Stable human platelet lysate compositions, methods and uses thereof

Abstract

The present disclosure relates to a stable human platelet lysate composition; preferably a stable heparin-free human platelet lysate composition; the invention also relates to a method for obtaining the composition and to the use thereof.

Description

Technical Field

[0001] The present disclosure relates to stable human platelet lysate compositions; methods for obtaining the compositions and uses thereof. Background Art

[0002] The demand for standardized and safe culture systems, including cell culture supplements, is growing rapidly worldwide. In this regard, human platelet lysate (hPL) has attracted a lot of attention as a raw material / auxiliary material for biotechnological applications (at both research and clinical levels). Prepared from human plasma or platelet concentrates, hPL contains high concentrations of growth factors that enhance cell proliferation and is superior to fetal bovine serum (FBS), the gold standard for cell culture supplementation.

[0003] While the requirements for basic research are lower, clinical cell manufacturing has higher regulatory and compliance requirements. The continued advancement and clinical translation of cell therapies requires xeno-free cell culture media produced according to GMP standards. Although hPL is a human-derived product, its wider application is limited by the high fibrinogen content, which binds to the calcium present in the cell culture medium and induces the formation of fibrin clots, necessitating the addition of heparin (anticoagulant) to the cell culture medium supplemented with hPL (to prevent clotting). Non-xeno-free cell culture processes can be achieved using porcine-derived heparin. In this case, heparin supplementation can negatively affect the quality of the product because (i) heparin may directly affect cell growth due to interactions with multiple growth factors, (ii) recombinant heparin (i.e., an alternative to porcine reagents) is not as efficient in preventing clot formation (i.e., small clots formed when hPL is thawed), and (iii) the addition of heparin is an additional step in the preparation of cell culture media [1]. Therefore, in order to provide a more user-friendly and quality-enhanced cell culture supplement, fibrinogen should be removed from the final formulation.

[0004] The most effective mechanism for removing fibrinogen from hPL is the addition of Ca during manufacturing. 2+ (in the form of CaCl2 at a final concentration of 20 mM) to induce fibrin formation and subsequent clotting [2]. This approach ensures that the majority of fibrinogen, as well as other clotting factors, are removed (as evidenced by the lack of clotting when used as a supplement to cell culture media), while minimizing the impact on the distribution and content of growth factors in the resulting hPL product.

[0005] Ideally, methods for removing the fibrinogen content of hPL should be performed below the maximum physiological temperature to maintain the content and quality of the growth factors, minimize or even avoid the use of reagents of animal or recombinant origin, provide a product free of visible particles and low levels of microscopic particles, and be able to meet industry and regulatory standards, including those of good manufacturing practices.

[0006] Document EP2723354 [3] describes a method for preparing an hPL composition comprising the following steps: (a) lysing platelets to provide a lysate; (b) removing cell debris; and (c) removing fibrinogen by adding a metal salt such as calcium chloride and a composition comprising a heparinized platelet lysate from which fibrinogen has been removed to form a removable material.

[0007] Document EP3638263 [4] relates to a method for obtaining a mixed human platelet lysate, the mixed human platelet lysate itself and its use for treating neurological diseases such as neurodegenerative diseases, neuroinflammatory diseases, neurodevelopmental diseases and / or neurovascular diseases. EP3638263 relates to a method for preparing a heat-treated mixed human platelet lysate, the method comprising the following steps: a) providing a mixed human platelet lysate (pHPL), b) heat-treating the mixed human platelet lysate at a temperature of 50°C to 70°C for 20 to 40 minutes, and c) purifying the heat-treated mixed human platelet lysate of step b) using glass beads and CaCl2.

[0008] The document DOI: 10.1039 / C9TB01764J [5] describes a method for obtaining a mixed platelet lysate, which is itself used in a culture medium for the expansion / culture of human mesenchymal stem cells. DOI: 10.1039 / C9TB01764J [5] describes a method comprising the following steps: a) freeze and thaw cycles (-80°C / 37°C) and centrifugation (3000 x g for 30 min at 20°C, b) activation of platelets with calcium chloride (23 mM) in the presence of glass beads at 24-26°C and removal of fibrin, and c) centrifugation of the suspension and heat treatment of the supernatant at 56°C for 30 min followed by centrifugation to separate insoluble proteins.

[0009] However, EP3638263 [4] used heparin, while EP3638263 [4] and DOI: 10.1039 / C9TB01764J [5] used heat treatment of hPL to reduce the fibrinogen content. EP3638263 [4] and DOI: 10.1039 / C9TB01764J [5] did not report the critical quality attributes of hPL, including the composition of particulate matter and growth factors.

[0010] These facts are disclosed to illustrate the technical problems solved by the present disclosure. Summary of the invention

[0011] The present disclosure relates to a method for producing xeno-free human platelet lysate (further referred to as XF-hPL). Using the method described in the present disclosure, an hPL product with low fibrinogen content, high growth factor content and quality can be produced, which can meet performance and regulatory requirements, whether as a raw material for cell expansion or advanced therapeutic drug production, or as a raw material for the production of biopharmaceutical products.

[0012] A stable human platelet lysate composition comprising less than 5 μg / ml of fibrinogen and a mass ratio of PDGF-BB growth factor / fibrinogen content of not less than 6 ng / μg. Unexpectedly, the composition of the present disclosure avoids the need to add heparin because it does not coagulate and / or does not induce clot formation. The composition of the present disclosure is suitable for use as a heparin-free composition as an improved cell culture medium.

[0013] In one embodiment, the composition of the present disclosure is a heparin-free composition.

[0014] In one embodiment, the composition of the present disclosure is a xeno-free composition.

[0015] In one embodiment, the compositions of the present disclosure may comprise less than 4 μg / ml of fibrinogen; preferably less than 3 μg / ml of fibrinogen.

[0016] In one embodiment, the mass ratio of PDGF-BB growth factor / fibrinogen content is not less than 8 ng / μg; preferably not less than 10 ng / μg.

[0017] In one embodiment, the growth factor may be selected from the list consisting of BDNF, EGF, FGF-2, GM-CSF, HGF, IL-1β, IL-8, PDGF-AB, PDGF-BB, VEGF, and combinations thereof.

[0018] In one embodiment, the mass ratio of growth factor (nanograms) per microgram of fibrinogen (wherein the growth factor is selected from BDNF, EGF, VEGF-A or a combination thereof) ranges from 0.5 to 2.0 ng. 生长因子 / μg 纤维蛋白原 , preferably 1.0-1.5ng 生长因子 / μg 纤维蛋白原 .

[0019] In one embodiment, the mass ratio of growth factor (nanogram) per microgram of fibrinogen (wherein the growth factor is selected from FGF-2, GM-CSF, HGF, IL-1β or a combination thereof) ranges from 0.05-0.5 ng 生长因子 / μg 纤维蛋白原, preferably 0.1-0.4 ng 生长因子 / μg 纤维蛋白原 .

[0020] In one embodiment, the mass ratio of growth factor (nanograms) per microgram of fibrinogen (wherein the growth factor is selected from PDGF-AB, PDGF-BB or a combination thereof) ranges from 4 to 12 ng. 生长因子 / μg 纤维蛋白原 , preferably 6-8 ng 生长因子 / μg 纤维蛋白原 .

[0021] In one embodiment, the composition of the present disclosure may comprise a ratio of growth factor (nanograms) per microgram of fibrinogen in the following range: 0.5-2.0 ng 生长因子 / μg 纤维蛋白原 (Growth factors include BDNF, EGF, VEGF-A), 0.05-0.5 ng 生长因子 / μg 纤维蛋白原 , (wherein the growth factors are FGF-2, GM-CSF, HGF, IL-1β), and 4-12 ng 生长因子 / μg 纤维蛋白原 , (wherein the growth factors are PDGF-AB, PDGF-BB), preferably 1.0-1.5 ng 生长因子 / μg 纤维蛋白原 , (growth factors include BDNF, EGF, VEGF-A), 0.1-0.4 ng 生长因子 / μg 纤维蛋白原 , (wherein the growth factors are FGF-2, GM-CSF, HGF, IL-1β), and 6.0-8.0 ng 生长因子 / μg 纤维蛋白原 , (the growth factors are PDGF-AB and PDGF-BB).

[0022] In one embodiment, the composition of the present disclosure has a transparent yellow color with no visible particles detectable by visual observation, more preferably no insoluble visible particles.

[0023] In one embodiment, the compositions of the present disclosure may comprise a clarity and opalescence degree as measured by nephelometry of less than 400 NTU; preferably less than 350 NTU; more preferably less than 300 NTU.

[0024] In one embodiment, the compositions of the present disclosure may comprise clarity and opalescence in the range of 100-350 NTU; more preferably 150-200 NTU as measured by nephelometric method.

[0025] In one embodiment, the composition of the present disclosure may contain sub-visible particles, as measured by flow imaging microscopy (FIM), the number of sub-visible particles up to 25 μm in the composition of the present disclosure is less than 2.0 x 10 7 particles / mL, preferably less than 1.0 x 10 7 particles / mL, more preferably less than 5.0 x 10 6 Particles / mL.

[0026] In one embodiment, the composition of the present disclosure may comprise microscopic particles, as measured by flow imaging microscopy (FIM), with the number of particles less than 25 μm being 5.0 x 10 6 In another embodiment, the number of particles less than 5 μm in the composition of the present disclosure is 3.9x10 6 In another embodiment, the composition of the present disclosure comprises less than 1.3 x 10 5 Microscopic particles with a size of 5-10 µm were detected at 100 particles / mL.

[0027] In one embodiment, the compositions of the present disclosure may comprise undetectable amounts of fibrinogen beta chains as measured by proteomic analysis using an LC-MS method.

[0028] In one embodiment, the composition of the present disclosure may be stable for up to 6 months under refrigerated conditions of -80°C to 5°C, preferably -20°C to 4°C.

[0029] Another aspect of the present disclosure relates to a cell culture medium comprising the stable human platelet lysate composition of the present disclosure.

[0030] Another aspect of the present disclosure relates to a pharmaceutical product incorporating the stable human platelet lysate composition of the present disclosure.

[0031] Another aspect of the present disclosure relates to a method for obtaining a stable human platelet lysate, comprising the following steps:

[0032] - Obtain frozen human platelet units;

[0033] - subjecting the human platelet units to thaw / freeze cycles to disrupt the platelet membranes;

[0034] - adding silicon dioxide and calcium chloride to the mixture obtained in the previous step to induce clot formation;

[0035] - incubating the sample at a temperature ranging from 35°C to 39°C for up to 1 hour to 4 hours;

[0036] - Manual clot disruption;

[0037] - Centrifuge the sample and collect the resulting supernatant;

[0038] - Filtering the supernatant to obtain a stabilized human platelet lysate; preferably a stabilized heparin-free human platelet lysate.

[0039] In one embodiment, a combination of 0.005-0.04% (m / v) silicon dioxide and 0.5-1.0% calcium chloride is added.

[0040] Another aspect of the present disclosure relates to a heparin-free human platelet lysate obtained by the method of the present disclosure.

[0041] In one embodiment, the methods disclosed in the present disclosure produce an improved XF-hPL compared to the prior art. That is, the product is clear and free of visible particulate matter, the fibrinogen content is consistently less than 5 µg / mL, and proteomic analysis does not detect fibrinogen beta chains; the final product does not clot after reconstitution, i.e., is easier to handle; does not contain heparin during the manufacturing or reconstitution process; and it does not contain heparin substitutes or any additional animal-derived components.

[0042] In one embodiment, the starting materials for the XF-hPL formulation include human platelet units (hPU), including hPU that can no longer be used for transfusion purposes, stored at -80°C to -20°C, and the following reagents are added to the mixed starting materials during the manufacturing process: calcium chloride (aqueous solution, 1 M concentration); and silicon dioxide (high purity grade, containing 3-7% (m / m) water).

[0043] In one embodiment, the stable human platelet lysate composition of the present disclosure can be obtained by the disclosed method and is:

[0044] - Xeno-free products, no heparin or any other animal-derived substitutes;

[0045] - Very low fibrinogen content due to the fibrinogen removal process;

[0046] -High content of growth factors at levels similar to or higher than non-xeno-free products or other commercially available xeno-free products;

[0047] -Has no detectable visible particles and reduced levels of microscopic particles that are smaller than other commercially available xeno-free products. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The following drawings are provided to illustrate preferred embodiments of the present disclosure and should not be considered to limit the scope of the present disclosure.

[0049] Figure 1 : Representative picture of primary packaging used for filling XF-hPL.

[0050] Figure 2 : Graphical representation of one embodiment of the XF-hPL final product quality control results (analytical parameters). Characteristics include total protein content, osmolarity, pH, fibrinogen content, and concentrations of the growth factors EGF (epidermal growth factor) and PDGF-AB (platelet-derived growth factor AB). Values ​​are expressed as mean ± standard deviation of at least three different batches. The dashed line represents the range defined in the QC panel.

[0051] Figure 3 : Graphic representation of one embodiment of XF-hPL final product quality control results (growth factor content determined by a multiplex bead-based assay).

[0052] Figure 4 : Graphic representation of one embodiment of XF-hPL final product quality control results (growth factor content normalized to fibrinogen content in the sample).

[0053] Figure 5 : Graphic representation of one embodiment of XF-hPL final product quality control results (product performance). Cell proliferation of hASCs and HFF-1 cells cultured in medium supplemented with XF-hPL (5% v / v; representative batch) or FBS (10% v / v) for 7 days, measured by DNA quantification using the PicoGreen assay. Values ​​are expressed as mean ± standard deviation of at least three (3) different batches. Statistical analysis was performed using the Kruskal-Wallis test followed by the Dunn's multiple comparison test. *p<0.05 compared to hASCs cultured in medium supplemented with 10% FBS.

[0054] Figure 6 : Graphical representation of one embodiment of product stability results under accelerated conditions (4°C), long-term storage conditions (-20°C, -80°C) and different time periods (1 month, 3 months and 6 months). The dotted lines represent the range defined in the QC panel.

[0055] Figure 7 : Description of the process used to manufacture XF-hPL. DETAILED DESCRIPTION

[0056] The present disclosure relates to a stable human platelet lysate composition; preferably a stable heparin-free human platelet lysate composition; a method for obtaining the composition and its use.

[0057] In one embodiment, the hPU undergoes up to three freeze-thaw cycles (at -80°C and 37°C, respectively) to disrupt the platelet membrane and release the cytoplasm and granule contents. The hPU is then transferred and mixed into a disposable sterile container (e.g., a large-volume sterile bag) using a plasma manual processor, and samples are collected for process control, including fibrinogen quantification (competitive ELISA assay) and total protein content (BCA assay).

[0058] In one embodiment, after control analysis, the mixed hPL is divided into smaller individual disposable sterile bags.

[0059] In one embodiment, calcium chloride and silicon dioxide are added to the mixed hPL to induce clot formation. The formation of a uniform clot can be confirmed by naked eye. In another embodiment, the final concentration of calcium chloride in the hPL solution ranges from 5.0 mM to 10 mM, preferably from 6.0 mM to 8.0 mM, and more preferably from 7.5 mM; and the final concentration of silicon dioxide in the hPL solution ranges from 0.005% to 0.04% (w / v), preferably from 0.01% to 0.03% (w / v), and more preferably from 0.02% (w / v).

[0060] In one embodiment, after adding calcium chloride and silicon dioxide, incubation is carried out at a temperature ranging from 35°C to 39°C, preferably 37°C, for up to 1 hour to 4 hours, preferably 2 hours, more preferably 1.5 hours.

[0061] In one embodiment, the clotted material is stored at a temperature in the range of 2°C to 25°C, preferably 15-23°C, for a period of up to 16 hours to 20 hours.

[0062] In one embodiment, the clotted material is manually disrupted and centrifuged at 3500 to 5000 x g, preferably 4000 x g, for 10-20 minutes, preferably 15 minutes at 15-25° C. In another embodiment, the resulting supernatant is collected and combined into disposable sterile bags; samples are collected for process control analysis, such as fibrinogen quantification (competitive ELISA assay), total protein content (BCA assay), and sterility assessment (BacT / Alert microbial detection system).

[0063] In one embodiment, the combined supernatants are terminally sterile filtered, preferably by filtration, more preferably by using a 0.22 µm filter (to remove bacteria with cell walls) and a 0.1 µm filter (to remove mycoplasmas, i.e. bacteria without cell walls). The sterile material is then stored in primary packaging at -80°C to -20°C, preferably -72°C to -78°C ( Figure 1 ) until further use.

[0064] In one embodiment, the appearance of the final product is visually assessed (i) after filling the primary package and (ii) after storage at -80°C and thawing at room temperature according to European Pharmacopoeia (Eur. Ph.) 2.2.1, Clarity and degree of opalescence of liquids (Visual method), and 2.9.20, Particle contamination: Visible particles.

[0065] In one embodiment, the clarity and opalescence of the final product are assessed by turbidity measurement according to Eur. Ph. 2.2.1, Clarity and degree of opalescence of liquids (Instrument method). In one embodiment, the presence of microscopic particles in the final product is assessed by imaging flow microscopy (FlowCam). This method can detect and characterize each individual particle detected in the sample by providing real-time high-resolution images along with particle size, count, shape and other parameters. The amount of microscopic particles was quantified for a representative batch of XF-hPL and a commercially available defibrinogen-free xeno-hPL reference sample (further referred to as sample A). The table below shows that XF-hPL has significantly less microscopic particles in all particle size ranges compared to sample A, with a focus on microscopic particles up to 25 µm.

[0066] Table 1 shows the number of microscopically visible particles in 1 ml of the sample of the present disclosure and 1 ml of sample A (i.e., a commercially available defibrinogen-free xeno-hPL reference sample).

[0067]

[0068] In one embodiment, sterility is assessed by detecting aerobic / anaerobic organisms in samples of the final product by inoculating the samples in BacT / Alert culture bottles and incubating them in the BacT / Alert Microbial Detection System for 14 days according to Eur. Ph. 5.1.6.

[0069] In one embodiment, mycoplasma contamination in samples of the final product is detected by quantitative real-time PCR (qRT-PCR) using the Venor® GeM qEP kit according to Eur. Ph. 2.6.7.

[0070] In one embodiment, endotoxin contamination in final product samples is detected by gel-clot assay according to Eur. Ph. 2.6.14.

[0071] In one embodiment, the pH of the final product sample is measured using a pH meter according to Eur. Ph. 2.2.3.

[0072] In one embodiment, the osmotic pressure level in the final product sample is measured using an osmometer according to Eur. Ph. 2.2.35.

[0073] In one embodiment, according to Eur. Ph. 2.5.33 and USP <1057> Protein determination: The total protein content in the final product samples was measured using the BCA assay (ThermoFisher).

[0074] In one embodiment, the human growth factors epidermal growth factor (EGF) and platelet-derived growth factor AB (PDGF-AB) are quantified in the final product sample by ELISA assay according to the manufacturer's instructions.

[0075] In one embodiment, human adipose-derived stromal / stem-like cells (hASC) and a human skin fibroblast cell line (HFF-1) were used to evaluate the performance of the final product samples as a cell culture medium supplement compared to non-xeno-free hPL and advanced FBS. Cell proliferation was measured after 7 days of expansion by DNA quantification using the PicoGreen kit.

[0076] In one embodiment, fibrinogen quantification is performed by competitive ELISA assay using a commercially available kit (Abnova, code KA0475) that allows the detection of fibrinogen in human plasma samples according to the manufacturer's instructions. Briefly, the kit uses a 96-well microplate (with removable strips) pre-coated with mouse antibodies specific for the protein. Fibrinogen in standards and samples competes with biotinylated human fibrinogen protein sandwiched between an immobilized antibody and a streptavidin-peroxidase (SP) conjugate. All unbound material is washed away and a peroxidase substrate is added. Color development is stopped and the color intensity (proportional to the fibrinogen concentration) is measured.

[0077] In one embodiment, human brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), basic fibroblast growth factor (FGF-2), granulocyte-macrophage colony stimulating factor (GM-CSF), hepatocyte growth factor (HGF), interleukin 1β (IL-1β), interleukin 8 (IL-8), platelet-derived growth factor BB (PDGF-BB), and vascular endothelial growth factor A (VEGF-A) are quantified in the final product sample by a bead-based immunoassay using a 9-Plex Human ProcartaPlex Multiplex Plate (ThermoFisher Scientific) compatible with the MAGPIX Luminex instrument according to the manufacturer's instructions. The ProcartaPlex immunoassay is based on the principle of a sandwich ELISA, using two highly specific antibodies that bind to different epitopes of a protein to quantify all protein targets simultaneously. In contrast to ELISA assays, capture antibodies (specific for a single target protein) are coupled to spectrally unique beads (not adsorbed to the microplate wells), and bound proteins are identified with biotinylated antibodies and streptavidin-R-phycoerythrin (RPE). Coupling of protein-specific antibodies to different beads allows analysis of multiple targets in a single well.

[0078] In one embodiment, the protein content of the final product samples is analyzed by liquid chromatography-mass spectrometry (LC-MS) [6]. Briefly, the samples were diluted in ultrapure water to a final concentration of 10 µg / mL, mixed with 4x Laemmli sample buffer (Bio-Rad) to a final concentration of 1x, incubated at 95°C for 5 minutes, cooled to room temperature and stored at -20°C. The samples were analyzed on a NanoLC™ 425 system (Eksigent) coupled to a Triple TOF™ 6600 mass spectrometer (Sciex) and an ionization source (ESI DuoSpray™ source).

[0079] In one embodiment, silica and calcium in the final product sample are quantified by inductively coupled plasma optical emission spectroscopy (ICP-OES). Briefly, the sample is first digested with sulfuric acid (H2SO4) and perchloric acid (HClO4), and then digested with hydrofluoric acid (HF). The digested solution is quantified according to the standard curves of silica and calcium.

[0080] In one embodiment, scalability from a small laboratory scale (50-500 mL) to an industrial scale (50 L) was evaluated, and quality control (QC) testing showed that the final product had a clear yellow appearance (without any visible particles or clots) and analytical parameters were very consistent between batches ( Figure 2 ),Right now:

[0081] -Total protein: 5.26 ± 0.52 g / dL;

[0082] -Osmolality: 320.5 ± 3.6 mOsm / kg;

[0083] -pH: 7.83 ± 0.15;

[0084] - Epidermal growth factor (EGF) content: 2455 ± 302 pg / mL (ELISA-based assay);

[0085] - Platelet-derived growth factor AB (PDGF-AB) content: 31027 ± 9354 pg / mL (ELISA-based assay).

[0086] The fibrinogen levels measured for all batches produced by the described method were less than 5 µg / mL (3.05 ± 1.04 µg / mL) ( Figure 2 ), confirming that the silica-based approach can consistently remove fibrinogen from hPL, regardless of the scale of production. Furthermore, residual levels of silica in the final product (approximately 3 ppm) were similar to basal plasma levels, indicating that the clot and subsequent processing (including filtration) removes all silica added during manufacturing.

[0087] In one embodiment, a total of nine (9) growth factors and cytokines (BDNF, EGF, FGF-2, GM-CSF, HGF, IL-1β, IL-8, PDGF-BB, and VEGF-A) were quantified in samples of the final product by a bead-based multiplex assay using a 9-Plex multiplex plate compatible with the MAGPIX Luminex instrument. Different samples were used for comparison, namely commercially available xeno-free hPL depleted of fibrinogen (sample A) and commercially available xeno-free hPL containing fibrinogen (further referred to as sample B and sample C). In this regard, it was found that the growth factor levels ( Figure 3 ) and the amount of growth factor (ng) per microgram of fibrinogen (in each sample) ( Figure 4 ) were similar or higher than the reference samples included in the assay. Importantly, when relative fibrinogen levels were considered, the proposed method produced higher concentrations of growth factors ( Figure 4 ).

[0088] In one embodiment, the absorbance of a 200 μl volume of the hPL sample at a wavelength of 410 nm is measured using the device Synerg HTX and the software GEN5. The optical density of each sample is calculated.

[0089] In one embodiment, product characterization includes performance assays using two different cell types, human adipose-derived stem / stromal-like cells (hASC) and human dermal fibroblasts (HFF-1). Product performance is assessed by measuring the proliferation of hASC or HFF-1 cells expanded in medium supplemented with XF-hPL (at 5% v / v) and compared to the proliferation of cells cultured in medium supplemented with 10% FBS (i.e., the standard supplement for these cells). Cell proliferation is measured by quantifying DNA extracted from cells in the culture dish (using the Quant-iT™ PicoGreen™ double-stranded DNA (dsDNA) assay), with the amount of DNA extracted being proportional to the total number of cells in the culture surface. As Figure 5 As shown, enhanced proliferation of hASCs and HFF-1 cells cultured in medium supplemented with 5% XF-hPL for 7 days was detected compared to that detected for cells expanded in medium supplemented with 10% FBS.

[0090] In another embodiment, detailed characterization of the protein content provides further analytical data to confirm the biochemical characteristics of the product. In contrast to chemically defined media, the composition of hPL products is not defined. As cell therapy manufacturers move toward more defined cell therapy products, more extensive and detailed characterization of media supplements is highly desirable. Therefore, proteomic analysis of XF-hPL samples was performed by liquid chromatography-mass spectrometry (LC-MS) [6]; commercially available XF-hPL (sample A) was used as a reference for comparison. Immunoglobulins were the most represented protein family detected (N = 57, including immunoglobulin chains), followed by apolipoproteins and complement cascade members (N = 12). Cytoskeletal and cytoskeletal-associated proteins (e.g., actin, tubulin, filamin, talin, myosin, focal adhesion proteins), oxygen and iron / copper transporters (hemoglobin, transferrin, ceruloplasmin), and albumin were also identified in all final product samples. Interestingly, while both α and β chains of fibrinogen were detected in the reference sample, only the α chain was detected in XF-hPL, indicating that the residual level of fibrinogen in the final product (quantified by ELISA assay) was derived from its α chain. The low fibrinogen content, as well as the absence of fibrin in the final product, is expected to improve the safety of XF-hPL, as fibrinogen and fibrin have been implicated in the regulation of inflammation [7].

[0091] Furthermore, data analysis allowed the identification of proteins present in XF-hPL at much higher levels than in reference sample A (ratio > 2), including (i) chaperones (endoplasmic reticulum chaperone protein BiP, heat shock protein HSP90-α), (ii) regulators of actin function (coronin-1A), (iii) protease inhibitors (human kallikrein binding protein), (iv) immunoglobulins (immunoglobulin heavy variant 3-43), and (v) integrin-associated proteins (integrin-linked protein kinase).

[0092] In one embodiment, stability studies have shown that the final XF-hPL product can maintain its properties after storage under refrigerated conditions (4°C, -20°C, -80°C) for up to 6 months, such as Figure 6 shown.

[0093] The results presented here demonstrate a novel method for producing ready-to-use XF-hPL without the use of heparin or any animal-derived alternatives, which results in very low fibrinogen content, a high growth factor / fibrinogen content ratio, and excellent performance as a cell culture supplement.

[0094] The term "comprising", whenever used in this document, is intended to indicate the presence of stated features, integers, steps, components, but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0095] The present disclosure should not in any way be considered limited to the described embodiments, and a person skilled in the art will foresee many possibilities for modifications thereof.The above embodiments are combinable.

[0096] The following claims further set forth specific embodiments of the present disclosure.

[0097] References

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[0099] [2] US 9688959B2, Compositions, uses, and preparation of plateletlysates.

[0100] [3] EP2723354, Compositions, uses, and preparation of plateletlysates.

[0101] [4] EP3638263, Process for preparing a pooled human platelet lysate,pooled human platelet lysate and its use for treating neurological disorders.

[0102] [5] Yan Gao, Nien-Ju Ku, Tzu-Cheng Sung, Akon Higuchi, Chi-ShengHung, Henry Hsin-Chung Lee, Qing-Dong Ling, Nai-Chen Cheng, Akihiro Umezawa,Lassina Barro, Thierry Burnouf, Qingsong Ye, Hao Chen. The effect of humanplatelet lysate on the differentiation ability of human adipose-derived stemcells cultured on ECM-coated surfaces. J Mater Chem B. 2019 Dec 7;7(45):7110-7119. doi: 10.1039 / c9tb01764j.

[0103] [6] Castander-Olarieta A, Pereira C, Montalban IA, Mendes VM, CorreiaS, Suarez-Alvarez S, Manadas B, Canhoto J, Moncalean P. Proteome-WideAnalysis of Heat-Stress in Pinus radiata Somatic Embryos Reveals a CombinedResponse of Sugar Metabolism and Translational Regulation Mechanisms. FrontPlant Sci. 2021 Apr 12; 12:631239; doi: 10.3389 / fpls.2021.631239.

[0104] [7] Jennewein C, Tran N, Paulus P, Ellinghaus P, Eble JA, ZacharowskiK. Novel aspects of fibrin(ogen) fragments during inflammation. Mol Med. 2011May-June; 17(5-6):568-73; doi: 10.2119 / molmed.2010.00146.

Claims

1. A stable human platelet lysate composition comprising less than 5 μg / ml of fibrinogen and not less than 6 ng 生长因子 / μg 纤维蛋白原 The mass ratio of PDGF-BB growth factor / fibrinogen content is as follows; preferably, the composition is a heparin-free composition and / or a xeno-free composition.

2. A stable human platelet lysate composition according to any one of the preceding claims, comprising less than 4 µg / ml fibrinogen.

3. A stable human platelet lysate composition according to the preceding claim, comprising less than 3 µg / ml of fibrinogen.

4. A stable human platelet lysate composition according to any one of the preceding claims, wherein The mass ratio of the PDGF-BB growth factor / fibrinogen content is not less than 8 ng 生长因子 / μg 纤维蛋白原 .

5. A stable human platelet lysate composition according to the preceding claim, wherein: The mass ratio of the PDGF-BB growth factor / fibrinogen content is not less than 10 ng 生长因子 / μg 纤维蛋白原 .

6. A stable human platelet lysate composition according to the preceding claim, wherein: The growth factor is selected from the list consisting of BDNF, EGF, FGF-2, GM-CSF, HGF, IL-1β, IL-8, PDGF-AB, PDGF-BB, VEGF, and combinations thereof.

7. A stable human platelet lysate composition according to any one of the preceding claims, wherein The mass ratio of growth factor per unit mass of fibrinogen ranges from 0.5 to 2.0 ng 生长因子 / μg 纤维蛋白原 , preferably 1.0-1.5ng 生长因子 / μg 纤维蛋白原 , and wherein the growth factor is selected from BDNF, EGF, VEGF-A, or a combination thereof.

8. A stable human platelet lysate composition according to any one of the preceding claims, wherein The mass ratio of growth factor per unit mass of fibrinogen ranges from 0.05 to 0.5 ng 生长因子 / μg 纤维蛋白原 , preferably 0.1-0.4ng 生长因子 / μg 纤维蛋白原 , and wherein the growth factor is selected from FGF-2, GM-CSF, HGF, IL-1β, or a combination thereof.

9. A stable human platelet lysate composition according to any one of the preceding claims, wherein The mass ratio of growth factor per unit mass of fibrinogen ranges from 4 to 12 ng 生长因子 / μg 纤维蛋白原 , preferably 6-8 ng 生长因子 / μg 纤维蛋白原 , and wherein the growth factor is selected from PDGF-AB, PDGF-BB, or a combination thereof.

10. A stable human platelet lysate composition according to any one of the preceding claims, wherein The composition had a transparent yellow color with no visible particles detectable by visual observation.

11. A stable human platelet lysate composition according to the preceding claim, wherein: The composition had a transparent yellow color with no undissolved visible particles.

12. A stable human platelet lysate composition according to any one of the preceding claims, wherein The clarity and opalescence measured by turbidity measurement is less than 400 NTU.

13. A stable human platelet lysate composition according to the preceding claims, wherein: The clarity and opalescence are less than 350 NTU; preferably less than 300 NTU.

14. A stable human platelet lysate composition according to any one of the preceding claims, wherein The optical density is less than 1.

15. A stable human platelet lysate composition according to the preceding claim, wherein: The optical density is less than 0.9, preferably less than 0.

8.

16. A stable human platelet lysate composition according to any one of the preceding claims, wherein The number of microscopic particles up to 25 µm in size is less than 2.0 x 10 7 Particles / mL.

17. Where the optical density is less than 1.0 x 10 7 particles / mL, more preferably less than 5.0 x 10 6 Particles / mL.

18. A stable human platelet lysate composition according to any one of the preceding claims, wherein Proteomic analysis using LC-MS could not detect the amount of fibrinogen β chain.

19. A stable human platelet lysate composition according to any one of the preceding claims, wherein: The composition is stable when stored at refrigerated conditions of -80°C to 5°C, preferably at -20°C to 4°C, for up to 6 months.

20. A stable human platelet lysate composition according to any one of the preceding claims for use in medicine or veterinary medicine and / or as a raw material in cell culture supplementation and / or as a raw material in the production of advanced therapeutic pharmaceuticals.

21. A method for obtaining a stable human platelet lysate, comprising the following steps: Obtain frozen human platelet units; subjecting the human platelet units to thaw / freeze cycles to disrupt platelet membranes; Adding silicon dioxide and calcium chloride to the mixture obtained in the previous step to induce clot formation; incubating the sample at a temperature in the range of 35°C to 39°C for up to 1 hour to 4 hours, preferably 2 hours; The incubated samples were centrifuged and the obtained supernatant was collected; Purifying the supernatant to obtain a stable human platelet lysate; preferably a stable heparin-free human platelet lysate; optionally comprising adding a combination of 0.005-0.04% (w / v) silicon dioxide and 0.5-1.0% (w / v) calcium chloride.

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