Product suitable for treating tear film deficiency of xerophthalmia and preparation method thereof

Abstract

The invention relates to a product suitable for treating tear film defects of xerophthalmia and a manufacturing method of the product. The invention also relates to a product for treating dry eye tear film deficiency. The product has an effect of improving wound healing in xerophthalmia and other diseases.

Description

Technical Field

[0001] The present invention relates to a product suitable for treating tear film defects and corneal epithelial regeneration in dry eye and a method for preparing the same. The present invention also relates to a product in medical applications and a product for treating dry eye, which improves wound healing in dry eye and other diseases. Background Art

[0002] The lack of the aqueous phase of the tear film is the main pathological factor leading to tear film breakup in dry eye disease. Oxidative stress occurs due to the disruption of the balance between the antioxidant system and the pro-oxidant system. Based on the elevated inflammatory activity during the onset of dry eye disease, the level of oxidized proteins will increase, and it is speculated that oxidative stress will damage the ocular surface and play a key role in dry eye disease. In addition, the loss of corneal epithelial integrity will lead to the influx of water into the stroma.

[0003] Serum has a wide range of applications in diagnostics and is also used in medical treatments. Serum refers to the soluble component of whole blood from which the coagulation factors and all types of cells have been removed, e.g. by coagulation of a blood sample and subsequent centrifugation and filtration. Serum therefore includes essentially all proteins that are not involved in blood clotting, as well as electrolytes, antibodies, antigens and hormones.

[0004] Tears of healthy subjects contain a variety of antioxidants, such as ascorbic acid, lactoferrin, uric acid, and cysteine, to protect the ocular surface from certain free radicals. It has been hypothesized that components present in human serum may have antioxidant effects and could be used to treat tear film deficiency in dry eye.

[0005] WO 2022 / 029109 A1 describes compositions and bioactive blood fractions for treating damaged epithelial surfaces associated with eye diseases. WO 2022 / 029109 A1 also describes a corresponding preparation method, i.e. obtaining these fractions by ultracentrifugation and subsequent separation from total serum or total plasma. The obtained blood fractions are formulated into eye drops. It was found that the blood fractions obtained after serum ultracentrifugation are biologically active and have a healing effect on damaged epithelial cells. The obtained serum fractions are substantially free of lipids and lipoproteins, can improve fluidity and facilitate packaging, and can be sterile filtered to remove pathogens such as bacteria.

[0006] The healthy cornea has a limited influx (leakage) of water into the stroma, which is balanced by the outflow of water caused by bicarbonate, lactate and NaCl solutes on the Descemet's membrane and endothelial monolayer. Oxidative stress occurs due to the breakup of the tear film and the subsequent disruption of the balance between the antioxidant and pro-oxidant systems and damages the ocular surface, playing a crucial role in dry eye, as the levels of oxidized proteins increase with the increase in inflammatory activity during these diseases. In addition, the loss of corneal epithelial integrity leads to an influx of water into the stroma. The conditions need to be adequately and effectively treated, in particular the epithelial defects need to be closed rapidly and the tear film needs to be stabilized to avoid long-term damage and worsening of dry eye.

[0007] However, pharmaceutical products known in the art do not provide adequate regenerative capacity, i.e., by treating corneal epithelial defects.Therefore, there is an unmet clinical need for new products that are more effective in treating dry eye and related pathologies.

[0008] The above objectives can be achieved through the above claims and the following specific technical solutions. Summary of the invention

[0009] In a first aspect, the present invention relates to a multi-donor human serum preparation comprising insulin-like growth factors (IGFs) bound in a macromolecular complex with a molecular size of at least 30 kDa, the preparation also comprising at least 20 ng / mL of IGF-1, and less than 70 mg / dL of monosaccharides, the preparation optionally being an isotonic solution or an isotonic gel.

[0010] In a related aspect, the present invention relates to a human serum preparation comprising insulin-like growth factors (IGFs) bound in a macromolecular complex with a molecular size of at least 30 kDa, the preparation comprising 20 ng / mL-100 ng / mL of IGF-1, and less than 70 mg / dL of monosaccharides, the preparation optionally being an isotonic solution or an isotonic gel.

[0011] The inventors have found that human serum contains insulin-like growth factors (IGFs) bound in macromolecular complexes of at least 30 kDa in size, and that these macromolecular complexes, when intact and incorporated into pharmaceuticals, can improve wound healing. In addition to high concentrations of proteins such as albumin and fibronectin, human serum also contains specific epithelial-promoting factors such as IGF-1, EGF, etc. Serum can directly support the proliferation and migration of epithelial cells, or indirectly increase the survival rate of epithelial cells by binding and neutralizing inflammatory cytokines. Growth factors and cytokines are important regulatory factors that stimulate cell growth, proliferation, migration, differentiation, adhesion, ECM deposition, and protease regulation during wound healing.

[0012] In a second aspect, the present invention relates to the medical application of human serum products.

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

[0014] In a third aspect, the present invention relates to a method for producing a human serum product, comprising the following steps:

[0015] - Provide human allogeneic blood volume fractions from multiple donors,

[0016] - selecting a volume fraction from at least two donors,

[0017] - extracting serum from selected volume fractions,

[0018] - combining all sera to obtain a pooled serum, optionally from donors all aged 45 years or less;

[0019] - obtaining an intermediate preparation from the pooled serum, said intermediate preparation comprising at least 100 ng / mL IGF-1; and

[0020] - Obtaining a multi-donor human serum preparation from the intermediate preparation of the present invention.

[0021] The method relies on providing whole blood volume fractions from a large number of donors to ensure sufficient IGF concentration. The IGF-1 concentration in human blood varies from person to person. Some volume fractions may have very high IGF-1, while others have very low IGF-1 concentrations. By selecting volume fractions with sufficient IGF-1 concentrations, 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 life span increases. Therefore, limiting the age of blood donors to 45 years or younger helps to improve production efficiency because fewer blood portions need to be discarded. The method provides a method for obtaining an intermediate product, wherein the intermediate product contains 100 ng / mL or more of IGF-1. The obtained product contains insulin-like growth factors (IGFs) bound to macromolecular complexes with a molecular size of 30 kDa or greater. Advantageously, the method is universal and can be used to obtain the product of the present invention from the intermediate product by selecting variants, parameters and conditions, so the present invention provides a product with improved therapeutic properties, such as for repairing corneal epithelial defects and anti-inflammatory.

[0022] In an aspect related to the human serum product, the present invention also provides an intermediate product comprising insulin-like growth factors (IGFs) bound in a macromolecular complex with a molecular weight of 30 kDa or greater, wherein the intermediate product contains 100-500 ng / mL of IGF-1.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1A and Figure 1B Age-dependent IGF-1 and IGF-BP3 concentrations in human serum are shown, respectively. Age is in years.

[0025] Figure 2A and Figure 2B The activity of serum fractions in an in vitro wound closure assay was shown.

[0026] FIG. 3A to FIG. 3D The effects of four different human recombinant (hr) growth factors, EGF, IGF-1, PDGF and KGF, on wound closure when combined with either the high molecular weight fraction or the low molecular weight fraction of ultrafiltered multi-donor human serum are shown.

[0027] Figure 4 The anti-inflammatory effects of the three high molecular weight fraction samples in the (LPS)-TNF-α inhibition assay are shown. DETAILED DESCRIPTION

[0028] Products

[0029] In a first aspect, the present invention relates to a multi-donor human serum preparation comprising insulin-like growth factors (IGFs) bound in a macromolecular complex with a molecular size of at least 30 kDa, the preparation also comprising at least 20 ng / mL of IGF-1, and less than 70 mg / dL of monosaccharides, the preparation optionally being an isotonic solution or an isotonic gel.

[0030] In a related aspect, the present invention relates to a human serum preparation comprising insulin-like growth factors (IGFs) bound in a macromolecular complex with a molecular size of at least 30 kDa, the product containing 20-100 ng / mL of IGF-1, and less than 70 mg / dL of monosaccharides, optionally, the preparation is an isotonic solution or an isotonic gel.

[0031] The "human serum products" involved in the present invention also refer to "multi-donor human serum products" and "human serum products".

[0032] 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 quantified experimentally. The inventors have found that human serum contains insulin-like growth factors bound in macromolecular complexes with a molecular weight greater than 30 kDa, and that when these macromolecular complex substances remain intact and are incorporated into pharmaceutical products, they can improve wound healing. The molecular weights of IGF-1 and IGF-2 are 7.65 kDa and 7.5 kDa, respectively (referring to human proteins). The macromolecular complexes contain IGF-1 and / or IGF-2 and their binding proteins, thus reaching a molecular weight of 30 kDa or more. It has been determined and / or confirmed that the macromolecular complexes remain intact in the absence of proteases and under neutral conditions, i.e., at a pH of about 7. High salt conditions, the addition of organic solvents or the addition of protease inhibitors will destroy the macromolecular complexes.

[0033] In one embodiment, the molecular size of the macromolecular complex is 40-50 kDa. In one embodiment, the molecular size of the macromolecular complex is 150 kDa.

[0034] IGFs bound in macromolecular complexes also provide continuous availability of active growth factors and improve product storage stability. IGFs in monomeric form are more susceptible to degradation due to the lack of native form.

[0035] In one embodiment, the human serum preparation contains less than 70 mg / dL monosaccharides, less than 40 mg / dL monosaccharides, or less than 30 mg / dL monosaccharides. Advantageously, the human serum preparation contains IGFs bound in macromolecular complexes, which, combined with a small amount of low-molecular components (i.e., monosaccharides) typically found in human serum, can accelerate wound healing. For example, as a reference, whole blood from a healthy donor includes a blood glucose level of 60 to 140 mg / dL (<5.6 to 7.8 mmol / L). Low-molecular components are generally considered to have a molecular weight below 900 Da. For example, the concentration of low-molecular components in serum is reduced in the filtrate obtained after ultracentrifugation, or in the human serum preparation content obtained by diluting human serum.

[0036] In one embodiment, the human serum preparation comprises less than 40mg / dL of total cholesterol, wherein total cholesterol is the sum of LDL (low-density lipoprotein) and HDL (high-density lipoprotein). Advantageously, the human serum preparation comprises IGF bound in a macromolecular complex, which is combined with a certain amount of high-molecular components (e.g., cholesterol) commonly found in human serum, and can accelerate wound healing. For example, as a reference, the whole blood of a healthy donor contains about 150mg / dL of total cholesterol. The high-molecular components in the serum are enriched at least to a certain extent relative to the concentration in the retentate obtained after ultracentrifugation, relative to their concentration in the original serum. In the human serum preparation process obtained by diluting human serum, the concentration of these high-molecular components is diluted accordingly.

[0037] In one embodiment, the human serum preparation comprises IGFs bound in macromolecular complexes having a molecular weight of 30 kDa or greater, the product comprises 20 ng / mL or more of IGF-1, and comprises 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.

[0038] In one embodiment, the human serum preparation comprises IGFs bound in macromolecular complexes having a molecular weight of 30 kDa or greater, the product comprising 20 to 100 ng / mL of IGF-1, and comprising 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.

[0039] In the context of the present disclosure, total cholesterol and urea are components of human serum that do not play a role or contribute to the specific functions of human serum products. If the whole blood comes from a healthy donor, the content of total cholesterol and urea in human serum products is considered to be harmless.

[0040] In one embodiment, the human serum preparation contains less than 40 mg / dL of total cholesterol, less than 35 mg / dL of total cholesterol, or less than 30 mg / dL of total cholesterol, wherein total cholesterol is the sum of LDL (low-density lipoprotein) and HDL (high-density lipoprotein). In one embodiment, the preparation contains 5 mg / dL or more, 10 mg / dL or more, or 15 mg / dL or more of total cholesterol. In one embodiment, the preparation 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.

[0041] In one embodiment, the human serum preparation contains less than 70 mg / dL of LDL and / or less than 30 mg / dL of HDL. In one embodiment, the preparation contains 2-70 mg / dL of low-density lipoprotein (LDL) and / or 1-30 mg / dL of high-density lipoprotein (HDL). In one embodiment, the preparation contains 2-70 mg / dL of LDL, 5-60 mg / dL of LDL, 10-50 mg / dL of LDL, or 15-40 mg / dL of LDL. In one embodiment, the preparation contains 1-30 mg / dL HDL, 2-25 mg / dL HDL, or 5-20 mg / dL HDL.

[0042] In one embodiment, the human serum preparation comprises less than 100 mg / dL urea, or less than 40 mg / dL urea, or less than 10 mg / dL urea, or less than 3 mg / dL urea.

[0043] Human serum products can be derived from human serum, preferably from human allogeneic blood. Therefore, human serum products are easy to manufacture and require only a limited number of additives. In one embodiment, the human serum product contains added ingredients and / or additives, such as vitamins, growth factors, pH regulators, buffers, surfactants, viscosity modifiers, and / or contains a certain amount of NaCl to make the product isotonic in the intended pharmaceutical application.

[0044] In one embodiment, human serum products are substantially free of stabilizers. In one embodiment, human serum products are substantially free of preservatives. Those skilled in the art will appreciate that stabilizers or preservatives may be added if desired. Human serum products require no stabilizers, and no preservatives, meaning that human serum products are neither allowed to add stabilizers nor preservatives. Although typical pharmaceutical products may require the addition of preservatives, such as benzalkonium chloride (irritating to the eyes), human serum products according to the present invention can also work in the absence of these preservatives. Due to the manufacturing method obtained from human allogeneic blood, intermediate products are not susceptible to microbial contamination, and therefore can be manufactured, further processed and stored in the absence of preservatives.

[0045] 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 the application of preparing drugs for pharmaceutical and / or medical use. In particular, for the treatment of eye-related diseases, the tension or osmotic pressure of the human serum product must be provided and / or maintained at a physiological level. Therefore, those skilled in the art understand that referring to an isotonic solution or an isotonic gel means that the effective osmotic pressure concentration of the human serum product is substantially the same as the osmotic pressure concentration of the relevant body fluid. Therefore, the solute concentration of the human serum product is adjusted to an isotonic solution or an isotonic gel, corresponding to the solute concentration in human cells or human tissues.

[0046] Osmolality refers to the concentration of osmotically active solutes in a solution, while tonicity refers to the corresponding physiological effect in the body, also known as "effective osmotic concentration."

[0047] In one embodiment, the pH of the human serum preparation is between 5.6 and 10.5, 7.3 and 9.7, or about 7.4.

[0048] In one embodiment, the tonicity of the human serum preparation is in the range of 127-475 mosmol / kg, 222-445 mosmol / kg, 250-320 mosmol / kg or about 285 mosmol / kg.

[0049] In one embodiment, the pH value of the human serum preparation is between 5.6 and 10.5, and the tonicity is 127 to 475 mosmol / kg. In one embodiment, the pH value of the human serum preparation is between 7.3 and 9.7, and the tonicity is 222 to 445 mosmol / kg. In one embodiment, the pH value of the human serum preparation is about 7.4, and the tonicity is about 285 mosmol / kg. A pH value of about 7.4 and a tonicity of about 285 mosmol / kg are generally considered physiological concentrations, thereby providing good physiological tolerability for ophthalmic solutions.

[0050] In one embodiment, the human serum preparation comprises 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 preparation comprises 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 preparation comprises IGF-1 at a concentration of 20 to 600 ng / mL, 30 to 500 ng / mL, 50 to 400 ng / mL, or 100 to 300 ng / mL. The presence of IGF-1 in the human serum preparation contributes to wound healing of the product.

[0051] 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-BP to 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, in the extracellular environment, and inside cells. It is considered the major IGF transporter in the blood, where the growth factors it carries are mainly present in the form of stable complexes that contain the binding proteins IGF-1 or IGF-2, and a third protein called the acid-labile subunit (ALS).

[0052] In one embodiment, the human serum preparation comprises 1300 ng / mL or more, 2000 ng / mL or more, or 3000 ng / mL or more IGF-BP3. In one embodiment, the human serum preparation comprises 8000 ng / mL or less, 7000 ng / mL or less, or 6000 ng / mL or less IGF-BP3. In one embodiment, the product comprises 1300-8000 ng / mL, 2000-7000 ng / mL, or 3000-6000 ng / mL IGF-BP3.

[0053] In one embodiment, the human serum preparation comprises a molar ratio of monomeric (i.e., free or unbound) IGF-1 to IGF-1 bound in macromolecular complexes (molecular weight of 30 kDa or greater) of 1 / 1000 to 20 / 1000, or 2 / 1000 to 15 / 1000, or 3 / 1000 to 10 / 1000. In one embodiment, the human serum preparation comprises a molar ratio of monomeric (i.e., free or unbound) IGF-1 to IGF-1 bound in macromolecular complexes (molecular weight of 30 kDa or greater) of 20 / 1000 or less, 15 / 1000 or less, or 10 / 1000 or less.

[0054] In one embodiment, the human serum preparation comprises IGF bound in a macromolecular complex having a molecular size of 30 kDa or greater, the product comprising IGF-1 bound to IGF-BP3 at a concentration of 20 to 600 ng / mL IGF-1 and 1300 to 8000 ng / mL IGF-BP3, 2000 to 7000 ng / mL IGF-BP3 or 3000 to 6000 ng / mL IGF-BP-BP3.

[0055] In one embodiment, the human serum preparation comprises IGFs bound in macromolecular complexes with a molecular weight of 30 kDa or greater, and the preparation comprises IGF-1 bound to IGF-BP3 molecules, wherein the IGF-1 concentration is 20-600 ng / mL, 30-500 ng / mL, 50-400 ng / mL or 100-300 ng / mL, and the IGF-BP3 concentration is 1300-8000 ng / mL, wherein the concentration of IGF-1 refers to the total concentration of IGF-1 protein in the human serum preparation, that is, the sum of the concentrations of bound and unbound (free) forms.

[0056] In one embodiment, in an in vitro wound closure test, based on the wound area as a standard, at 35 hours, the wound closure performance of the human serum product reaches 50% or higher, that is, after 35 hours, 50% or more of the initial wound area is closed.

[0057] In one embodiment, in an in vitro wound closure test, based on the wound area as a standard, at 21 hours, the wound closure performance of the human serum product reaches 50% or more, that is, after 21 hours, 50% or more of the initial wound area is closed.

[0058] Wound closure performance is based on an in vitro wound closure assay that monitors human corneal epithelial cells (HCE-T cells) over time, where the readout of wound area reduction (in %) is based on HCE-T cells with defined microinjuries (scratches). A detailed description of the invention is given in the Examples section.

[0059] The in vitro wound closure assay can be used to simulate, for example, dry eye and other pathological conditions and monitor wound closure of negative controls based on serum-free KGM (Keratinocyte Growth Medium-2) and positive controls based on KGM supplemented with bovine pituitary extract (i.e., 10 μg / mL).

[0060] In one embodiment, the in vitro wound closure assay comprises the following steps: (a) providing an in vitro cell culture comprising HCE-T cells, (b) applying micro-injury, such as scratching on the HCE-T (monolayer) cells, (c) using the human serum preparation of the present invention, (d) monitoring the in vitro cell culture, such as by optical microscopy, (e) quantifying the time-dependent wound closure as wound closure performance by calculating the percentage difference of wound area at different time points.

[0061] Quantification of time-dependent wound closure can be performed or assisted by, for example, image analysis software.

[0062] In one embodiment, in an in vitro wound closure test, based on the wound area, at 35 hours, the wound closure performance of the human serum product reaches 50% or more, 55% or more, 60% or more, or 65% or more. In one embodiment, in an in vitro wound closure test, based on the wound area, at 35 hours, the wound closure performance of the human serum product is 90% or less, 85% or less, 80% or less, or 75% or less. In one embodiment, in an in vitro wound closure test, based on the wound area, at 35 hours, the wound closure performance of the human serum product is 50% to 90%, 55% to 85%, 60% to 80%, or 65% to 75%.

[0063] In one embodiment, in an in vitro wound closure test, based on the wound area, at 21 hours, the wound closure performance of the human serum product is 50% or more, 55% or more, 60% or more, or 65% or more. In one embodiment, in an in vitro wound closure test, based on the wound area, at 21 hours, the wound closure performance of the human serum product is 90% or less, 85% or less, 80% or less, or 75% or less, based on the wound area. In one embodiment, in an in vitro wound closure test, based on the wound area, at 21 hours, the wound closure performance of the human serum product is 50% to 90%, 55% to 85%, 60% to 80%, or 65% to 75% at 21 hours.

[0064] In one embodiment, the human serum preparation has anti-inflammatory properties in an inflammation assay, wherein the anti-inflammatory property is standardized to the value of LPS-induced TNF-α (as a reference base value), the anti-inflammatory capacity is 50% or higher, and / or the human serum preparation contains less than 0.2 mg / dL of C-reactive protein (CRP).

[0065] The anti-inflammatory properties in the inflammation test were determined as follows:

[0066] - Provide macrophages from at least five donors;

[0067] - Pooling of isolated macrophages;

[0068] - LPS (lipopolysaccharide) stimulation combined with macrophage induction of TNF-α secretion, used as a reference value for inflammation;

[0069] - Stimulate the combined macrophages with LPS and measure the anti-inflammatory properties of human serum products, with 3 parallel samples;

[0070] - when the combined macrophages are stimulated with LPS, the value of prednisolone, preferably prednisolone acetate (1.0% by weight), is determined as a positive control, taking as a 100% anti-inflammatory property;

[0071] - the value of the anti-inflammatory property of the culture medium when the combined macrophages were stimulated with LPS was determined as 0% anti-inflammatory property, as a negative control; and

[0072] - Quantification of the anti-inflammatory properties of human serum preparations as percentages, normalized to positive and negative controls.

[0073] In one embodiment, the human serum preparation contains less than 0.2 mg / dL CRP, less than 0.06 mg / dL CRP, or less than 0.02 mg / dL CRP. In one embodiment, the human serum preparation contains 0.0002 mg / dL or more, 0.0006 mg / dL or more, or 0.002 mg / dL or more CRP. In one embodiment, the product contains 0.0002-0.2 mg / dL CRP, 0.0006-0.06 mg / dL CRP, or 0.002-0.02 mg / dL CRP.

[0074] When the body is inflamed, CRP can be detected in blood and serum. In particular, after inflammation, the CRP level in human (or mammal) blood increases. Advantageously, the product contains a low amount of CRP, avoiding and / or alleviating the triggering of inflammatory reactions.

[0075] The human serum composition can be derived from human serum, preferably from human allogeneic blood. Multiple methods can determine that the serum product is obtained from multiple donors, that is, it is a multi-donor serum product. In the present invention, reference to a "mixed" serum product means that it comes from multiple donors. The multi-donor human serum product is characterized by one or more of the following criteria:

[0076] - According to the International Society of Blood Transfusion (ISBT) classification, the presence of antigens indicates that the human serum components are from two or more donors;

[0077] - the presence of soluble HLA class I molecules indicates that the human serum components are from two or more donors; and

[0078] -The presence of glycolipids indicates that the human serum composition is from two or more donors.

[0079] In one embodiment, the presence of HLA class I molecules indicates that the human serum preparation is from two or more donors, based on a method comprising the steps of:

[0080] -Provide human serum products;

[0081] - purifying soluble HLA class I molecules, such as sHLA-A, sHLA-B, sHLA-C, from a human serum composition by affinity chromatography using polyclonal anti-HLA-A, anti-HLA-B and anti-HLA-C antibodies to obtain an HLA eluate;

[0082] - purifying the HLA eluate, for example by gel permeation chromatography, to obtain a purified eluate;

[0083] - detection of soluble HLA class I molecules by Western blotting using anti-HLA-A, anti-HLA-B and anti-HLA-C antibodies;

[0084] - Trypsin digestion of soluble HLA class I molecules;

[0085] -optionally, extracting trypsin-digested soluble HLA class I molecules; and

[0086] - Analysis of trypsin-digested soluble HLA class I molecules by mass spectrometry (MS).

[0087] Those skilled in the art are familiar with methods for protein extraction, separation and purification. Those skilled in the art also know that Western blotting requires pre-separation according to the size of the protein or peptide, such as by SDS-PAGE. In one embodiment, the extract of the tryptic peptides is dried by rapid vacuum drying and resuspended in a buffer suitable for MS analysis.

[0088] Advantageously, in multi-donor human serum products, the amounts of all serum components are balanced, and fluctuations in serum components between donors are also balanced. Therefore, potential, harmful serum components, such as donor antibodies (which may trigger patient reactions) will be diluted, thereby minimizing potential risks and / or adverse reactions faced by patients. Therefore, a more standardized product can be obtained or provided than similar products based on human serum compositions from only one donor. In terms of product preparation, the use of human serum from two or more donors also allows for the expansion of production scale to produce larger quantities of products. Starting from multi-donor serum, it is beneficial to manufacture and store serum-based eye drops as finished drugs.

[0089] It is also advantageous that serum obtained from healthy donors can be screened and / or optimized with respect to serum composition, for example with respect to tolerability and efficacy, for example with respect to establishing a well-defined concentration range for growth factors.

[0090] There are several observable indicators that can assess and distinguish whether a human serum composition comes from two or more donors, such as the presence of various antigens that indicate donors with different blood groups. For example, based on antigen analysis, the International Society of Blood Transfusion currently recognizes 43 different blood group systems. In addition, the presence of glycolipids may indicate that a human serum composition comes from two or more donors.

[0091] In one embodiment, the human serum preparation comprises one or more growth factors selected from EGF, IGF-1, IGF-BP3, IGF-2, PDGF-BB, α2-macroglobulin, KGF and PEDF. Advantageously, the growth factors can stimulate cell proliferation, particularly wound healing. The growth factors can be added to the human serum preparation as recombinant proteins. However, these growth factors may be naturally present in human serum components.

[0092] In one embodiment, the human serum preparation comprises one or more, in particular all, of the following growth factors within the following concentration ranges:

[0093] - 50-500 pg / mL of EGF, or 20-200 pg / mL of EGF, optionally quantified according to the EGF-Quantikine Elisa assay of Bio-Techne Ltd. (Catalog No. DEG00);

[0094] -50-250 ng / mL IGF-1, or 20-100 ng / mL IGF-1, optionally quantified by the Diasorin Liaison IGF-1 chemiluminescent assay;

[0095] - 650 to 4000 ng / mL of IGF-BP3, or 260 to 1600 ng / mL of IGF-BP3, optionally quantified according to a ligand binding assay;

[0096] - 450-6000 ng / mL ALS, or 180-2400 ng / mL ALS, optionally quantified according to the R&D Ltd. IGFBP-ALSElisa kit (my bioresearch, catalog number MBS450295);

[0097] - 0.37-1.49 g / l α2-macroglobulin, or 0.15-0.6 g / l α2-microglobulin, optionally quantified according to Elisa IMD Potsdam;

[0098] - 471 to 3683 pg / mL PDGF-BB, or 188.4 to 1473.2 pg / mL PDGF-BB, optionally quantified according to the Quantitikine Elisa kit (Catalog No. DBB00) of Bio-Techne Co., Ltd.;

[0099] -2~100pg / mL KGF, optionally, according to R&D Co., Ltd. ELISA kit for quantification (Catalog No. DKG00);

[0100] -1 to 30 ng / mL TGF-β1, or 0.4 to 12 ng / mL TGF-β-1, which can be quantified according to the IMD Berlin TGF-β-1IVD assay;

[0101] -30 to 222.5 pg / mL VEGF or 12 to 89 pg / mL VEGF, optionally quantified according to IMD Berlin;

[0102] - 60-5000 ng / mL PEDF or 24-2000 ng / mL PEDF, optionally quantified according to an ELISA kit from R&DR&D Ltd.; and

[0103] - 1750-2600 mg / dL albumin, or 700-1040 mg / dL albumin, optionally quantified according to MVZ Labor-Volkmann Karlsruhe.

[0104] In one embodiment, the human serum preparation contains 50-500 pg / mL EGF, or 100-250 pg / mL EGF, or 20-200 pg / mL EGF, or 40-150 pg / mL EGF, optionally quantified according to the EGF-Quantikine Elisa Kit (Catalog No. DEG00) of Bio-Techne Co., Ltd.

[0105] In one embodiment, the human serum preparation comprises 50-250 ng / mL IGF-1, or 70-200 ng / mL IGF-1, or 20-100 ng / mL IGF-1, or 30-80 ng / mL IGF-1, optionally quantified according to the Diasorin Liaison IGF-1 chemiluminescent assay.

[0106] In one embodiment, the human serum preparation comprises 650-4000 ng / mL of IGF-BP3, 1000-3000 ng / mL of IGF-PP3, 260-1600 ng / mL of IGF-PB3, or 500-1200 ng / mL of IGF-BB3, optionally quantified according to a ligand binding assay.

[0107] In one embodiment, the human serum preparation comprises 450-6000 ng / mL ALS, 1000-4000 ng / mL ALS, 180-2400 ng / mL ALS or 500-1500 ng / mL ALS, optionally quantified according to the R&D Ltd. IGFBP-ALS Elisa kit (mybioresearch, catalog number MBS450295).

[0108] In one embodiment, the human serum preparation comprises 0.37-1.49 g / l α2-macroglobulin, 0.55-1.20 g / l α2-microglobulin, 0.15-0.6 g / l α-2-macroglobulin or 0.2-0.5 g / l α2-macroglobulin, optionally quantified according to Elisa IMD Potsdam.

[0109] In one embodiment, the human serum preparation comprises 471-3683 pg / mL PDGF-BB, 1000-3000 pg / mL PDGF-BB, 188.4-1473.2 pg / mL PDGF-BB or 400-1200 pg / mL PDGF-B, optionally quantified according to the Quantitikine Elisa kit (Catalog No. DBB00) of Bio-Techne Co., Ltd.

[0110] In one embodiment, the human serum preparation comprises 2-100 pg / mL KGF, 5-70 pg / mL KGG or 10-50 pg / mL KGE, optionally according to R&D Co., Ltd. ELISA kit (Cat. No. DKG00) was used for quantification.

[0111] In one embodiment, the human serum preparation comprises 1-30 ng / mL TGF-β1, 3-20 ng / mL TGF-β2, 0.4-12 ng / mL TGF-β3, or 1.0-8 ng / mL TGF-β4, optionally quantified according to the IMD Berlin TGF-β1 IVD kit.

[0112] In one embodiment, the human serum preparation comprises 30-222.5 pg / mL VEGF, 50-150 pg / mL VEGF, 12-89 pg / mL VEGF or 20-60 pg / mL VEGF, optionally quantified according to IMD Berlin.

[0113] In one embodiment, the human serum preparation contains 60-5000 ng / mL PEDF, 300-3000 ng / mL PEDF, 24-2000 ng / mL PEDF or 120-1200 ng / mL PEDF, optionally quantified according to an ELISA kit of R&D Co., Ltd.

[0114] In one embodiment, the human serum preparation comprises 1750-2600 mg / dL albumin or 700-1040 mg / dL albumin, optionally quantified according to MVZ Labor-Volkmann Karlsruhe.

[0115] As those skilled in the art will appreciate, ELISA is the result of deliberate design choices coupled with many other (sometimes elusive) factors. Therefore, a standard curve must be generated in order to normalize, i.e., calibrate, the readings.

[0116] Advantageously, albumin prevents precipitation of proteins (such as growth factors) in aqueous solutions and has antioxidant properties, acting as a free radical scavenger during storage. Albumin can also prevent hydrophobic interactions between serum components and on the surface of drug containers.

[0117] In one embodiment, a multi-donor human serum preparation comprises insulin-like growth factors (IGFs) bound in a macromolecular complex with a molecular weight of 30 kDa or greater, the product comprises 20 ng / mL or more of IGF-1, less than 70 mg / dL of monosaccharides, optionally in an isotonic solution or isotonic gel, and in an in vitro wound closure test, based on the wound area as a standard, the wound closure performance of the product is 50% or more at 35 hours, wherein the product comprises one or more, in particular all of the following growth factors within the following concentration ranges:

[0118] - 50 to 500 pg / mL of EGF, optionally quantified according to the EGF-Quantikine Elisa assay of Bio-Techne Ltd. (Catalog No. DEG00);

[0119] -50 to 250 ng / mL IGF-1, optionally quantified by the Diasorin Liaison IGF-1 chemiluminescent assay;

[0120] - 650 to 4000 ng / mL of IGF-BP3, optionally quantified by ligand binding assay;

[0121] -450-6000 ng / mL ALS, optionally quantified according to the R&D Ltd. IGFBP-ALSElisa kit (mybioresearch, catalog number MBS450295);

[0122] - 0.37 to 1.49 g / l α2-macroglobulin, optionally quantified according to Elisa IMD Potsdam;

[0123] -471 to 3683 pg / mL PDGF-BB, optionally quantified according to the Quantitikine Elisa kit (Catalog No. DBB00) of Bio-Techne Co., Ltd.;

[0124] -2~100pg / mL KGF, optionally, according to R&D Co., Ltd. ELISA kit for quantification (Catalog No. DKG00);

[0125] -1~30ng / mL TGF-β1, which can be quantified according to the IMD Berlin TGF-β-1IVD assay;

[0126] -30 to 222.5 pg / mL VEGF, optionally quantified according to IMD Berlin;

[0127] -60~5000ng / mL PEDF, which can be quantified according to the ELISA kit of R&DR&D Co., Ltd.; and

[0128] - 1750-2600 mg / dL albumin, optionally quantified according to MVZ Labor-Volkmann Karlsruhe.

[0129] In one embodiment, a multi-donor human serum preparation comprises insulin-like growth factors (IGFs) bound in a macromolecular complex with a molecular weight of 30 kDa or more, the product comprises 20 ng / mL or more of IGF-1, less than 70 mg / dL of monosaccharides, optionally in an isotonic solution or isotonic gel, and in an in vitro wound closure test, based on the wound area, at 35 hours, the wound closure performance of the preparation is 50% or more, wherein the preparation comprises one or more, in particular all of the following growth factors within the following concentration ranges:

[0130] 20-200 pg / mL of EGF, optionally quantified according to the EGF-Quantikine Elisa assay (Catalog No. DEG00) of Bio-Techne Co., Ltd.;

[0131] 20-100 ng / mL IGF-1, optionally quantified by Diasorin Liaison IGF-1 chemiluminescence assay;

[0132] 260-1600 ng / mL IGF-BB3, optionally quantified by ligand binding assay;

[0133] 180-2400 ng / mL ALS, optionally quantified according to the R&D Co., Ltd. IGFBP-ALSElisa kit (mybioresearch, catalog number MBS450295);

[0134] 0.15-0.6 g / l α2-microglobulin, optionally quantified according to ElisaIMD Potsdam;

[0135] -188.4 to 1473.2 pg / mL PDGF-BB, optionally quantified according to the Quantitikine Elisa kit (Catalog No. DBB00) of Bio-Techne Co., Ltd.;

[0136] -2~100pg / mL KGF, optionally, according to R&D Co., Ltd. ELISA kit for quantification (Catalog No. DKG00);

[0137] -0.4~12ng / mL TGF-β1, which can be quantified according to the IMD Berlin TGF-β-1 IVD assay;

[0138] -12 to 89 pg / mL VEGF, optionally quantified according to IMD Berlin;

[0139] 24-2000 ng / mL PEDF, which can be quantified using the ELISA kit of R&DR&D Co., Ltd.; and

[0140] 700-1040 mg / dL albumin, optionally quantified according to MVZ Labor-VolkmannKarlsruhe.

[0141] In one embodiment, a human serum preparation comprises insulin-like growth factors (IGFs) bound in a macromolecular complex with a molecular weight of 30 kDa or more, the preparation comprises 20-100 ng / mL of IGF-1, and less than 70 mg / dL of monosaccharides, optionally in an isotonic solution or isotonic gel, and has a wound closure performance of 50% or more in an in vitro wound closure test, relative to the area of ​​injury, at 35 hours, based on the wound area as standard. The preparation comprises one or more, in particular all of the following growth factors within the following concentration ranges.

[0142] Medical Applications

[0143] In a second aspect, the invention relates to the medical use of the preparation.

[0144] Due to its composition, the human serum product of the present invention has the property of stimulating cell proliferation and promoting the healing of epithelial defects. Therefore, the human serum product can be used in medical and pharmaceutical applications, for example, after inflammation and / or injury. In addition, the human serum product can be used after surgery to assist and / or accelerate recovery.

[0145] In a related aspect, the present invention relates to the use of human serum products in the treatment of dry eye.

[0146] In one embodiment, the human serum preparation of the present invention is used to treat macular degeneration, corneal epithelial damage, diseases caused by or after corneal transplantation, diseases related to or caused by wound healing disorders, or diseases related to or caused by ophthalmic laser treatment.

[0147] The inventors found that the human serum preparation is superior to existing compositions in performance and has improved epithelial defect healing performance ( Figures 2A to 4 The described experiment has been shown to be a useful model for dry eye 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). Therefore, the human serum preparation can be used for improved therapies for dry eye and its related conditions.

[0148] In one embodiment, the human serum product is used as a medicine or for treating dry eye, and the human serum product is derived from allogeneic blood.

[0149] In one embodiment, the human serum product is used to treat macular degeneration, corneal epithelial damage, conditions caused by or following corneal transplantation, conditions associated with or caused by impaired wound healing, or conditions associated with or caused by ophthalmic laser treatment, and the human serum product is derived from allogeneic blood.

[0150] Allogeneic blood can be obtained by providing and / or merging blood from a number of donors. Such allogeneic blood is beneficial for the production of drugs (i.e., pharmaceuticals) because they balance the compositional variation between donor blood and allow for scale-up of production. In addition, blood from multiple donors can be selected to obtain the desired concentrations described herein. For example, if there is a minimum IGF level, the IGF content in the blood and / or serum of a single donor can be checked and used for merging.

[0151] In one aspect, the present invention relates to an ophthalmic preparation, such as eye drops or gel, containing the above-mentioned preparation. Eye drops and gels are typical preparations that can be used to treat dry eye disease or its related conditions or similar conditions.

[0152] Product manufacturing method

[0153] In one aspect, the present invention relates to a method for preparing a human serum product, comprising the following steps:

[0154] - providing human allogeneic blood volume fractions from multiple donors,

[0155] - selecting a volume fraction from at least two donors,

[0156] - extracting serum from selected volume fractions,

[0157] - combining all sera to obtain a pooled serum, optionally from donors all aged 45 years or less;

[0158] - obtaining an intermediate preparation from the pooled serum, said intermediate preparation comprising at least 100 ng / mL IGF-1; and

[0159] - Obtaining the multi-donor human serum preparation of the present invention from the intermediate preparation.

[0160] The method relies on providing whole blood from a large number of human donors that are selected and pooled to ensure adequate IGF concentrations such that the intermediate product contains 100 ng / mL or more of IGF-1. It has been determined that IGF-1 concentrations in human allogeneic blood vary significantly and decrease with increasing donor age ( Figure 1A ). Therefore, limiting the age of blood donors to 45 years or younger is an efficient production method because less blood needs to be discarded. This selection step ensures that the human serum product has an adequate concentration of IGF-1.

[0161] In one embodiment of the method, the intermediate product is serum, wherein obtaining the intermediate product comprises preparing serum from human allogeneic blood.

[0162] In one embodiment of the method, the intermediate product is a retentate of an ultrafiltration process, wherein obtaining the intermediate product comprises preparing serum from human allogeneic blood, ultrafiltering the serum to obtain a filtrate and a retentate, and selecting the retentate.

[0163] In one embodiment of the method, obtaining the human serum preparation comprises diluting the intermediate preparation.

[0164] In one embodiment, the dilution of the intermediate product comprises diluting the intermediate product, such as serum (from human allogeneic blood), with a common isotonic solution (such as NaCl solution) to a volume percentage of 10-30%, or 15-25%, or about 20%.

[0165] In one embodiment, the product can be obtained by adding recombinant proteins, such as recombinant growth factors.

[0166] In one embodiment of the method, the ultrafiltration process uses a molecular cut-off membrane with a molecular weight cut-off of at least 20 kDa, or at least 30 kDa, or at least 50 kDa, or at least 100 kDa. Studies have found that the retentate contains insulin-like growth factors (IGFs) bound in macromolecular complexes with a molecular size of 30 kDa or greater. The method advantageously provides a product with improved therapeutic efficacy (e.g., for wound healing) and anti-inflammatory efficacy.

[0167] In one embodiment of this method, the product obtained has anti-inflammatory properties, wherein the anti-inflammatory property is expressed as a normalized LPS-induced TNF-α value (as a reference base value), wherein the anti-inflammatory property is 50% or higher, and / or wherein the product contains less than 0.2 mg / dL of C-reactive protein (CRP).

[0168] In one embodiment, obtaining serum from human allogeneic blood includes allowing the blood to coagulate, also known as clotting, and removing the clots and all cells present in the blood. Alternatively, the clots and all cells present in the blood are removed by centrifugation, and the remaining yellowish supernatant is serum.

[0169] In an alternative embodiment, obtaining serum from human allogeneic blood includes providing human allogeneic blood volume fractions from a plurality of donors, selecting volume fractions from at least two donors, obtaining serum fractions from the selected volume fractions, and pooling the selected serum fractions, wherein optionally, all selected volume fractions are from donors aged 45 years or less. In this alternative embodiment, serum is not pooled but blood. Except for this modification, the remaining method steps are the same as the methods in other embodiments of the present invention.

[0170] In one embodiment, obtaining the human serum preparation comprises adding a solution to the intermediate preparation, wherein the solution optionally comprises components and / or additives, such as vitamins, growth factors, pH adjusters, buffers, surfactants, viscosity adjusters, and / or wherein the solution comprises NaCl to make the preparation isotonic with the drug to be applied.

[0171] The method according to the present invention has the advantage that the obtained product does not require the addition of stabilizers during the manufacturing process. Although typical pharmaceutical products may require preservatives, such as benzalkonium chloride (irritating to the eyes), the method according to the present invention can be carried out without such agents. According to the preparation method of the present invention, the intermediate product obtained from human allogeneic blood is not susceptible to microbial contamination and can therefore be manufactured, further processed and stored without preservatives.

[0172] In one embodiment, a method for manufacturing an article is provided, comprising the following steps:

[0173] - Provide human allogeneic blood volume fractions from multiple donors,

[0174] - selecting a volume fraction from at least two donors,

[0175] - extracting serum from selected volume fractions,

[0176] - combining all sera to obtain a pooled serum, optionally from donors all aged 45 years or less;

[0177] - obtaining an intermediate preparation from the pooled serum, said intermediate preparation comprising at least 100 ng / mL IGF-1; and

[0178] - Obtaining the multi-donor human serum preparation of the present invention from the intermediate preparation.

[0179] Advantageously, alginate dressings allow for a controlled slow release of active serum components and / or recombinant growth factors, so these components and factors can be added to alginate dressings. Alginate dressings are a natural wound dressing that contains carbohydrates that cannot be metabolized by the human body. Alginate is a type of isotonic gel that is soft and thixotropic. Cells and / or growth factors can be embedded and / or incorporated into alginate gels, respectively. In order to maintain a constant dose distribution in alginate, the use of ocular implants ( provides a feasible method for delivering active ingredients to the eye in a controlled manner over several hours. The implant is placed in the lower eye. Sodium alginate is preferred as the matrix material because of its solubility in water and its ability to be stabilized at room temperature by the addition of Ca 2+ Ions are easily cross-linked.

[0180] In one embodiment of the method, the majority of donors are 45 years old or younger, 40 years old or younger, preferably 30 years old or younger. In one embodiment of the method, the average age of the majority of donors is 18 years old or older, or 20 years old or older. In one embodiment of the method, the majority of donors are 18 to 45 years old, 18 to 40 years old, or 20 to 30 years old. Age selection is helpful to ensure that the donor serum has a sufficiently high IGF-1 concentration (see Figure 1A ).

[0181] In one embodiment of the method, the intermediate product contains less than 140 mg / dL of monosaccharides and / or 10-200 mg / dL of total cholesterol, wherein total cholesterol is the sum of LDL (low-density lipoprotein), HDL (high-density lipoprotein) and VLDL (very low-density lipoprotein).

[0182] In one embodiment, the method comprises the step of removing viruses, for example by filtering and / or inactivating the viruses.

[0183] Intermediate products

[0184] In an aspect related to human serum products, the present invention provides an intermediate product comprising insulin-like growth factors (IGFs) bound in a macromolecular complex with a molecular weight of 30 kDa or greater, wherein the intermediate product contains 100-500 ng / mL of IGF-1.

[0185] In one embodiment, the intermediate product comprises IGF-1 at a concentration of 100 ng / mL or more, 150 ng / mL or more, 250 ng / mL or more, 500 ng / mL or more. In one embodiment, the intermediate product comprises IGF-1 at a concentration of 3000 ng / mL or less, 2500 ng / mL or less, 2000 ng / mL or less, 1500 ng / mL or less. In one embodiment, the intermediate product comprises IGF-1 at a concentration of 100-3000 ng / mL, 150-2500 ng / mL, 250-2000 ng / mL, or 500-1500 ng / mL.

[0186] In one embodiment, the intermediate preparation comprises 6500 ng / mL or more, 10000 ng / mL or more, or 15000 ng / mL or more IGF-BP3. In one embodiment, the intermediate preparation comprises 40000 ng / mL or less, 35000 ng / mL or less, or 30000 ng / mL or less IGF-BP3. In one embodiment, the intermediate preparation comprises 6500-40000 ng / mL IGF-BP3, 10000-35000 ng / mL IGF-BP3, or 15000-30000 ng / mL IGF-BP3.

[0187] In one embodiment, the intermediate product comprises IGFs bound in a macromolecular complex with a molecular size of 30 kDa or greater, the product comprising IGF-1 bound to IGF binding protein 3 (IGF-BP3), the concentration of IGF-1 being 100 to 3000 ng / mL, and the concentration of IGF-BP3 being 6500 to 40000 ng / mL, 10000 to 35000 ng / mL or 15000 to 30000 ng / mL.

[0188] In one embodiment, the intermediate product comprises IGFs bound in a macromolecular complex with a molecular size of 30 kDa or greater, the product comprises IGF-1 bound to IGF binding protein 3 (IGF-BP3), the concentration of IGF-1 is 100-3000 ng / mL, 150-2500 ng / mL, 250-2000 ng / mL or 500-1500 ng / mL, and the concentration of IGF-BP3 is 6500-40000 ng / mL.

[0189] Detailed description of the drawings

[0190] Figure 1A and Figure 1B The age-dependent IGF-1 concentration and IGF-BP3 concentration in human serum are shown separately, proving that the IGF-1 concentration in human serum decreases with the increase of donor age, while IGF-BP3 only decreases slightly with age. Age is in years. Circles represent clinical diagnosis data, and squares represent published data (Growth Hormone & IGF Research 18 (2008): 228-237). These data show that the age of blood donors is required to be limited to under 45 years old. The concentration of mixed serum IGF-1 is adjusted to 130-280 ng / mL by monitoring the concentration of active IGF-1 (+ / - 20%), and the stability is up to 24 months.

[0191] Figure 2 shows the activity of serum fractions in an in vitro wound closure assay. Human corneal epithelial cells (HCE-T cells) were monitored over time in an in vitro wound closure assay, where the readout of wound area reduction (in %) was based on a defined microinjury (scratch) of the HCE-T cells. Pooled serum was ultrafiltered to produce a separation of high molecular weight fractions (in the retentate) from low molecular weight fractions (in the filtrate). The data showed that, in addition to serum, high molecular weight fractions showed better wound healing than low molecular weight fractions, i.e., wounds healed faster ( Figure 2A Two independent experiments 1 and 2 confirmed the different activities between the high molecular weight fraction and the low molecular weight fraction ( Figure 2B ).

[0192] Figure 3 shows 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, on wound closure. The effects were determined over the following 50 hours by wound closure. The results showed that the serum and HMW fractions were similar in their ability to induce corneal epithelial cell migration and proliferation, with 90% wound closure observed at the 28-hour time point, while the LMW fraction had no effect on wound closure. These results suggest that the bioactive components are primarily present in the HMW fraction.

[0193] The individual rh-growth factors, in order of increasing effect on wound closure, were - PDGF; KGF; IGF-1 and EGF. Wound closure was further accelerated when the rh-growth factors were combined with HMW and, surprisingly, also with LMW, with EGF having the highest closure rate. These results suggest that, with the exception of PDGF, HMW and LMW components act synergistically with all the rh-growth factors analyzed.

[0194] Figure 4 The anti-inflammatory effect of three high molecular weight fraction samples in a lipopolysaccharide (LPS)-TNF-alfa inhibition assay compared to prednisolone (positive control) after storage at -20°C for 6 years is shown. Monocytes were isolated from heparinized blood and stimulated with lipopolysaccharide. Lipopolysaccharide is a surface molecule of Gram-negative bacteria that induces a significant inflammatory response and TNF-α secretion by binding to CD14 molecules. The standardized expression of LPS-induced TNF-α was set as the basal value. Duplicate controls were set and LPS-induced TNF-α secretion was analyzed under the conditions of adding each component. Readings below the baseline value indicate an anti-inflammatory effect, and readings above the baseline value indicate that the corresponding component has an in vitro pro-inflammatory effect. Prednisone acetate (1.0wt.%) (glucocorticoid) was used as a positive control.

[0195] method

[0196] Heterogeneous blood and serum

[0197] After obtaining written informed consent and approval from the ethics committee, venous blood was collected from healthy volunteers by venipuncture, with a maximum of 500 mL per donation, placed in blood bags without anticoagulants. Serum was prepared by placing the collected whole blood at room temperature for at least 2 hours, tested by PTT (partial thromboplastin time) to achieve complete coagulation, and then stored at 4-8°C for 24 hours. The coagulated blood was centrifuged at 3000g for 10 minutes at room temperature, twice. The resulting supernatant (serum) was separated from the blood cake using a manual sampler, combined, and diluted with isotonic NaCl solution to a final concentration of 20 vol.%. After filtering viruses and inactivating pathogens using a 3k filter sterilizer, the serum was divided into 5 or 10 mL aliquots.

[0198] Combined serum ultrafiltration

[0199] The pooled serum (1 L) was diluted with NaCL (0.9%) and ultrafiltered at a flow rate of 100 mL / min using 30 kDa and 100 kDa polyethersulfone (PES) membranes (PALL Centramate T series, Dreieich, Germany) via an Almatechnik (Zeiningen, CH) multipurpose pump. Ultrafiltration was stopped when the retentate volume was 10% of the starting volume.

[0200] Wound closure test

[0201] An in vitro model of immortalized human corneal epithelial cells (HCE-T) in monolayer culture was used to measure corneal epithelial injury and regeneration. Briefly, 4x10 4 Cells were seeded in 24-well plates and cultured in serum-free growth medium (KGM) at 37°C, 5% CO2 humidified conditions until the cells formed an optically confluent monolayer. Frozen serum samples, KGM (keratinocyte growth medium-2, Lonza), supplements, and PBS (phosphate-buffered saline) were placed at room temperature. A sterile 200 μL plastic pipette tip was then used to scrape the wound through the center of the well to form a cell-free wound of 800 to 900 μm, and the wound was washed twice with PBS (1 mL per well) to remove floating cells and destroy the epithelial cell model by mechanical injury. The size of the injury was detected by optical microscopy (Feng Ye et al., Epithelial Wound Healing on Keratin Film, Amniotic Membrane and Polystyrene In Vitro, Current Eye Research 2014; 39 (6): 561-570). Epithelial cells were further cultured on serum-free KGM. Wound healing was evaluated using micrographs. Six photos were taken at each time point for up to 60 hours. The regeneration enhancement effect was calculated as the percentage difference in wound width at different time points. HCE-T cells cultured in KGM served as a negative control. HCE-T cells cultured in KGM supplemented with bovine pituitary extract (10 μg / mL) served as a positive control.

[0202] Inflammation detection

[0203] The anti-inflammatory properties of the serum preparation were determined by a TNF-α inhibition test. Tumor necrosis factor alpha (TNF-α) is a cytokine involved in almost all inflammatory reactions. Macrophages were isolated from at least five donors and pooled and stimulated with LPS (lipopolysaccharide). LPS is a surface molecule of Gram-negative bacteria that can induce a significant inflammatory response, namely, by binding to the CD14 receptor molecule of macrophages, stimulating the secretion of TNF-α in macrophages. A standardized LPS-induced TNF-α release was used as a reference value for inflammation. The preparations described in the present invention were measured (n=3) and compared with the LPS-induced TNF-α secretion. TNF-α secretion concentration values ​​below the reference value are considered to have an anti-inflammatory effect; values ​​greater than this value are considered to have a pro-inflammatory effect. Prednisolone acetate (1.0wt.%) was used as a positive control and culture medium was used as a negative control, respectively, indicating a 100% and 0% reduction in inflammation, respectively.

[0204] HLA class I testing

[0205] Trypsin digestion of HLA class I molecules provides approximately 90% sequence coverage, thereby distinguishing HLA class I molecules from different serum donors. HLA class I molecules are highly polymorphic molecules that vary greatly among human individuals in the population. Therefore, methods for detecting soluble HLA-class I (sHLA) molecules in serum have been developed. The entire HLA gene complex contains approximately 4.000 kb and is generally divided into two genetic regions, where class I genes include sHLA-A, sHLA-B, and sHLA-C. sHLA-A, sHLA-B, and sHLA-C serum molecules are purified from 10 mL of serum by affinity chromatography using anti-HLA-A, anti-HLA-B, and anti-HLA-C polyclonal antibodies. Subsequently, the eluate is further purified by gel permeation chromatography, and the combined fractions are detected by SDS-PAGE, followed by Western blotting using anti-HLA-A, anti-HLA-B, and anti-HLA-C antibodies. 0.5% (w / v) PVP-40 solution was blocked for 30 minutes at room temperature. After spot hybridization of the PVDF membrane with HLA-A, HLA-B and HLA-C proteins, the PVDF membrane was wetted with methanol and washed with water 10 times. Subsequently, it was reduced and alkylated with 10mM DTT and 55mM IDA, respectively. The PVDF membrane was then immersed in 25μL 25mM NH4HCO3 and heated at 95°C for 5 minutes to denature the protein. Subsequently, 25μL trypsin solution (10μL 40 ng / μL trypsin, 5μL 5% Tween-20 and 10μL acetonitrile) was added and incubated at 37°C overnight. The trypsin-digested peptides were extracted from the PVDF membrane three times with 40μL of 5% (v / v) trifluoroacetic acid and 45% H2050% (v / v) ACN at room temperature for 1 hour. The extracts were dried by speed vacuum and resuspended in 20 μL 0.1% TFA for mass spectrometry analysis.

[0206] The mass spectrometry sequence analysis of HLA class I molecules was based on the detection of tryptic peptides. The samples were measured using an LTQ Orbitrap VelosPro system (Thermo Fisher Scientific, Bremen, Germany) connected online to a U3000 RSLCnano (Thermo Fisher Scientific, Idstein, Germany) using an Acclaim PepMap analytical column (75 μm × 500 mm, 2 μm, Sequester software 2.2SP1.48 (Thermo Fisher Scientific, Bremen, Germany) was used for data-dependent tandem mass spectrometry (MS / MS) analysis (https: / / www.sciencedirect.com / topics / biochemistry-genetics-and-molecular-biology / sequest). Extracted ion chromatograms (XICs) of standard and analytical peptides were generated using Qual Browser embedded 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 / ).

[0207] Example

[0208] Preparation of multi-donor human serum eye drops

[0209] Briefly, up to 500 mL of venous whole blood was collected from male donors aged 18 to 40 years (n=5). The blood was collected in multi-bag blood collection bags without anticoagulant (Compoflex Fresenius). The blood was placed at 18-22°C (RT) for at least 2 hours and then stored at 2-8°C. Within 24 hours after blood collection, the supernatant (serum) was extracted by centrifugation at 3000 x g for 10 minutes at RT twice using a manual extractor. The serum (1 L) was pooled to obtain an intermediate product and sampled for quality control analysis. The blood was collected using an Aero Pump Ophthalmic Multi-Dose System under grade A / B clean conditions. The pooled serum was adjusted to the corresponding concentration [diluted to 50% or 20% with NaCl (0.9%)] using a graduated syringe with a filter (0.22 μm) in the system and divided into 5 mL aliquots, followed by release control. Subsequently, the eye drop bottles were frozen and stored at -20°C or lower for future use.

[0210] Prepare intermediate products by ultrafiltration to obtain high molecular weight components and low molecular weight components

[0211] The combined intermediate serum product (1 L) obtained in Example 1 was separated into low molecular weight (LMWF) and high molecular weight (HMWF) parts by ultrafiltration. That is, the combined serum was diluted with 0.9% NaCl and ultrafiltered using a multipurpose pump of Almatechnik (Zeiningen, CH) at a flow rate of 100 mL / min using a polyethersulfone (PES) membrane (PALL Centramate T series, Dreieich, Germany) with a molecular weight cutoff of 30 kDa and 100 kDa, respectively. When the retention volume reached 10% of the starting volume, the ultrafiltration was stopped. Samples were taken from the mixed serum and the corresponding HMWF and LMWF, and a more detailed analysis was performed by testing the regenerative epithelial efficacy and anti-inflammatory efficacy.

[0212] Comparison of the re-epithelialization efficacy of low molecular weight fraction (LMWF) and high molecular weight fraction (HMWF) and unfiltered multi-donor pooled serum in corneal HCE-T cell assay

[0213] Figure 2 shows the activity of serum fractions in an in vitro wound closure assay. Human corneal epithelial cells (HCE-T cells) were monitored over time in an in vitro wound closure assay, and the readout of wound area reduction (in %) was based on HCE-T cells with defined microinjuries (scratches). Ultrafiltration of pooled serum allowed separation of high molecular weight fractions (in the retentate) from low molecular weight fractions (in the filtrate). The data showed that, in addition to serum, high molecular weight fractions had better wound healing than low molecular weight fractions, i.e., wound healing was faster ( Figure 2A Two independent experiments 1 and 2 confirmed the different activities between the high molecular weight fraction and the low molecular weight fraction ( Figure 2B ).

[0214] Detection of bound IGF-1 in macromolecular complexes of multi-donor human serum preparations in the HMWF fraction (retentate)

[0215] HMWF samples were measured using the LTQ Orbitrap Velos Pro system (see Methods, section “HLA-class I Assay”). This method allowed the identification of albumin, IGF-1BP3-ALS complex, α-2 macroglobulin, PDGF-BB, TGF-β1, VEGF, and PEDF. The concentrations of selected physiological components in the corresponding filtrates (30 kDa and 100 kDa) were analyzed as shown in the table below.

[0216] Characterization of intermediate products

[0217] In the first approach, the concentrations of the components were determined experimentally under laboratory conditions. Next, the concentrations of the selected components were determined under contract in a certified diagnostic laboratory (MVZ Karlsruhe). The results are shown in Table 1. The regenerative capacity to close epithelial defects was analyzed in an in vitro wound healing test (HCE-T cell test). The anti-inflammatory capacity was determined in a TNF-α inhibition test.

[0218] Table 1: Determination of component concentrations of multi-donor intermediate products

[0219]

[0220]

[0221] nd: below the detection limit

[0222] Product formula and characteristics

[0223] The intermediate preparation (pooled serum) was adjusted to the desired concentration using NaCl (0.9%). Table 2 lists the component contents and additional quality control analyses performed to characterize the product.

[0224] Table 2

[0225]

[0226]

Claims

1. A multi-donor human serum product, wherein: The preparation comprises insulin-like growth factors (IGFs) bound in a macromolecular complex having a molecular size of at least 30 kDa, at least 20 ng / mL of IGF-1, and less than 70 mg / dL of monosaccharides; The preparation is an isotonic solution or an isotonic gel.

2. The preparation according to claim 1, comprising less than 40 mg / dL of total cholesterol, wherein total cholesterol is the sum of LdL (low-density lipoprotein) and HdL (high-density lipoprotein).

3. The product according to claim 1 or 2, wherein In an in vitro wound closure test, the preparation has a wound closure performance of 50% or more at 35 hours based on the wound area.

4. The product according to any one of claims 1 to 3, wherein The preparation has anti-inflammatory properties in an inflammation test, wherein the anti-inflammatory properties are standardized and expressed as LPS-induced TNF-α values ​​(as a reference base value), wherein the anti-inflammatory properties are 50% or more, And / or the preparation comprises less than 0.2 mg / dL of C-reactive protein (CRP).

5. The preparation according to any one of claims 1 to 4, comprising one or more growth factors selected from the group consisting of EGF, IGF-1, IGF-BP3, IGF-2, PDGF-BB, α2-macroglobulin, KGF and PEDF.

6. The preparation according to any one of claims 1 to 5, comprising one or more growth factors within the following concentration ranges: -50~500pg / mL EGF or 20~200pg / mL EGF; -50 to 250 ng / mL IGF-1 or 20 to 50 ng / mL IGF-1; -650 to 4000 ng / mL of IGF-BP3 or 260 to 1600 ng / mL of IGF-BB3; -450 to 6000 ng / mL ALS or 180 to 2400 ng / mL ALS; - 0.37 to 1.49 g / L α2-macroglobulin or 0.15 to 0.6 g / L α2-macroglobulin; -471 to 3683 pg / mL or 188.4 to 1473.2 pg / mL of PDGF-BB; -2 to 100 pg / mL KGF; -1~30ng / mL TGF-β1 or 0.4~12ng / mL TGF-β1; -30 to 222.5 pg / mL VEGF or 12 to 89 pg / mL VEGF; - 60 to 5000 ng / mL PEDF or 24 to 2000 ng / mL PEDF; and -1750 to 2600 mg / dL albumin or 700 to 1040 mg / dL albumin.

7. Use of the product according to any one of claims 1 to 6 in medicine.

8. Use of the preparation according to any one of claims 1 to 6 in treating dry eye.

9. The product for medical use according to claim 7, or the product for use in treating dry eye according to claim 8, wherein: The human serum composition is derived from allogeneic blood.

10. An ophthalmic composition, such as an eye drop or a gel, wherein: The ophthalmic composition comprises the preparation according to any one of claims 1 to 6.

11. A method for preparing a product, comprising the following steps: - Provide human allogeneic blood volume fractions from multiple donors, - selecting a volume fraction from at least two donors, - extracting serum from selected volume fractions, - combining all sera to obtain a pooled serum, optionally from donors all aged 45 years or less; - obtaining an intermediate preparation from the pooled serum, said intermediate preparation comprising at least 100 ng / mL IGF-1; and - Obtaining a multi-donor human serum preparation as claimed in any one of claims 1 to 6 from said intermediate preparation.

12. The method according to claim 11, wherein: (i) the intermediate product is serum, and obtaining the intermediate product comprises preparing serum from human allogeneic blood; or (ii) the intermediate product is a retentate of an ultrafiltration process, and obtaining the intermediate product comprises preparing serum from human allogeneic blood, ultrafiltering the serum to obtain a filtrate and a retentate, and selecting the retentate.

13. The method according to claim 12, wherein: The ultrafiltration process of step (ii) is performed using an ultrafiltration membrane with a molecular weight cutoff of at least 20 kDa, or at least 30 kDa, or at least 50 kDa or at least 100 kDa, or wherein the step of obtaining a human serum product further comprises diluting the intermediate product.

14. The method according to any one of claims 11 to 13, wherein: The intermediate preparation contains less than 140 mg / dL of monosaccharides, and / or 10 to 200 mg / dL of total cholesterol, wherein total cholesterol is the sum of LdL (low density lipoprotein), HdL (high density lipoprotein) and VLdL (very low density lipoprotein).

15. An intermediate preparation comprising insulin-like growth factor (IGF) bound in a macromolecular complex having a molecular size of 30 kDa or greater, the intermediate preparation comprising 100-500 ng / mL of IGF-1.

Images