing acts as a natural wound dressing and contains a carbohydrate source that cannot be metabolized by humans. The alginate is in the form of an isotonic gel, the alginate dressing is soft, and has thixotropic properties. Cells and / or growth factors can be encapsulated and / or incorporated into the isotonic gel, respectively. To maintain a certain dosing profile in the alginate, the use of an ocular insert provides a feasible way to deliver the active ingredient to the eye over a period of several hours in a controlled manner. The insert is placed in the lower part of the eye. Due to its solubility in water and the ease of cross-linking at room temperature through the addition of Ca 2+ Sodium alginate is preferably selected as the matrix material due to its solubility in water and the ease of cross-linking at room temperature through the addition of Ca ions.

[0105] In one embodiment of the method, a number of donors are all under 45 years old, under 40 years old, preferably under 30 years old. In one embodiment of the method, a number of donors are all 18 years old or older or 20 years old or older. In one embodiment of the method, a number of donors are all 18 to 45 years old, 18 to 40 years old or 20 to 30 years old. It is advantageous to select donors who have a sufficiently high IGF-1 concentration in their human serum due to the donor's age (see Figure 1A).

[0106] In one embodiment of the method, the intermediate product contains monosaccharides less than 140 mg / dl and / or total cholesterol of 10 to 200 mg / dl, and the total cholesterol is the sum of LDL (low-density lipoprotein), HDL (high-density lipoprotein) and VLDL (very-low-density lipoprotein).

[0107] In one embodiment, the method includes a virus removal step by, for example, filtration and / or inactivation of the virus.

[0108] Intermediate product In one aspect regarding the human serum product, the present disclosure provides an intermediate product containing insulin-like growth factor (IGF) bound in a macromolecular complex with a molecular size of 30 kDa or more, and containing IGF-1 of 100 to 500 ng / ml.

[0109] In one embodiment, the intermediate product contains IGF-1 at a concentration of 100 ng / ml or more, 150 ng / ml or more, 250 ng / ml or more, or 500 ng / ml or more. In one embodiment, the intermediate product contains IGF-1 at a concentration of 3000 ng / ml or less, 2500 ng / ml or less, 2000 ng / ml or less, or 1500 ng / ml or less. In one embodiment, the intermediate product contains IGF-1 at a concentration of 100 to 3000 ng / ml, 150 to 2500 ng / ml, 250 to 2000 ng / ml, or 500 to 1500 ng / ml.

[0110] In one embodiment, the intermediate product contains IGF-BP3 of 6500 ng / ml or more, 10000 ng / ml or more, or 15000 ng / ml or more. In one embodiment, the intermediate product contains IGF-BP3 of 40000 ng / ml or less, 35000 ng / ml or less, or 30000 ng / ml or less. In one embodiment, the intermediate product contains IGF-BP3 of 6500 to 40000 ng / ml, 10000 to 35000 ng / ml, or 15000 to 30000 ng / ml.

[0111] In one embodiment, the intermediate product comprises IGF bound in a macromolecular complex having a molecular size of 30 kDa or more, and the product comprises IGF-1 at a concentration of 100 to 3000 ng / ml and IGF-BP3 at a concentration of 6500 to 40000 ng / ml, 10000 to 35000 ng / ml or 15000 to 30000 ng / ml, and comprises IGF-1 bound to IGF-BP3.

[0112] In one embodiment, the intermediate product comprises IGF bound in a macromolecular complex having a molecular size of 30 kDa or more, and the product comprises IGF-1 at a concentration of 100 to 3000 ng / ml, 150 to 2500 ng / ml, 250 to 2000 ng / ml or 500 to 1500 ng / ml and IGF-BP3 at a concentration of 6500 to 40000 ng / ml, and comprises IGF-1 bound to IGF-BP3.

[0113] Detailed Description of the Drawings Figures 1A and 1B respectively show the age-dependent IGF-1 concentration and IGF-BP3 concentration in human serum, demonstrating that as the lifespan of the donor increases, the IGF-1 concentration in human serum decreases, while IGF-BP3 decreases only slightly throughout age. Age is indicated in years. The round circles show clinical diagnostic data, and the squares show published data (Growth Hormone & IGF Research 18 (2008): 228-237). This data advocates restricting the age of blood donors to less than 45 years old. Serum pools adjusted to a concentration of IGF-1 from over 130 ng / ml to 280 ng / ml of IGF-1 were stable up to 24 months when monitored by the concentration of active IGF-1 (+ / -20%).

[0114] Figure 2 shows the activity of serum fractions in an in vitro wound closure assay. The in vitro wound closure assay monitors human corneal epithelial cells (HCE-T cells) over time, and the readout for reduction in wound area (% units) is based on HCE-T cells with well-defined microlesions (scratches). Ultrafiltration of pooled serum separated low molecular weight contents (in the filtrate) from high molecular weight contents (in the retentate). The data show that, compared to serum-free, unfiltered serum and negative controls, the high molecular weight fraction results in better, i.e., faster, wound healing than the low molecular weight fraction (Figure 2A). Two independent experiments 1 and 2 confirmed different activities between the high molecular weight and low molecular weight fractions (Figure 2B).

[0115] Figure 3 shows the effect on wound closure of five different (recombinant) human (rh) growth factors, namely EGF (Figure 3A), IGF-1 (Figure 3B), PDGF (Figure 3C) and KGF (Figure 3D), when combined with either the high molecular weight (HMW) fraction or the low molecular weight fraction (LMW) of ultrafiltered pooled human serum, respectively. The effect was determined as wound closure over the next 50 hours. The results show that the potency to induce corneal epithelial cell migration and proliferation is similar for serum and the HMW fraction (90% wound closure was observed at the 28-hour time point), but no wound closure effect was observed for the LMW fraction. These results indicate that the biologically active components are mainly present in the HMW fraction.

[0116] List the single rh-growth factors in increasing order of their effect on wound closure - PDGF; KGF; IGF-1 and EGF. Wound closure is further promoted when the rh-growth factors are combined with HMW and, surprisingly, also with LMW, with the highest effect in the case of EGF. These results indicate that the HMW and LMW components act synergistically with all rh-growth factors analyzed, except PDGF.

[0117] Figure 4 shows the anti-inflammatory effects of three high molecular weight fraction samples after storage at -20°C for 6 years in a lipopolysaccharide (LPS)-TNF-alpha inhibition assay compared to prednisolone (positive control). Monocytes are isolated from heparinized blood and stimulated with lipopolysaccharide. Lipopolysaccharide is a surface molecule of gram-negative bacteria, which induces a significant inflammatory response and TNF-alpha secretion by binding to its CD14 molecule. The normalized expression of LPS-induced TNF-alpha (as a reference value) is used as a reference. In the repeated control, LPS-induced TNF-alpha secretion is analyzed under the influence of the added preparation. Readings below the reference value indicate an anti-inflammatory effect. Values above the reference value indicate the pro-inflammatory in vitro effect of each preparation. Prednisolone acetate (1.0 wt%) (a glucocorticoid) was used as the positive control.

[0118] Method Allogeneic blood and serum After written informed consent and approval by the ethics committee, venous blood was collected from healthy volunteers by venipuncture into blood bags without anticoagulants at a maximum of 500 ml per donation. Serum was prepared by maintaining the collected whole blood at room temperature for at least 2 hours, confirming with a PTT (partial thromboplastin time) assay, and allowing it to clot completely, followed by storage at 4 - 8°C for a maximum of 24 hours. The clotted blood was centrifuged twice at 3000 g for 10 minutes at room temperature. The resulting supernatant (serum) was separated from the clot using a manual extraction device, pooled, and diluted to a final concentration of 20 vol% in isotonic NaCl solution. The serum was pathogen-inactivated by viral filtration and filter sterilization with a 3K-multi-dose applicator and then aliquoted into 5 or 10 ml aliquots.

[0119] Ultrafiltration of pooled serum The pooled serum (1 L) was diluted with NaCl (0.9%) and ultrafiltered using a 30 kDa and 100 kDa polyethersulfone (PES) membrane (PALL Centramate T-series, Dreieich, Germany) with a flow rate of 100 ml / min using a multi-purpose pump from Almatechnik (Zeiningen, CH). The ultrafiltration was stopped when the retentate volume corresponded to 10% of the starting volume.

[0120] Wound closure assay An in vitro model of immortalized human corneal epithelial cells (HCE-T) grown as a monolayer cell culture was used to assay corneal epithelial injury stimulation and its regeneration. Briefly, 4×10 4Cells were seeded in 24-well plates and cultured in a humidified environment containing 5% CO2 at 37 °C until the cells formed an optically confluent monolayer using serum-free growth medium (KGM). Frozen serum samples, KGM (Keratinocyte Growth Medium-2, Lonza), supplements, and PBS (phosphate-buffered saline) were thawed to room temperature. Subsequently, a sterile 200 μl plastic pipette tip was used to scratch the center of the well to create a cell-free wound of 800 - 900 μm, damaging the epithelial cell model by mechanical injury, and washed twice with PBS (1 ml per well) to remove floating cells. The size of the injury was confirmed by light microscopy (Feng Y et al., Epithelial Wound Healing on Keratin Film, Amniotic Membrane and Polystyrene In Vitro, Current Eye Research 2014; 39(6):561 - 570). Subsequently, the epithelial cell model was further cultured using serum-free KGM supplemented with various components, namely serum, high molecular weight fraction, low molecular weight fraction, and recombinant human growth factor. Wound healing was evaluated using a microscope camera. Six pictures were taken per time point over a period of up to 60 hours. The regeneration enhancement effect was calculated as the difference in the percentage of wound width at various time points. HCE-T cells cultured in KGM were treated as a negative control. HCE-T cells cultured in KGM supplemented with bovine pituitary extract (10 μg / ml) were treated as a positive control.

[0121] Inflammatory assay The anti-inflammatory properties of the serum preparation were assayed by TNF-α inhibition test. Tumor necrosis factor alpha (TNF-α) is a cytokine involved in almost all inflammatory responses. Macrophages were isolated from at least five donors. The combined macrophages were stimulated with LPS (lipopolysaccharide). LPS is a surface molecule of Gram-negative bacteria that induces a significant inflammatory response, namely the secretion of TNF-α in macrophages, by binding to its CD14 receptor molecule. This standardized LPS-induced TNF-α release was used as a reference value for inflammation. The product according to the present invention was assayed and compared with respect to the secretion of LPS-induced TNF-α (n = 3). TNF-α secretion concentration values below the reference value were considered to have an anti-inflammatory effect; values greater than that were considered to have a pro-inflammatory effect. Prednisolone acetate (1.0 wt%) was used as a positive control, and the medium was used as a negative control, showing 100% and 0% inflammation reduction, respectively.

[0122] Assay of HLA-class I Trypsin digestion of HLA-class I molecules results in approximately 90% sequence coverage and thus differentiates HLA-class I molecules from different serum donors. HLA-class I molecules are highly polymorphic molecules that vary greatly among human individuals within a population. Therefore, a method as described was developed for detecting soluble HLA-class I (sHLA) molecules in serum. The entire HLA gene complex contains approximately 4,000 kilobases and is generally divided into two gene regions, and the class I genes therein include sHLA-A, sHLA-B, and sHLA-C. sHLA-A, sHLA-B, and sHLA-C serum molecules were purified from 10 ml of serum by affinity chromatography using polyclonal anti-HLA-A, anti-HLA-B, and anti-HLA-C antibodies. Subsequently, the eluate was further purified by gel permeation chromatography, and the pooled fractions were detected by SDS-PAGE and subsequent Western blotting using anti-HLA-A, anti-HLA-B, and anti-HLA-C antibodies. The PVDF blot was blocked in 0.5% (w / v) PVP-40 at room temperature for 30 minutes. After cutting out the PVDF membrane containing the spots of HLA-A, HLA-B, and HLA-C proteins, the PVDF membrane was moistened with methanol and washed 10 times with water. Subsequently, the blot was successively reduced and alkylated with 10 mM DTT and 55 mM IDA, respectively. Then, the blot was immersed in 25 μl of 25 mM NH4HCO3 and heated at 95 °C for 5 minutes to denature the protein. Subsequently, 25 μl of trypsin solution (10 μl of 40 ng / μl trypsin, 5 μl of 5% Tween-20, and 10 μl of acetonitrile) was added and incubated overnight at 37 °C. The trypsin-digested peptides were extracted from the blot three times at room temperature for 1 hour using 40 μl of 5% (v / v) trifluoroacetic acid and 45% H2O, 50% (v / v) ACN. The extract was dried by speed vacuum and resuspended in 20 μl of 0.1% TFA for mass spectrometry.

[0123] Mass spectrometric sequence analysis of HLA-class I molecules is based on the detection of tryptic peptides. Samples were measured using an LTQ Orbitrap Velos Pro system (Thermo Fisher Scientific, Bremen, Germany) online connected to a U3000 RSLCnano (Thermo Fisher Scientific, Idstein, Germany) using an Acclaim PepMap column for analysis (75 μm × 500 mm, 2 μm, 100 Å, Thermo Fisher Scientific, Bremen, Germany) at a flow rate of 250 nl / min. MS / MS measurements were performed after carbamidomethylation and digestion with trypsin for protein fragment sequencing. Separation was performed using an acetonitrile linear gradient. Data-dependent tandem mass spectrometry (MS / MS) analysis (https: / / www.sciencedirect.com / topics / biochemistry-genetics-and-molecular-biology / sequest) was performed using software 2.2 SP1.48 (Thermo Fisher Scientific, Bremen, Germany). Extracted ion chromatograms (XIC) for standard peptides and analyte peptides were generated using the Qual Browser incorporated in the XCalibur software suite. For peptide identification, MS / MS spectra were correlated with the UniProt human reference proteome set (http: / / www.uniprot.org). Theoretical average molecular weights were calculated with ProtParam (https: / / web.expasy.org / protparam / ).

Example

[0124] Generation of multi-donor human serum eye drops Briefly, a maximum of 500 ml of whole venous blood was collected from male donors (n = 5) aged 18 - 40 years. The blood was collected into a multi - bag collection bag without an anticoagulant (Compoflex Fresenius). The blood was stored at 18 - 22 °C (room temperature) for at least 2 hours and then allowed to clot while stored at 2 - 8 °C. Within 24 hours after blood collection, serum was separated from the clot by centrifuging twice at 3000×g for 10 minutes at room temperature. The obtained supernatant (serum) was extracted using a manual extraction device. The serum was pooled (1 L) to obtain an intermediate product, and the sample was subjected to quality control analysis. The intermediate serum pool was adjusted to the corresponding concentration [(diluted with NaCl (0.9%) to 50% or 20%], and using an Aero Pump ophthalmic multidose 3K (registered trademark) system under clean room conditions class A / B, a graduated syringe with a filter (0.22 μm) was used to dispense into 5 ml aliquots, and then release control was continued. Subsequently, the eye drop bottles were frozen and stored at a temperature of - 20 °C or lower until use.

[0125] Generation of intermediate products by ultrafiltration to obtain high - molecular - weight and low - molecular - weight fractions As described in Example 1), a pooled intermediate serum product (1 L) was obtained. Subsequently, the serum pool was separated into a low - molecular - weight fraction (LMWF) and a high - molecular - weight fraction (HMWF) by ultrafiltration. Thus, the pooled serum was diluted with 0.9% NaCl and ultrafiltered using a polyethersulfone (PES) membrane (PALL Centramate T - series, Dreieich, Germany) with cut - offs of 30 kDa and 100 kDa respectively, using a multi - purpose pump from Almatechnik (Zeiningen, CH) at a flow rate of 100 ml / min. Ultrafiltration was stopped when the retentate volume corresponded to 10% of the starting volume. Samples were obtained from the serum pool as well as the corresponding HMWF and LMWF and analyzed in more detail by testing aliquots for epithelial regeneration efficacy and anti - inflammatory efficacy.

[0126] Comparison of the epithelial regeneration efficacy of unfiltered multi-donor serum pool with low molecular weight fraction (LMWF) and high molecular weight fraction (HMWF) in corneal HCE-T cell assay Figure 2 shows the activity of serum fractions in an in vitro wound closure assay. The in vitro wound closure assay monitors human corneal epithelial cells (HCE-T cells) over time, and the readout for the reduction (%) of the wound area is based on HCE-T cells with well-defined microlesions (scratch wounds). Ultrafiltration of the pooled serum separated the low molecular weight content (in the filtrate) from the high molecular weight content (in the retentate). The data show that, compared to serum-free, unfiltered serum and negative controls, the high molecular weight fraction results in better, i.e., faster, wound healing than the low molecular weight fraction (Figure 2A). Two independent experiments 1 and 2 confirmed different activities between the high molecular weight fraction and the low molecular weight fraction (Figure 2B).

[0127] Detection of IGF-1 bound in the macromolecular complex of multi-donor human serum products in the HMWF fraction (retentate) HMWF samples were measured using an LTQ Orbitrap Velos Pro system (see the method section "HLA-class I assay"). This approach identified albumin, IGF-1 BP3 ALS complex, alpha-2 macroglobulin, PDGF-BB, TGF-beta1, VEGF, and PEDF. As shown in the following table, the corresponding filtrates (30 kDa and 100 kDa) were analyzed for the concentration of selected physiological components.

[0128] Characterization of intermediate products In the first approach, the concentrations of the components mentioned were experimentally determined under laboratory conditions. Next, under a contract with an accredited diagnostic laboratory (MVZ Karlsruhe), the concentrations of the selected components were determined. The results are presented in Table 1. The regenerative ability to close epithelial defects was analyzed in an in vitro wound healing assay (HCE-T cell assay). The anti-inflammatory ability was determined in a TNF-alpha inhibition test.

[0129]

Table 1

[0130] Formulation and Characterization of the Product The intermediate (serum pool) was adjusted to the required concentration using NaCl (0.9%). Table 2 presents the content of the resulting components and the additional QC analyses performed to characterize the product.

[0131]

Table 2

Claims

1. A multi-donor human serum product containing insulin-like growth factor (IGF) bound in a macromolecular complex with a molecular size of 30 kDa or more, and containing IGF-1 at a concentration of 20 ng / ml or higher. Contains monosaccharides in amounts less than 70 mg / dl, Multi-donor human serum products, which may be isotonic solutions or gels depending on the case.

2. The product according to claim 1, comprising less than 40 mg / dl of total cholesterol, wherein the total cholesterol is the sum of LDL (low-density lipoprotein) and HDL (high-density lipoprotein).

3. The product according to claim 1, which, in an in vitro wound closure assay, has a wound closure ability of 50% or more after 35 hours, normalized to the area of ​​injury.

4. In inflammation assays, the substance exhibits anti-inflammatory activity, and this anti-inflammatory activity is 50% or more when normalized to the standardized expression of LPS-induced TNF-α (as a reference value). and / or the product according to claim 1, comprising less than 0.2 mg / dl of C-reactive protein (CRP).

5. The product according to claim 1, comprising one or more growth factors selected from the list of EGF, IGF-1, IGF-BP3, IGF-2, PDGF-BB, alpha-2-macroglobulin, KGF, and PEDF.

6. The product according to claim 1, comprising one or more growth factors within the following concentration ranges: - EGF at 50-500 pg / ml or 20-200 pg / ml; - IGF-1 at 50-250 ng / ml or IGF-1 at 20-100 ng / ml; - IGF-BP3 at 650–4000 ng / ml or IGF-BP3 at 260–1600 ng / ml; - ALS with a blood glucose level of 450–6000 ng / ml or 180–2400 ng / ml; - 0.37–1.49 g / l of alpha-2-macroglobulin or 0.15–0.6 g / l of alpha-2-macroglobulin; - PDGF-BB at 471–3683 pg / ml or PDGF-BB at 188.4–1473.2 pg / ml; - KGF in concentrations of 2-100 pg / ml; - TGF-β1 in concentrations of 1–30 ng / ml or 0.4–12 ng / ml; - VEGF at 30–222.5 pg / ml or VEGF at 12–89 pg / ml; - PEDF of 60 to 5000 ng / ml or PEDF of 24 to 2000 ng / ml; and - Albumin at 1750–2600 mg / dl or 700–1040 mg / dl.

7. A product according to any one of claims 1 to 6, for use in pharmaceuticals.

8. A product according to any one of claims 1 to 6, for use in the treatment of dry eye disease.

9. The product according to claim 7, for use in pharmaceuticals, wherein the human serum composition is derived from allogeneic blood.

10. The product according to claim 8, wherein the human serum composition is derived from allogeneic blood, for use in the treatment of dry eye disease.

11. An ophthalmic composition, such as eye drops or a gel, comprising the product described in any one of claims 1 to 6.

12. - A step of preparing a volume portion of human allogeneic blood from multiple donors, - A step of selecting volume portions from at least two donors, - A step of taking serum from the selected volume portion to obtain the serum portion, - A step of pooling selected serum portions to obtain pooled serum, wherein, in some cases, all of the selected serum portions are derived from donors aged 45 years or younger. - A step of obtaining an intermediate product from the pooled serum, wherein the intermediate product contains 100 ng / ml or more of IGF-1, - A step of obtaining the human serum product according to any one of claims 1 to 6 from the intermediate product. A method for producing a product that includes [the specified product].

13. (i) The intermediate product is serum, and the step of obtaining the intermediate product includes the step of preparing serum from the human allogeneic blood; or (ii) The intermediate product is a retaining solution of the ultrafiltration process, and the step of obtaining the intermediate product includes the steps of preparing serum from the human allogeneic blood, ultrafiltration the serum to obtain a filtrate and a retaining solution, and selecting the retaining solution. The method according to claim 12.

14. The method according to claim 13, wherein the ultrafiltration process of step (ii) is carried out using a molecular cutoff membrane of at least 20 kDa, at least 30 kDa, at least 50 kDa, or at least 100 kDa, or the step of obtaining the human serum product further includes a step of diluting the intermediate product.

15. The aforementioned intermediate product contains less than 140 mg / dl of monosaccharides and / or 10 to 200 mg / dl of total cholesterol, wherein the total cholesterol is the sum of LDL (low-density lipoprotein), HDL (high-density lipoprotein), and VLDL (very low-density lipoprotein). The method according to claim 12.

16. An intermediate product containing insulin-like growth factor (IGF) bound in a macromolecular complex with a molecular size of 30 kDa or more, and containing 100-500 ng / ml of IGF-1.

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