Laminin extraction, purification and polymerization methods, uses, polylaminin and kits

Abstract

Described herein are methods of extracting and purifying laminin from human placenta, wherein the laminin has a retained acid polymerization ability to produce polylaminin. The present application also describes an acid polymerization process of the purified laminin, the use of the purified laminin in the preparation of a pharmaceutical composition of polylaminin, and a kit for the immediate preparation of said composition, where the compositions and kits are suitable for the treatment of central nervous system injury, predominantly spinal cord injury.

Description

Technical Field

[0001] The present invention belongs to the field of methods for protein extraction and purification for therapeutic purposes. More precisely, the present invention relates to the extraction and purification of laminin from human placenta, which has retained acid polymerization ability to produce polylaminin. The present invention also relates to the acid polymerization method of the purified laminin, the use of the purified laminin in the preparation of a pharmaceutical composition of polylaminin, and a kit for the immediate preparation of said composition, wherein the composition and the kit are suitable for the treatment of central nervous system injuries, mainly spinal cord injuries. Background Art

[0002] According to the Ministry of Health (MoH) of Brazil, "Spinal cord injury is one of the most severe and acute injuries that can affect humans, with huge physical, psychological, and social consequences. We refer to spinal cord injury as any injury to the structures within the spinal canal (medulla, conus medullaris, and cauda equina), which can lead to motor, sensory, autonomic, and psycho-emotional alterations". In 2013 and 2015, the MoH stated that the annual incidence of spinal trauma worldwide was approximately 15 to 40 cases per million inhabitants, and in Brazil, it was estimated that more than 10,000 new cases of spinal cord injury occurred annually, with trauma being the main cause (Brazil, Ministry of Health, Secretariat of Health Care, Department of Planning and Strategic Actions, Guidelines for the Care of Patients with Spinal Cord Injury / Ministry of Health, Secretariat of Health Care, Department of Program Strategic Actions, and Department of Specialized Care. 2 nd .ed - Brasilia: Ministry of Health, 2015. 68p.: il. ISBN 978 - 85 - 334 - 2229 - 2; Brazil, Ministry of Health, Secretariat of Health Care, Department of Planning and Strategic Actions, Guidelines for the Care of Patients with Spinal Cord Injury / Ministry of Health, Secretariat of Health Care, Department of Program Strategic Actions, and Department of Specialized Care, - - Brasilia: Ministry of Health, 2013. 68p.: il. ISBN 978 - 85 - 334 - 2025 - 0). The World Health Organization (WHO) estimates that worldwide, 250,000 to 500,000 people suffer spinal cord injury each year. Besides the obvious physical and psychological damage to the individual, including a 2 - to 5 - fold increased likelihood of premature death, especially in low - and middle - income countries, this type of injury also results in huge personal and social costs (https: / / www.who.int / news - room / fact - sheets / detail / spinal - cord - injury, accessed on July 11, 2022).

[0003] The MoH also recommends that any patient with multiple injuries requires special care from the first aid at the accident scene, where he / she must be fully immobilized as he / she is considered suspected of spinal cord injury. Regarding drug treatment, although the administration of methylprednisolone has been reported in some studies, its use is not currently recommended for patients with spinal trauma. After initial care (such as the use of a cranial halo in cervical fractures / dislocations and rest in thoracolumbar fractures), definitive surgical treatment of the fracture is recommended as soon as possible for clinically stable patients.

[0004] In view of this situation, it is clear that the treatment of central nervous system injuries (mainly spinal cord injuries) remains challenging because there is no spontaneous regeneration process and essentially no treatment strategies as there is currently no treatment capable of reversing spinal cord injuries.

[0005] To overcome this deficiency, various methods have been identified in the prior art, attempting to regenerate the traumatized neural tissue and promote the reconstruction of spinal cord injuries. Some of these efforts include, for example, the therapeutic application of the protein laminin.

[0006] Laminin or laminins are large extracellular glycoproteins from a family of structurally homologous subtypes that form the major components of the basement membrane. Laminin has a cross-shaped shape composed of three different polypeptide chains (α-α, β-β, γ-γ), which are bound together by α-helical interactions and disulfide bonds and have the ability to self-organize. Different combinations of the α, β, and γ chains produce 15 different known laminin subtypes, thus conferring specificity to the tissues containing them.

[0007] Properties observed in laminin that can be exploited for therapeutic applications are the ability to polymerize to form polymers or protein aggregates. While laminin polymerization is promoted in vivo and is related to its ability to adhere to the plasma membrane surface by binding to homologous integrins, α-dystroglycan, and sulfated glycolipids, laminin has been observed to polymerize in vitro under specific conditions. Yurchenco et al. (1985) described the strict requirement for a critical laminin concentration of 140 nM (0.1 mg / mL) for self-polymerization and formation of large three-dimensional networks in vitro, although subsequently, other authors have demonstrated polymerization at subcritical concentrations and specific pH conditions, more precisely in acidic media (Yurchenco, P.D., Tsilibary, E.C., Charonis, A.S., & Furthmayr, H. (1985). Laminin polymerization in vitro. Evidence for a two-step assembly with domain specificity. The Journal of Biological Chemistry, 260(12), 7636-7644). Generally, in the literature, the terms "polymerization" and "polymer" are used to refer to these structures with several associated laminin units (Yurchenco, P.D., & Cheng, Y.-S. (1993). Self-assembly and Calcium-binding Sites in Laminin. The Journal of Biological Chemistry, 268(23), 17286-17299).

[0008] In the prior art, Freire and Coelho-Sampaio in 2000 (Freire, E., & Coelho-Sampaio, T. (2000). Self-assembly of Laminin Induced by Acidic pH. Journal of Biological Chemistry, 275(2), 817-822) described that the laminin polymer was obtained from a mixture of laminin in an acidic medium independent of the critical concentration. In this article, the authors examined the relationship between the lipid surface charge and laminin polymerization. During the study, they observed that in the presence of divalent cations, acidic pH induced the aggregation of laminin in solution, which was an organized process rather than a simple non-specific precipitation of the protein at its isoelectric point, even in the absence of a solid substrate for adsorption. In 2002, Freire correlated the structural properties of the laminin matrix specifically polymerized in an acidic medium with neuritogenesis and neuroplasticity (Freire, E. (2002). Structure of laminin substrate modulates cellular signaling for neuritogenesis. Journal of Cell Science, 115(24), 4867-4876). According to this literature, these matrices were morphologically similar to those found on surfaces that allow axon growth in vivo, favored neuritogenesis rather than cell proliferation, and pointed out the possibility of using acidic laminin matrices to stimulate axon regeneration.

[0009] However, the above-mentioned literature failed to describe the therapeutic use of a pharmaceutical composition comprising laminin and laminin polymers in nerve tissue injury, mainly spinal cord injury. In this regard, the Brazilian patent application PI 0805852-0 entitled "Protein Acid Polymers, Production Method, Uses of Protein Acid Polymers, Pharmaceutical Compositions and Therapeutic Methods" relates to the application of laminin polymers in pharmaceutical compositions having regenerative and anti-inflammatory activities, particularly for treating animals affected by traumatic, degenerative or inflammatory tissue injuries in nerve, muscle, epithelial and connective tissues. Regarding the composition, this literature includes, in an acidic medium, in the presence of divalent cations (preferably Ca 2+) In the presence of [specific conditions], the polymerization of laminin, and subsequent injection into the damaged area to provide the claimed therapeutic activity. The acidic polymer thus formed will have regenerative and anti-inflammatory capabilities, and in addition to promoting the morphological regeneration of nerve tissue and the functional recovery of mammalian spinal cord injuries, it is also capable of restoring the neuronal plasticity lost during the development of cerebral cortex explants of neonatal animals. According to this literature, laminin can be extracted from murine EHS (Engelbreth-Holm-Swarm) sarcoma, or it can be recombinant human laminin, or laminin extracted from human placenta, or even a combination of these laminins. However, this literature does not provide information on the methodology for extracting and purifying laminin. The laminin polymer formed under the conditions disclosed in this patent literature was later named poly-laminin by the inventors and is currently undergoing clinical research in Brazil for the treatment of acute spinal cord injuries (https: / / ensaiosclinicos.gov.br / rg / RBR-9dfvgpm).

[0010] Therefore, there is potentially a need for an effective method for the large-scale extraction and purification of laminin, which can be applied on an industrial scale, and the method allows the protein to be obtained with a relatively high yield and purity, enabling laminin to be applied in the preparation of compositions for therapeutic use.

[0011] The first method for laminin extraction recognized in the prior art dates back to 1979, when Timpl et al. identified and named laminin as the major component of the tumor stroma of rat EHS sarcoma and, in normal tissues, localized it in the basement membrane (Timpl, R., Rohde, H., Robey, P.G., Rennard, S.I., Foidart, J.M., Martin, G.R. (1979). Laminin--a glycoprotein from basement membranes. The Journal of Biological Chemistry, 254(19), 9933-9937). The authors described a method for obtaining laminin from this tumor, which included an extraction step consisting of homogenizing the material in a neutral buffer and NaCl (3.4 M NaCl, 0.05 M Tris-HCl, pH 7.4, 4 °C), followed by centrifugation and further homogenization (10 to 20 volumes (w / v) of 0.5 M NaCl, 0.05 M Tris-HCl, pH 7.4). Next, a step of precipitating impurities (especially collagen) was carried out by adding 1.7 M NaCl. Finally, in the purification step, the supernatant was subjected to dialysis (2 M urea; 0.05 M Tris-HCl, pH 8.6), anion-exchange chromatography (DEAE-cellulose), concentration by ultrafiltration, size-exclusion chromatography (agarose A), further dialysis (0.05% acetic acid or 0.4 M NaCl, 0.05 M Tris-HCl, pH 7.4), concentration, and lyophilization.

[0012] Palm e Furcht (Palm, S.L., & Furcht, L.T. (1983)) has identified other methods in the prior art for extracting laminin from EHS sarcoma (Palm, S.L., & Furcht, L.T. (1983). Production of laminin and fibronectin by Schwannoma cells: cell - protein interactions in vitro and protein localization in peripheral nerve in vivo. The Journal of Cell Biology, 96(5), 1218 - 1226). Similar to the previous method, this method includes extraction steps with homogenization (3.4M NaCl, 0.01M phosphate buffer, pH 7.4, 4°C), centrifugation, and further homogenization (0.5M NaCl, 0.01M phosphate, pH 7.4), followed by an impurity precipitation step with 1.7M NaCl and a laminin precipitation with 30% ammonium sulfate. The centrifuged precipitate is resuspended and dialyzed (0.5M NaCl, 0.01M phosphate, pH 7.4), subjected to size - exclusion chromatography (Sephacryl S - 300), dialyzed (0.14M NaCl, 0.01M phosphate buffer, pH 7.4), affinity chromatography (heparin - agarose), further dialyzed (0.5M NaCl, 0.01M phosphate, pH 7.4), and finally filtered through a 0.45 μM filter and stored at - 70°C.Although the above methods can be used to provide laminin from rat EHS sarcoma, their efficiency may not necessarily extrapolate to the extraction of laminin from other tissues (such as human placenta, a material that is considered clinical waste and is regarded as a source of laminin, especially laminin composed of A, M, β1, and β2 subunits, such as subtypes 111, 211, and 221 (Rohde, H., Wick, G., & Timpl, R. (1979). Immunochemical characterization of the basement membrane glycoprotein laminin. European Journal of Biochemistry, 102(1), 195 - 201; Foidart, J. M., Bere, E. W., Jr, Yaar, M., Rennard, S. I., Gullino, M., Martin, G. R., & Katz, S. I. (1980). Distribution and immunoelectron microscopic localization of laminin, a noncollagenous basement membrane glycoprotein. Laboratory Investigation, 42(3), 336 - 342; Ohno, M., Martinez - Hernandez, A., Ohno, N., & Kefalides, N. A. (1983). Isolation of laminin from human placental basement membranes: amnion, chorion and chorionic microvessels. Biochemical and Biophysical Research Communications, 112(3), 1091 - 1098)).In this regard, according to Risteli and Timpl (1981), although the use of neutral buffer results in the extraction of approximately 80% of intact laminin in its native state from the EHS sarcoma matrix, due to the presence of covalent and non-covalent interactions between basement membrane structures, it may not be true for other tissues to achieve the same effect (Risteli, L., & Timpl, R. (1981). Isolation and characterization of pepsin fragments of laminin from human placental and renal basement membranes. The Biochemical Journal, 193(3), 749-755. https: / / doi.org / 10.1042 / bj1930749). Therefore, to extract laminin from human placenta, the authors used a pepsin digestion step to dissolve the P1 fragment of laminin. Next, the method included precipitation with NaCl (1.2 M to 2 M to 4 M), purification by anion-exchange chromatography (DEAE-cellulose), dialysis, removal of collagen by the action of collagenase, and size-exclusion chromatography.

[0013] However, the disadvantage of the method described by Risteli e Timpl (1981) is that it cannot recover laminin intact, only fragments recovered from pepsin digestion. On the other hand, from the perspective of identifying common features, it has been observed that the methods described so far for extraction from EHS sarcoma and from placenta have focused on selective precipitation techniques using different NaCl concentrations (salting-in and salting-out), size-exclusion chromatography, and anion-exchange chromatography.

[0014] To overcome the problem of recovering laminin fragments from human placenta, Ohno et al. (1983) developed a non-degrading method for recovering from the placental basement membrane of anatomical tissues of amnion, chorion, and chorionic microvessels, which does not require prior protease digestion. After preparing the three tissues with NaCl and EDTA, laminin was extracted by sequentially treating with the following in the presence of protease inhibitors phenylmethylsulfonyl fluoride (PMSF) and N-ethylmaleimide (NEM): 1) 0.5 M NaCl containing 5 mM PMSF and 1 mM NEM; 2) 8 M urea in 0.05 M phosphate buffer, pH 7.0; and 3) 8 M urea with 2% 2-mercaptoethanol and 2% sodium dodecyl sulfate, with centrifugation and dialysis performed between each treatment. The authors managed to extract intact laminin from the three tissues; however, there are potential toxic substances for drug use, such as protease inhibitors and reducing agents. For example, NEM can inconveniently and irreversibly alter the native structure of proteins containing reduced cysteine in solution.

[0015] Dixit (1985) also developed an extraction method in which intact form of laminin could be obtained from human placenta in the presence of 10 mM EDTA (Dixit S.N. (1985). Isolation, purification and characterization of intact and pepsin-derived fragments of laminin from human placenta. Connective Tissue Research, 14(1), 31-40. https: / / doi.org / 10.3109 / 03008208509089841). In this method, the pre-washed placenta was homogenized in a neutral buffer (0.02-M Tris-HCl pH 7.4), 1 M NaCl and 3% Triton X-100 in the presence of 10 mM EDTA, which solubilized laminin. The supernatant was subjected to two rounds of salting out with 4 M NaCl. The re-suspended and dialyzed precipitate was subjected to the steps of anion-exchange chromatography (DEAE-cellulose), size-exclusion chromatography (Sepharose A-5M-void), dialysis and concentration (PM10 Diaflo) for purification. According to this study, 17 mg to 20 mg of purified laminin was recovered from 1 kg of wet placental tissue, indicating that placenta could be a good source of human laminin.

[0016] In 1987, Paulsson et al. also applied EDTA as an extractant for laminin from rat EHS sarcoma, in which laminin was conjugated with nidogen (another characteristic protein of basement membrane), excluding the salting out step and maintaining size-exclusion chromatography (Bio-Gel A5m or Sepharose Cl-6B) (Bio-Gel A5m or Sepharose Cl-6B) (Paulsson, M., Aumailley, M., Deutzmann, R., Timpl, R., Beck, K., & Engel, J. (1987). Laminin-nidogen complex. Extraction with chelating agents and structural characterization. European Journal of Biochemistry, 166(1), 11-19).

[0017] Returning to human placenta, the patent document WO1991011462A2, entitled "Merosin, Encoding Nucleic Acids, Fragments Thereof and Uses", describes a method for extracting merosin (a laminin isoform) from human placenta, which includes: a protein solubilization step in a neutral buffer containing 10 mM EDTA, and precipitation with 4 M NaCl or saturated concentration of 40% ammonium sulfate. The precipitate is subjected to size exclusion chromatography (Sepharose 6B) and anion exchange chromatography (DEAE cellulose) steps, where merosin is eluted with 0.2 M NaCl.

[0018] U.S. Patent 5,019,087, entitled "Nerve Regeneration Conduit", relates to a tubular device containing human placenta laminin, which can be used for in vivo regeneration of nerve tissue or damaged nerves, and describes a method for extracting the laminin. The method is carried out at 4 °C, and low concentration of EDTA together with NEM is used as a protease inhibitor. After washing the placenta with NaCl and Tris, extraction is immediately carried out with urea (a chaotropic agent) to remove blood components. Then, the dialysis centrifuged supernatant is used, and the extract is concentrated using affinity chromatography (heparin) and precipitated with saturated concentration of 30% ammonium sulfate.

[0019] In 2000, Champliaud et al. described a method for obtaining laminin from the placenta, which included extraction in the presence of protease inhibitors PMSF and NEM, precipitation and purification with ammonium sulfate at a saturation concentration of 30%, where purification included steps of anion exchange chromatography (DEAE cellulose), size exclusion chromatography (gelatin - agarose), and immunoaffinity chromatography (pAb G4 - G5 agarose → laminin α - 1; MAb 545 agarose → laminin β - 1) (Champliaud, M.F., Virtanen, I., Tiger, C.F., Korhonen, M., Burgeson, R., & Gullberg, D. (2000). Posttranslational modifications and beta / gamma chain associations of human laminin alpha1 and laminin alpha5 chains: purification of laminin - 3 from placenta. Experimental Cell Research, 259(2), 326 - 335). It is important to emphasize that although immunoaffinity chromatography is widely present in small - scale laminin purification methods in the literature, from an industrial production perspective, the use of immunoaffinity chromatography is not the most appropriate. In addition, due to the toxicity of protease inhibitors, the presence of protease inhibitors in the culture medium is not desirable in methods for obtaining products for pharmaceutical applications.

[0020] Gorelik et al. (2001) further extracted laminin from the placenta using neutral buffer, EDTA, and NaCl, precipitated it with 4M NaCl, and purified it using only size-exclusion chromatography (Sephacryl S-300). Although they did not report the quality of the laminin obtained from the extract, the authors emphasized that, in addition to the bands of laminin and nestin components that already appeared in the gels stained with Coomassie blue, the results of SDS-PAGE analysis stained with silver revealed some other bands (Gorelik, J.V., Cherepanova, O.A., Voronkina, I.V., Diakonov, I.A., Blinova, M.I., & Pinaev, G.P. (2001). Laminin-2 / 4 from human placenta is a better adhesion agent for primary keratinocytes than laminin-1 from EHS sarcoma. Cell Biology International, 25(5), 395-402).

[0021] Hackethal et al. (2018) developed a method for isolating laminin isoform 111 that included an extraction step with Tris-NaCl buffer, combined with a rapid step of non-denaturing precipitation of the protein with 30% ammonium sulfate and tangential flow filtration (Hackethal, J., Schuh, C.M.A.P., Hofer, A., Meixner, B., Hennerbichler, S., Redl, H., & Teuschl, A.H. (2018). Human Placenta Laminin-111 as a Multifunctional Protein for Tissue Engineering and Regenerative Medicine. Advances in Experimental Medicine and Biology, 1077, 3-17). According to the article, the average amount of laminin 111 recovered after isolation was 175 ± 35 mg of laminin per 100 g of wet basal tissue. A major drawback of this method is the need to remove the chorion, which is the serous membrane attached to the placental tissue and whose removal is difficult and laborious, hindering its scale-up and application on an industrial scale.

[0022] Analysis of the prior art clearly shows a trend towards using some main processes for the extraction and purification of laminin from the placenta. Besides selective precipitation (especially salting out with saturated concentrations of ammonium sulfate (about 30% to 40%) or sodium chloride (about 3.4 M to 4 M) or salting in with sodium chloride (1.7 M) especially for removing collagen), chromatographic techniques are widely used in the purification step. For example, size exclusion chromatography stands out due to the unique size of laminin, but if applied without combination with another chromatographic technique, it results in unsatisfactory purity. Affinity chromatography, especially immunoaffinity chromatography, is also one of the preferred chromatographies in the prior art; however, they involve the use of large amounts of antibodies, which makes the method more laborious and more expensive. In addition, chromatographic resins functionalized with chemical groups instead of antibodies are easier to maintain, which makes the method more convenient to implement on an industrial scale. Ion exchange chromatography is also a commonly used technique in laminin purification methods, and in a convergent manner, the selection of anion exchange chromatography instead of cation exchange chromatography seems to be the consensus for laminin purification.

[0023] In clear contrast to this trend, the patent document WO 1997 / 47652 entitled "Purification of Soluble Laminin 5" discloses a method for purifying laminin that includes a step of cation exchange chromatography. However, in fact, the described protocol clearly follows the teachings of the prior art, since anion exchange chromatography techniques are also used as expected in conventional laminin purification methods.

[0024] However, the present inventors have observed that laminin extracted from human placenta does not retain its acid polymerization ability if purified by anion exchange chromatography methods, and thus the resulting product is not suitable for the treatment of central nervous system injuries (mainly spinal cord injuries) in accordance with the concept of the present invention. Therefore, there is a need for a new method to obtain laminin with retained acid polymerization ability.

[0025] In this regard, the present invention aims to provide a method for extracting and purifying laminin that overcomes the need in the prior art for a method with good yield and industrial application, and mainly produces a purified product that is laminin with retained acid polymerization characteristics, since this property is essential for achieving its therapeutic applications, including in pharmaceutical compositions and kits for treating central nervous system injuries (mainly spinal cord injuries). Summary of the Invention

[0026] The present invention relates to a method for extracting laminin from human placenta and a purification method, which aims to obtain purified laminin with good yield and purity, enabling it to be used in the preparation of compositions for therapeutic use, and which retains the acid polymerization ability to produce poly-laminin. The present invention also relates to an acid polymerization method of the purified laminin obtained by the method disclosed in the present invention and the poly-laminin obtained therefrom. In addition, the present invention relates to the use of the purified laminin obtained by the purification method in the preparation of pharmaceutical compositions, and a kit capable of instantaneously preparing poly-laminin for treating central nervous system injuries (mainly spinal cord injuries).

[0027] The first embodiment of the present invention relates to a method for extracting laminin from human placenta to obtain a protein extract rich in laminin, wherein the method comprises the following steps: treating the placenta to obtain clean placental tissue without the need to remove the chorion; homogenizing the placental tissue; extracting laminin with an extraction buffer; optionally filtering; and precipitating laminin by salting out to recover a protein extract rich in laminin.

[0028] The second embodiment of the present invention relates to a method for purifying laminin from a protein extract rich in laminin, wherein the method comprises the steps of dissolving the extract in a buffer containing a chaotropic agent and obtaining purified laminin by cation exchange chromatography and size exclusion chromatography. In addition, this embodiment of the present invention includes an optional step for final treatment, which includes one or more steps selected from concentration, filtration, and fractionation.

[0029] The third embodiment of the present invention relates to a method for polymerizing purified laminin, wherein the method is carried out on the purified laminin, which is obtained by the purification method disclosed in the present invention, or by combining the extraction method and purification method disclosed in the present invention, or even by combining a suitable extraction method in the prior art and the purification method disclosed herein, and the purified laminin retains their acid polymerization ability and therapeutic applications.

[0030] The fourth embodiment of the present invention relates to the use of purified laminin in the preparation of pharmaceutical compositions, wherein the purified laminin is obtained by the purification method disclosed in the present invention, or by combining the extraction method and purification method disclosed in the present invention, or even by combining a suitable extraction method in the prior art and the purification method disclosed herein, and the pharmaceutical compositions can be used to treat central nervous system injuries, mainly spinal cord injuries.

[0031] The fifth embodiment of the present invention relates to poly-laminin obtained by acid polymerization of purified laminin, which is produced by the purification methods disclosed herein, or by combining the extraction methods and purification methods disclosed herein, or even by combining suitable extraction methods in the prior art and the purification methods disclosed herein.

[0032] The sixth embodiment of the present invention relates to a kit, which includes: a first bottle containing purified laminin obtained by the purification method disclosed in the present invention, a second bottle containing an acidic preparation, and additionally, a divalent cation preparation contained in the first bottle, the second bottle or a third bottle, and additionally instructions for immediately preparing a poly-laminin composition for treating central nervous system injuries (especially for treating spinal cord injuries).

[0033] Surprisingly, the inventors have demonstrated that the extraction methods described herein are simpler and more labor-saving than those described in the prior art, and produce protein extracts rich in intact laminin and free of potentially toxic substances, the presence of which is taboo in products for therapeutic use. Furthermore, even more surprisingly and unexpectedly, the inventors have developed a purification method using cation exchange chromatography and have determined that even in the absence of an anion exchange chromatography step, this technique is sufficient to provide purified laminin, which contradicts the teachings of the prior art. Even more importantly, the inventors have detected that the use of anion exchange chromatography (commonly used in the prior art to purify placental laminin) alters the polymerization pattern of the purified laminin and results in a protein product unsuitable for therapeutic applications. On the other hand, the method of the present invention uses cation exchange chromatography (instead of anion exchange) as the sole chromatographic method for ionic interaction, producing a product with a relatively high yield and purity and retained acid polymerization ability.

[0034] The aspects and embodiments of the present invention will become clearer through the following description. Description of the Drawings

[0035] Figure 1 Shows Western Blots of samples of laminin evaluated by ammonium sulfate precipitation under conditions of 20%, 25% and 30% saturated salt concentration. In the image, the labels "γ1" and "α2C-term(80kDa)" refer to the regular chains in laminin subtypes 211 and 221. Note that, from left to right, 1: molecular weight marker; 2: standard laminin; 3: 30% precipitate; 4: 30% supernatant; 5: 25% precipitate; 6: 25% supernatant; 7: 20% precipitate; 8: 20% supernatant. It is observed in 6 and 8 that laminin is present in the supernatant fractions produced by precipitation at 25% and 20% respectively.

[0036] Figure 2 It is a comparative schematic diagram showing the variation of the extract quality (■) and the percentage of laminin in the extract (Δ) with the ammonium sulfate saturation concentration (%) used in the precipitation step. The obtained quality (■) increases linearly with the increase in the amount of salt used. Due to the increase of ammonium sulfate from 30% to 40%, the percentage of laminin in the extract (Δ) decreases from about 13% to 3%.

[0037] Figure 3 It is a diagram of the 6% SDS-PAGE analysis of the main intermediate fractions in the laminin purification method in a typical batch. Note that, from left to right, 1: molecular weight marker (mwm); 2: standard laminin (standard); 3: protein extract rich in laminin after resuspension, as described in Example 4.1 (resuspended); 4: filtered resuspended material, as described in Example 4.2 for filtration (filtered resuspended); Figure 5 : partially purified material obtained in the CEX step, as described in Example 4.3 for operation (CEX eluate); 6: purified laminin; Figure 7 : purified laminin after final treatment. Throughout the purification method, an increase in the purity of the material can be noted, which is demonstrated by the separation involving the laminin band, which is significant compared to the standard laminin.

[0038] Figure 4 It is a complete cation exchange chromatogram (CEX) involving the production of a typical batch, in which the injection step, column washing at 7.0 mS / cm - 7.1 mS / cm, elution at 14.0 mS / cm - 14.1 mS / cm, removal of remaining impurities at 48.0 mS / cm - 48.1 mS / cm, and cleaning with 1M NaOH (CIP) are indicated. In the figure, the solid line represents the absorbance at 280 nm (A 280 ), and the dashed line represents the measured conductivity.

[0039] Figure 5 It is a part of the cation exchange chromatography (CEX) involving the production of a typical batch, in which the laminin elution region is highlighted. In the figure, the solid line represents the absorbance at 280 nm (A 280 ), and the dashed line represents the measured conductivity. The arrow points to the increase in absorbance (A 280 ), indicating the start of fraction collection. The highlighted region represents the fractions collected and used for the next purification step.

[0040] Figure 6Is a complete size-exclusion chromatography (SEC) of a typical batch production, using a Cytiva HiLoad 16 / 600 Superose 6 pg column. The demarcated areas indicate the first fraction (F1) and the second fraction (F2) collected.

[0041] Figure 7 Is a comparative analysis of the elution curves of the first fraction (F1 - solid line) and the second fraction (F2 - dashed line) by size-exclusion chromatography (SEC), using a Cytiva Superose 6 Increase 10 / 300 gl column. Figure 7 A shows that in the batch production involved, the first fraction (F1) and the second fraction (F2) elute at similar retention times, so both are included in the laminin pool. Figure 7 B shows that in the batch production involved, the second fraction elutes at a retention time different from that of the first fraction, indicating a high impurity content and resulting in the discard of the second fraction (F2).

[0042] Figure 8 Is a chromatogram of cyclic SEC size-exclusion chromatography, using a Cytiva HiLoad 16 / 600 Superose 6 pg column, with 5 consecutive injections of 5 mL each. The chromatogram relates to the production of a typical batch, that is, its purification method is satisfactory. The highlighted area under the curve indicates the collected laminin-rich fraction to form the laminin pool.

[0043] Figure 9 Involves the evaluation of the application of an anion-exchange step (AEX) in the purification method of the present invention. Figure 9 A is the chromatogram obtained from the AEX step. The arrow indicates the elution peak of laminin. Figure 9 B shows the results of 6% SDS-PAGE analysis, in which a reduction in impurities in the sample after the anion-exchange step can be seen. Note, from left to right, 1: molecular weight marker (mwm); 2: standard laminin; 3: material eluted from the CEX step (material injected in the AEX step); 4: flow-through containing removed impurities (injected and collected, washed at 14 mS / cm); 5: material eluted from the AEX step at 28 mS / cm, containing laminin.

[0044] Figure 10 Shows the analysis of laminin aggregation obtained from the same protein extract with and without the AEX step in the purification. Figure 10 A shows the particle size distribution of the samples using this technique and DLS (dynamic light scattering) at neutral pH, and it can be observed that the two samples have similar average particle sizes and distributions.Figure 10 Panel B shows the particle size distribution of the sample in the DLS at acidic pH, and it can be observed that typical aggregated particle growth was only observed for laminin obtained in purification without the AEX step. In contrast, laminin subjected to the AEX step showed partial aggregation characteristics at acidic pH. Figure 10 Panel C shows the Pulldown analysis via 6% SDS-PAGE. In this analysis, "C" is the centrifuged (large centrifugable polymers), and "S" is the supernatant (trimeric laminin or small non-centrifugable oligomers). It should be noted that the proportion of centrifugable polymers at acidic pH decreased with the introduction of the AEX step. Detailed Description

[0045] The present invention relates to a method for extracting laminin from human placenta and a purification method, which aim to obtain laminin with retained acid polymerization ability to produce poly-laminin with a significant yield and purity. It also relates to the use of the purified laminin thus obtained in the preparation of a pharmaceutical composition for treating central nervous system injuries (mainly spinal cord injuries), and further relates to the acid polymerization method of the purified laminin of the present invention, as well as the poly-laminin obtained by acid polymerization and a kit, wherein the components of the kit include the purified laminin obtained according to the present invention, an acidic preparation, a divalent cation, and instructions for immediate preparation.

[0046] "Laminin" or "laminins" are synonymous and refer to the laminin isoform family. The term refers to a specific laminin isoform only when specified in this specification. In a preferred but non-limiting embodiment, the laminins are predominantly isoforms 221 and 211, the subunits of which have been detected in placental tissue (Rohde, H., Wick, G., & Timpl, R. (1979). Immunochemical characterization of the basement membrane glycoprotein laminin. European Journal of Biochemistry, 102(1), 195-201; Foidart, J.M., Bere, E.W., Jr, Yaar, M., Rennard, S.I., Gullino, M., Martin, G.R., & Katz, S.I. (1980). Distribution and immunoelectron microscopic localization of laminin, a noncollagenous basement membrane glycoprotein. Laboratory Investigation, 42(3), 336-342; Ohno, M., Martinez-Hernandez, A., Ohno, N., & Kefalides, N.A. (1983). Isolation of laminin from human placental basement membranes: amnion, chorion and chorionic microvessels. Biochemical and Biophysical Research Communications, 112(3), 1091-1098).

[0047] Within the scope of the present invention, the term "polymerization" is defined as the process of laminin aggregation (especially in vitro) and encompasses said aggregation process under different conditions, including as described in the prior art, either by reaching the critical concentration of laminin in solution, by laminin adhesion to a lipid surface, by laminin contact with an acidic medium in the presence or absence of divalent cations, or even according to other in vitro laminin aggregation methods known in the prior art.

[0048] Within the scope of the present invention, the term "acid polymerization" is defined as the polymerization of laminin only when subjected to an acidic medium. Specifically, it is characterized as the process of laminin aggregation, preferably in vitro, when mixed with an acidic preparation or when directly diluted in an acidic preparation, which causes the immediate formation of high molecular weight laminin aggregates. According to the present invention, the pH of the process must be acidic, preferably between 4.0 and 5.5, more preferably between 4.2 and 4.4. Even more preferably, acid polymerization occurs in the presence of divalent cations, and in a preferred embodiment of the present invention, the divalent cation is Ca 2+ .

[0049] Within the scope of the present invention, the term "laminin polymer" or "polymer" is defined as an aggregate of subunits, where the subunits are laminin molecules linked by non-covalent bonds. Thus, although not polymers from a chemical perspective, these terms are used herein to define the above-mentioned aggregates in accordance with their previous use in the scientific literature (Yurchenco, P.D., & Cheng, Y.S. (1993). Self-assembly and calcium-binding sites in laminin. A three-arm interaction model. The Journal of Biological Chemistry, 268(23), 17286-17299; Barroso, M.M., Freire, E., Limaverde, G.S., Rocha, G.M., Batista, E.J., Weissmüller, G., Andrade, L.R., & Coelho-Sampaio, T. (2008). Artificial laminin polymers assembled in acidic pH mimic basement membrane organization. The Journal of Biological Chemistry, 283(17), 11714-11720). In the context of obtaining them in an acidic medium, these terms are used to define the poly-laminin or the corresponding poly-laminin within the scope of the present invention.

[0050] Within the scope of the present invention, "poly-laminin" is defined as a nano- and micro-structured network formed in vitro through non-covalent interactions between laminins, having central nervous system regeneration and anti-inflammatory activities. Specifically, "poly-laminin" is defined as a laminin polymer obtained by acid polymerization. Generally, poly-laminins obtained in vitro mimic the laminin organization in the cell membrane in vivo, although they are not identical due to the lack of interaction with membrane receptors (Barroso, M.M., Freire, E., Limaverde, G.S., Rocha, G.M., Batista, E.J., Weissmüller, G., Andrade, L.R., & Coelho-Sampaio, T. (2008). Artificial laminin polymers assembled in acidic pH mimic basement membrane organization. The Journal of Biological Chemistry, 283(17), 11714-11720).

[0051] Still according to the present invention, the term "protein extract" or "laminin-rich protein extract" refers to an extract obtained by the extraction method described in the present invention, which is a precipitated material, usually wet and having a pasty appearance, suitable for use in subsequent purification steps and also included within the scope of the present invention. Although in the present specification the term is specifically applied to laminin extracts obtained from the extraction methods disclosed in the present invention, alternatively, a person skilled in the art may apply selective precipitation and extraction methods known in the prior art to obtain a laminin-rich protein extract that has not been fully purified and subject it to the purification methods disclosed herein. Specifically, protein extracts obtained by prior art methods that are considered suitable for the purification methods described herein are those that are rich in intact laminin and free of substances considered unacceptable for use in pharmaceutical compositions.

[0052] Within the scope of the present invention, "physiologically acceptable liquid" is defined as a solution that does not exhibit toxicity at the concentrations used and is suitable for the washing, resuspension, dissolution, and / or homogenization of cells, tissues, proteins, and biological materials, and which is preferably ultrapure water, a saline solution (0.9% w / v NaCl), or a physiologically acceptable buffer selected from the group consisting of phosphate, citrate, acetate, histidine, tris, and PBS (phosphate buffered saline) buffers.

[0053] A first embodiment of the present invention relates to a method for extracting laminin from human placenta to obtain a laminin-rich protein extract, wherein the method comprises the following steps:

[0054] (i) Treat the placenta to obtain clean placental tissue;

[0055] (ii) Homogenize the placental tissue and separate the homogenized tissue and the resulting fluid;

[0056] (iii) Extract laminin from the placental tissue;

[0057] (iv) Optionally, filter; and

[0058] (v) Selectively precipitate laminin in the solution obtained from step (iii) or step (iv) by salting out to obtain a laminin-rich protein extract.

[0059] The extraction method disclosed by the present invention effectively provides a protein extract rich in intact laminin from human placenta and is carried out faster and more simply than the methods of the prior art. In addition, the method disclosed herein provides an extract suitable for subsequent purification steps, which produces laminin suitable for therapeutic applications, particularly pharmaceutical compositions for treating central nervous system injuries, especially pharmaceutical compositions for treating spinal cord injuries. Next, the technical and inventive aspects related to the extraction method will become clear.

[0060] The placenta can be obtained, for example, through donations clearly authorized in advance by the parturient, regardless of whether the type of delivery is natural or surgical. After collection, the placenta can be stored and transported to the processing site in refrigerated or frozen form. Between collection and use, the placenta can be kept frozen, preferably at -20°C. The placenta subjected to the extraction method can be fresh, refrigerated or frozen, or they can be more than one of these conditions when subjected to the extraction method. However, the process of obtaining the placenta from the parturient is not part of the extraction method claimed herein.

[0061] If frozen placenta is used, the material must be thawed before homogenization, which can be carried out slowly under cooling or rapidly. Preferably, the placenta is thawed rapidly at room temperature.

[0062] Step (i), treating the placenta, includes the processes of removing unwanted components and washing the placental tissue. The treatment is carried out in a pollution-free environment, preferably in a laminar flow. This step is preferably carried out under cooling at a temperature of 2°C to 8°C, preferably 4°C.

[0063] Within the scope of the present invention, "undesired components" include the outer membrane, trace amounts of amnion, and umbilical cord, but do not include the chorion. The chorion is a serous membrane, the removal of which is difficult and laborious, but has been reported in prior art extraction methods. The inventors have surprisingly found that an extraction method that does not require the removal of the chorion in the placenta processing step can be carried out more easily and quickly, and produces laminin that is compatible with the desired quality and in a satisfactory amount, without the need for additional or alternative extraction steps.

[0064] The placental tissue is washed with a physiologically acceptable liquid, preferably with a saline solution (NaCl 0.9% w / v). Sub-preferably, ultrapure water is used as the physiologically acceptable liquid, as it may cause undesired coagulation in the material. The washing liquid, preferably the saline solution, is used at a cooling temperature preferably between 2°C and 8°C, most preferably at 4°C. Before being subjected to washing, the placental tissue is cut into a shape and size that allows the physiologically acceptable liquid to penetrate to remove blood, blood clots, and fluids released during the processing and cutting of the tissue. Preferably, the tissue is cut into cubes. In a preferred embodiment of the present invention, the tissue is cut into cubes of about 5 cm and washed with a saline solution. The washing is repeated twice, or until the tissue is free of blood and blood clots.

[0065] When the placental tissue is homogenized and the homogenized tissue and the resulting fluid are separated, step (ii) includes the homogenization of the tissue itself and its washing.

[0066] Perform a homogenization process using the processed tissue obtained in step (i), adding a physiologically acceptable liquid thereto at a cooling temperature preferably between 2 °C and 8 °C, most preferably 4 °C. The liquid is preferably ultrapure water, in a ratio of 2:1 (200 mL of water per 100 g of tissue), where the viscosity is optimized for homogenization, but the person skilled in the art can choose other ratios that allow suitable homogenization without an unwanted increase in material dilution. After adding the liquid, the suspended tissue is preferably homogenized in a blender. Alternatively, homogenization can be carried out by other devices known in the prior art that promote uniform fragmentation of the tissue. This process is carried out for at least 1.5 minutes, but preferably, this process is carried out for at least 4 minutes, which can be continuous or intermittent. In a preferred embodiment, this process is carried out in a blender with ultrapure water for 4 minutes, divided into 2-minute cycles. The inventors of the present invention have recognized that multiple, long, and intense cycles using a blender are unnecessary, and the period between 1.5 and 4 minutes is sufficient to satisfactorily break down the material and, mainly, not cause degradation of laminin due to over-homogenization. As an additional advantage of the method, although many methods have been proposed in the prior art, the inventors do not add toxic protease inhibitor substances during the extraction process, thus keeping the material free of potentially harmful pharmaceutical additives such as N-ethylmaleimide (NEM) and phenylmethylsulfonyl fluoride (PMSF). Despite the absence of these inhibitors, the developed method is capable of recovering intact laminin suitable for applications included within the scope of the present invention.

[0067] Perform a washing process to remove the fluid and blood released during homogenization of the placental tissue suspension. First, the material is centrifuged to separate the homogenized solid tissue from the fluid contained in the supernatant, which must be discarded. The homogenized solid tissue precipitated during centrifugation is resuspended in cold physiologically acceptable liquid, preferably ultrapure water. According to the present invention, the centrifugation / supernatant discard / resuspension cycle can be repeated to completely remove impurities in the supernatant, followed by a final centrifugation step. In a preferred aspect of the present invention, up to five centrifugation cycles (including the final centrifugation) are carried out. In an even more preferred aspect of the present invention, three centrifugation cycles are carried out, which include two centrifugation / supernatant discard / resuspension cycles and a final centrifugation. Centrifugation is preferably carried out at 3000×g at a low temperature, preferably between 2 and 8 °C, most preferably 4 °C for 5 minutes. During the development of the method of the present invention, the inventors found that continuous washing and centrifugation cycles provided removal of the fluid from the solid tissue. However, starting from the third centrifugation, laminin was gradually lost into the supernatant. Therefore, the inventors found that three centrifugation cycles provided a sufficient balance between the removal of the fluid and the minimal loss of laminin into the supernatant.

[0068] Step (iii) of extracting laminin from the placental tissue is applied to the homogenized solid tissue obtained in step (ii), and step (iii) includes resuspending the tissue in an extraction buffer, followed by stirring and centrifuging to obtain a laminin-rich supernatant.

[0069] According to the scope of the present invention, the "extraction buffer" contains Tris, NaCl, and EDTA. NaCl contributes to the dissolution of laminin through the salting-out phenomenon, while EDTA chelates this ion (which plays an important role in the formation and maintenance of laminin polymers in the basement membrane) from the medium as a Ca 2+ chelating agent, thereby releasing laminin into the solution to facilitate the dissolution. Specifically, the preferred buffer used in the present invention contains 50 mM Tris, 1.0 M NaCl, and 10 mM EDTA. The buffer pH is preferably between 7.3 and 8.1, more preferably pH 7.4. The extraction is carried out in the buffer at a low temperature, preferably between 2°C and 8°C, most preferably at 4°C.

[0070] For resuspension, the mass of the placental tissue and the volume of the buffer are preferably in a ratio of 1:2 (w / v). The extraction is carried out by stirring the homogenized tissue suspension in the extraction buffer for 8 to 16 hours at a temperature between 2°C and 8°C. Preferably, the stirring is slow. The term "slow stirring" refers to stirring without forming vortices in the suspension. During the development of this new method, the inventors found that vigorous stirring, i.e., under conditions where vortices are formed, has the disadvantage of causing laminin degradation and thus must be avoided.

[0071] The supernatant rich in soluble laminin is recovered by centrifugation. In a preferred embodiment of the method of the present invention, the suspension is centrifuged at 15000×g for 30 minutes at a low temperature, preferably between 2°C and 8°C, most preferably at 4°C.

[0072] Step (iv) is carried out by filtering the laminin-rich supernatant obtained in step (iii), which is optional and can be carried out to remove particulate material remaining in the laminin solution (as noted by the high turbidity of the solution). The inventors recognized during this development that the extraction method can be carried out without the filtration step (iv), but provides a material with residual solid residues. Therefore, the inclusion of the filtration step (iv) was evaluated in order to improve the quality of the laminin solution that will undergo the precipitation step (v). In a preferred embodiment of the present invention, filtration is carried out using a depth filter, and even more preferably, using a depth filter with a nominal filtration degree between 0.05 μm and 2 μm.

[0073] The final step of the extraction method, i.e., step (v), is the selective precipitation of laminin, which is applied to the laminin solution obtained from step (iii) or step (iv), and the protein extract rich in laminin is recovered. This step is carried out by salting out, where the addition of salt promotes aggregation and subsequent protein precipitation due to the selective reduction of solubility.

[0074] Laminin precipitation is carried out with ammonium sulfate salt. Alternatively, other salts known in the art for use in salting out can be used. Ammonium sulfate can be added in crystalline form or in saturated solution form.

[0075] The ammonium sulfate used in step (v) is ammonium sulfate with a saturation concentration between 20% and 80%, preferably between 20% and 40%, more preferably with a saturation concentration of 30%, where the inventors found that the precipitation of laminin in the extract is maximum while the precipitation of other proteins is reduced.

[0076] Precipitation is carried out by slowly adding the salt to the solution with slow stirring, ensuring homogenization without salt accumulation at the bottom of the container. This step is carried out at a low temperature, preferably between 2°C and 8°C, most preferably 4°C. The precipitation under these conditions is maintained for a period of time during which an increase in turbidity is visibly observed, indicating the precipitation of laminin. Specifically, to ensure the maximum precipitation of said laminin, the precipitation can be maintained for an extended period, such as 2 hours.

[0077] After the precipitation period, when the turbidity of the solution increases due to the presence of precipitated proteins (mainly laminin) in the suspension, the suspension is centrifuged. In a preferred embodiment of the present invention, the suspension is centrifuged at a low temperature, preferably between 2°C and 8°C, most preferably 4°C, at 15000×g for 30 minutes. The supernatant is discarded and the precipitate is recovered, which is referred to within the scope of the present invention as the protein extract rich in laminin, i.e., a pasty substance containing the precipitated protein. It has been found that this extract is suitable for the subsequent steps of the purification method, which can be carried out immediately, or alternatively, the extract can be frozen (preferably at -20°C) until needed for use.

[0078] Laminin-rich protein extracts can be characterized based on the presence of laminin and the laminin subtypes that compose it. Specifically, analytical techniques based on the specificity of antigen-antibody binding can be used for characterization. Preferably, techniques using polyclonal anti-laminin antibodies or monoclonal antibodies that bind specifically to a particular α, β, or γ chain can be used. More preferably, characterization can be accomplished by Western blotting. Alternatively, other analytical methods available in the prior art can be used. In addition, the extract can be characterized by a relative increase in the amount of laminin and a relative decrease in other proteins originally present in the starting material, thereby qualifying the extract as laminin-rich, or enriched in laminin, or an equivalent expression within the scope of the present invention that is laminin-rich. Preferably, protein detection and / or quantification techniques such as A 280 monitoring of protein profiles in two-dimensional or three-dimensional gels (such as SDS-PAGE), or even analytical size exclusion chromatography can be used. Alternatively, other analytical methods available in the prior art and whose applications are known to those skilled in the art can be used. In any case, within the scope of the present invention, detection can be substantially carried out to confirm that laminin is the major component of the protein extract when compared to the starting material, thereby characterizing it as an enriched extract.

[0079] A second embodiment of the present invention relates to a method for purifying laminin from a laminin-rich protein extract, wherein the method comprises the following steps:

[0080] (a) Dissolving the laminin-rich protein extract in a resuspension buffer in the presence of a chaotropic agent;

[0081] (b) Cation exchange chromatography; and

[0082] (c) Size exclusion chromatography to obtain purified laminin.

[0083] In addition, in the context of pharmaceutical applications and within the scope of the present invention, the purified laminin obtained in step (c) can be subjected to final processing steps. These procedures are not intended to actually purify laminin from proteinaceous impurities, and they are included to increase the final concentration of the purified laminin, reduce the volume of the purified laminin, and / or remove impurities (mainly non-proteinaceous impurities), such as reducing the microbial load.

[0084] Within the scope of the present invention, the term "purified laminin" refers to a solution containing laminin isolated by the methods disclosed herein, and the methods include maximizing the removal of other proteins considered to be proteinaceous impurities from the material, as well as possible non-proteinaceous impurities, while maintaining the maximum possible amount of laminin in the final material.

[0085] Contrary to the teachings of the prior art, the purification method disclosed by the present invention unexpectedly applies to purifying a large amount of laminin from human placenta, with a rather high yield and purity. The inventors of the present invention unexpectedly observed that the purified laminin by this new method (excluding the usual anion exchange chromatography step in the prior art) enables the obtaining of sufficiently purified laminin. Even more unexpectedly, the inventors confirmed that the purified laminin produced by the technique used in the present method retains the acid polymerization ability, which is different from the laminin obtained when using the usual prior art methods (more precisely, when using anion exchange chromatography technique). Acid polymerization is a necessary and fundamental feature for the therapeutic application of laminin in compositions and treatments for central nervous system injuries, especially spinal cord injuries within the scope of the present invention. Next, the technical and inventive aspects related to the purification method of the present invention will become clearer.

[0086] Perform step (a), i.e., in the presence of a chaotropic agent, dissolve the protein extract rich in laminin to reduce the conductivity of the protein solution and weaken the interaction between laminin and protein impurities in the solution.

[0087] The protein extract rich in laminin in this step is any extract obtained from human placenta by an extraction method or by an extraction and precipitation method (including similar methods known in the prior art), provided that the extract is rich in laminin. In a preferred embodiment of the present invention, the protein extract rich in laminin is obtained by the extraction method disclosed within the scope of the present invention. In the case of using an alternative protein extract rich in laminin (i.e., obtained by other methods described or commonly used in the prior art), according to the obvious knowledge of those skilled in the art regarding this issue, the dissolution of step (a) in the chaotropic agent can be omitted or adapted to the conditions for obtaining the alternative extract. For example, if the extract is diluted or lyophilized, in a non-paste form, has a high or low ionic strength, is obtained in the presence or absence of a chaotropic agent, such as as a result of an extraction step in the prior art, those skilled in the art can, according to their inherent knowledge, adjust the procedures of centrifugation, dissolution, and addition of the chaotropic agent to convert the said alternative extract into an extract suitable for the subsequent purification step (b) without compromising the scope claimed by the present invention.

[0088] Before and after each purification step, preferably, for example, by the absorbance of the sample at 280 nm (A 280) To evaluate or estimate the total protein concentration, or, less preferably, by other methods of the prior art, such as colorimetric methods, such as the Bradford method. In addition, preferably, the protein profile is evaluated by SDS-PAGE, and the presence of laminin is detected by immunodetection techniques, such as Western blotting. Chromatographic methods can also be used to monitor the purification steps, such as size exclusion chromatography (SEC), which can be combined with dynamic light scattering analysis techniques, such as SEC-MALS (MALS = multi-angle light scattering). Alternatively, those skilled in the art can monitor the method by applying other techniques available in the prior art.

[0089] In this step (a), a "resuspension buffer" is used, which is defined as a physiologically acceptable buffer containing a chaotropic agent. The physiologically acceptable buffer is preferably a Tris buffer. The chaotropic agent is preferably urea. The dissolution of the laminin-rich protein extract in the resuspension buffer promotes a decrease in the solution conductivity / salinity and facilitates the interaction between laminin and the cation exchange resin in the subsequent chromatographic step. In addition, the role of the chaotropic agent is to destabilize the protein structure and weaken the interaction between laminin and protein impurities, which will be undesirable in the next step. In a preferred embodiment of the present invention, the resuspension buffer is 20 mM Tris containing 2 M urea and preferably has a pH of 7.5.

[0090] The resuspension buffer is added to the laminin-rich protein extract, and the mixture is slowly stirred. Preferably, a magnetic stirrer is used to stir the mixture. Within the scope of the present invention, in a preferred embodiment, the resuspension buffer is added with stirring until the conductivity reaches a value below 7 mS / cm, and this value is maintained until the maximum dissolution of the precipitate. Preferably, the stirring is maintained for 15 minutes.

[0091] Step (b) is carried out, namely cation exchange chromatography to purify the laminin-rich solution, such as the solution obtained in step (a), which contains proteinaceous impurities and aims to improve the purity of the material by separating laminin from other proteins still present in the solution, thereby improving the quality of the material injected in the final purification. This separation is carried out based on the different interactions between the charges of different proteins and the charges of the cation exchange resin, which depends on various conditions of the ionic strength used in the method, which is regulated by the concentration of positive ions in the solution.

[0092] Most proteins, including laminin, have an overall negative charge at near-neutral pH. At first glance, under these pH conditions, this would favor the interaction of laminin with anion-exchange resins rather than cation-exchange resins. In fact, when there is a choice of ion-exchange chromatography in the purification of placental laminin, the prior art almost unanimously teaches the use of anion-exchange chromatography. However, contrary to the teachings of the prior art, the inventors have found that laminin not only interacts with cation-exchange resins, but that the anion-exchange step is unnecessary for its satisfactory purification. In addition, and related hereto, the present inventors have observed that, as taught by the prior art, the use of anion-exchange chromatography techniques to purify placental laminin, although a successful purification step is obtained, results in a reduced or lost acid polymerization ability of the recovered laminin. Thus, the laminin is not suitable for use in pharmaceutical compositions for the treatment of spinal cord injuries, which is another aspect of the present invention. On the other hand, the inventors have unexpectedly found that, in the present method, by omitting the anion-exchange chromatography step and retaining the cation-exchange chromatography step, not only is purified laminin obtained, but it has a retained acid polymerization ability. Next, the cation-exchange chromatography process of step (b) is detailed according to the scope of the present invention.

[0093] Before subjecting the laminin obtained in step (a) to the cation-exchange chromatography of step (b), the material obtained in step (a) can be prepared. Preparations known to those skilled in the art can be, for example, filtration steps. Thus, within the scope of the present invention, the resuspended protein extract obtained in step (a) or an equivalent material according to the previously described is filtered, with the aim of removing protein precipitates, cell debris, and residual tissue that may still be retained in the extract. This removal is necessary to ensure the quality of the subsequent steps and contributes to the lifespan of the chromatography resin. Thus, this preparation is not essential for increasing the degree of purification of the material related to laminin and is not considered to be the basis of the purification method itself.

[0094] Filtration is carried out on a filter of 0.5 μm or less. Preferably, filtration is carried out on a 0.1 μm filter. Even more preferably, filtration is carried out using a combination of a pre-filter and a filter. In a preferred embodiment of the present invention, filtration is carried out using a 0.5 μm pre-filter and a 0.1 μm filter. Filtration can be carried out with the aid of a peristaltic pump. After the filtration step, the solution must be clear and free of particles.

[0095] Thus, within the scope of the present invention, the material obtained in step (a) or an equivalent suitably prepared as known to the person skilled in the art, preferably the filtered material, is subjected to cation exchange chromatography in step (b). Preferably, a cation exchange resin suitable for separating large biomolecules such as laminin is used according to the instructions of the resin manufacturer. For example, the resin-based matrix is highly porous with large pore diameters, which reduces steric hindrance and facilitates the adsorption of macromolecules. In an embodiment of the present invention, a resin composed of a hydrophilic porous polymer or copolymer is used. In a preferred embodiment, a cross-linked copolymer of allyl dextran and N,N-methylenebisacrylamide or a cross-linked agarose matrix or a stabilized reinforced cellulose membrane is used. Regarding functionalization, the matrix is linked to a negatively charged group. Preferably, the matrix is functionalized with sulfopropyl (SP) or methylsulfonate group (S), which are strong cation exchangers. In a preferred embodiment of the present invention, the resin is a cross-linked copolymer of allyl dextran and N,N-methylenebisacrylamide, which is functionalized with -SO3 - and more preferably with sulfopropyl functionalization.

[0096] In the above chromatography step, two buffers are used. A resuspension buffer and an "elution buffer" as defined in this specification. The elution buffer is different from the resuspension buffer because it has a higher conductivity, which is required to elute the proteins bound to the chromatography resin. Preferably, the higher conductivity is obtained by increasing the ionic strength in the elution buffer and is due to the presence of NaCl. Preferably, the elution buffer contains Tris buffer, urea, and NaCl. In a preferred embodiment of the present invention, the elution buffer is 20 mM Tris, 2 M urea, 1 M NaCl, and pH 7.5.

[0097] The cation exchange chromatography step is carried out according to the knowledge of the person skilled in the art. The material obtained in step (a) or an equivalent suitably prepared as known to the person skilled in the art, preferably the filtered material, is introduced into a chromatography column in the resuspension buffer and eluted by gradually increasing the conductivity of the eluent, where the increase in the conductivity of the eluent is achieved by adding a predetermined amount of the elution buffer. Elution fractions are collected according to the increase in the total protein concentration, which can be evaluated by A 280 and those with a high laminin concentration relative to the total protein concentration are used for subsequent steps. Monitoring the laminin concentration in the above fractions to select the fractions suitable for the purification method is carried out by using techniques available in the prior art, preferably by analytical size exclusion chromatography of the samples.

[0098] In a preferred embodiment of the present invention, a chromatographic column is equilibrated with a buffer solution having a conductivity of 7.0 mS / cm to 7.1 mS / cm, wherein impurities with low interaction with the resin are eluted. Then, laminin is eluted at a conductivity of 14.0 mS / cm to 14.1 mS / cm. Finally, high-interaction impurities are eluted and the column is washed at a conductivity of 48.0 mS / cm to 48.1 mS / cm. In view of this non-limiting embodiment, a person skilled in the art can adjust the chromatographic conditions according to the selected resin.

[0099] The elution fraction having partially purified laminin can be stored under refrigerated conditions, preferably at a temperature of 2°C to 8°C, until used in subsequent purification steps.

[0100] In addition, but not necessarily, the elution fraction containing partially purified laminin obtained in step (b) of cation exchange chromatography can be concentrated to reduce its volume and facilitate subsequent steps. For example, when the A 280 absorbance of the elution fraction containing partially purified laminin is less than 1.00 AU (absorbance unit), the elution fraction can be concentrated.

[0101] For the material eluted in step (b) of cation exchange chromatography, i.e., the elution fraction containing partially purified laminin, step (c), size exclusion chromatography, is carried out. The purpose is to separate laminin from the impurities remaining in the solution according to the apparent size using an inert porous resin.

[0102] Preferably, according to the instructions of the resin manufacturer, a size exclusion resin suitable for separating large biomolecules such as laminin is used. Preferably, the resin has pores with a nominal separation range of 5 kDa to 5000 kDa. In an embodiment of the present invention, the resin is a hydrophilic porous polymer or copolymer. In a preferred embodiment of the present invention, the resin is a highly cross-linked agarose matrix.

[0103] In size exclusion chromatography, only buffer solution is used as the mobile phase, and its flow rate in the column is constant. Preferably, the buffer solution used is PBS.

[0104] Step (c), i.e., size exclusion chromatography, is carried out according to the knowledge of a person skilled in the art. The elution fraction containing partially purified laminin obtained in step (b) is inserted into a column already filled with equilibrated size exclusion resin. Then, buffer solution is injected at a constant flow rate, the eluted fractions are collected and tested for the presence of laminin and total protein. The monitoring of the estimated concentrations of total protein and laminin is carried out as explained in this specification. Those fractions in which the laminin concentration increases relative to the total protein concentration compared to the previous steps of the method are the fractions of interest.

[0105] The fraction collected as a result of step (c) comprises purified laminin.

[0106] The final treatment of the purified laminin is an additional purification process as its main purpose is not to increase the purity of the purified laminin with respect to protein properties. The final treatment is used to confer a higher concentration to the purified laminin and / or remove any residual impurities, especially impurities of a non - protein nature of the material, for example, to reduce the microbial load. The following description will make the purpose and conduct of the final treatment steps clear, where the person skilled in the art selects to apply the final treatment according to the needs observed during the conduct of the method and implements one or more of the aforementioned treatment steps.

[0107] The final treatment of the purified laminin may include a concentration step. The purpose of performing this step is to correct the concentration of the laminin solution to a target concentration, where the target concentration is higher than the concentration obtained as a result of step (c) of the purification method. Within the scope of the present invention, as part of the method of using laminin as an active ingredient in a pharmaceutical composition, the fraction eluted in the chromatography of step (c) is preferably subjected to concentration. To achieve this, a concentrator is used and the measured value A 280 is corrected to a value greater than 0.25 AU. The concentration method used is according to the description of the prior art and includes, for example, using a concentrator or tangential flow filtration (TFF). Preferably, a concentrator with a cut - off value of 30 kDa or 50 kDa is used. In a preferred embodiment of the present invention, a concentrator with a polyethersulfone (PES) membrane is used. At the end of the concentration step, the purified and concentrated laminin solution is recovered.

[0108] The final treatment of the purified laminin may include a filtration step. The purpose of performing this step is to reduce the microbial load of the material. The filtration step is carried out in a non - contaminated medium, preferably in a laminar flow, and a sterilizing filter, preferably a 0.2 μm filter, is used. In a preferred embodiment of the method of the present invention, filtration is carried out using a sterile polyethersulfone membrane filter.

[0109] The final treatment of the purified laminin may include a fractionation step. In this step, the purified, optionally concentrated, optionally filtered laminin is fractionated in a sterile container, preferably a polypropylene tube.

[0110] After the purification process, the purified laminin recovered in step (c), or alternatively the purified laminin that has undergone the final treatment, is analyzed for the total protein concentration (for example using A 280), and an analysis of the final laminin concentration is performed (e.g., using analytical SEC and SEC-MALS). The final yield of the purification method is also calculated. The material recovered in step (c), or alternatively in step (d), can then be stored frozen until use. Preferably, the material is stored at -80 °C.

[0111] As observed by the inventors, the purified laminins obtained by the purification methods disclosed herein are suitable for therapeutic applications for treating nervous system injuries, particularly for treating spinal cord injuries, because they retain their acid polymerization ability. For this property, the present invention is presented in other embodiments disclosed below, where, for the sake of simplicity, the term "purified laminin" is used to refer to laminins obtained by the purification method defined by steps (a) to (c), with or without undergoing a final treatment.

[0112] A third embodiment of the present invention relates to a method for the polymerization of purified laminin, wherein the method is carried out with purified laminin, which is a laminin obtained by the purification method disclosed in the present invention, or is carried out with a laminin obtained by combining the extraction method and the purification method disclosed in the present invention, or even is carried out with a laminin obtained by combining any suitable laminin extraction method in the prior art and the purification method disclosed herein.

[0113] The polymerization method is carried out by contacting the purified laminin with an acidic preparation, and the pH of the resulting composition is preferably between 4.0 and 5.5, more preferably the pH is between 4.2 and 4.4. In an embodiment of the polymerization method of the present invention, the purified laminin is mixed with an acetic acid preparation. In a preferred embodiment, the purified laminin is added to a 30 mM acetic acid preparation. The acidic preparation may also contain an osmotic pressure regulator in an amount sufficient to achieve a physiological osmotic pressure range suitable for local application in spinal cord injuries, preferably the osmotic pressure range is between 270 mOsmol / kg and 330 mOsmol / kg. A preferred osmotic concentration regulator is sodium chloride. In a preferred embodiment, the acidic preparation contains 144.87 mM sodium chloride. The inventors of the present invention have surprisingly observed that the purified laminins obtained by the method claimed in the present invention are suitable for the acid polymerization method, which is different from the laminins obtained by the purification methods usually involving an anion exchange chromatography step in the prior art.

[0114] An additional feature of the claimed polymerization method is the presence of cations in the polymerization medium. Preferably, the method is carried out in the presence of divalent cations. In a more preferred embodiment of the present invention, the divalent cation is Ca 2+In addition, in an embodiment of the present invention, the acidic preparation contains calcium ions. In a preferred embodiment, the acidic preparation comprises acetic acid and calcium chloride, and the latter preferably has a concentration of 1.2 mM. In a more preferred embodiment of the present invention, the acidic preparation comprises 30 mM acetic acid, 1.2 mM calcium chloride and 144.87 mM sodium chloride.

[0115] The fourth embodiment of the present invention relates to the use of purified laminin in the preparation of a polylaminin pharmaceutical composition, wherein the purified laminin is obtained by the purification method disclosed in the present invention or by a combination of the extraction and purification methods disclosed in the present invention, or alternatively by a combination of any suitable laminin extraction method in the prior art and the purification method disclosed herein. Since the laminin obtained by the improved purification method as described herein retains the acid polymerization property, the use of the purified laminin in the pharmaceutical polylaminin composition becomes feasible. Therefore, it is feasible to treat central nervous system injuries, mainly spinal cord injuries, with the above-mentioned pharmaceutical composition.

[0116] The fifth embodiment of the present invention relates to polylaminin obtained by acid polymerization of purified laminin, wherein the purified laminin is obtained by the purification method disclosed in the present invention, or by combining the extraction method and the purification method disclosed in the present invention, or even by a combination of a suitable laminin extraction method in the prior art and the purification method disclosed herein. The polylaminin is obtained by the polymerization method as described in the previous embodiments of the present invention, which comprises contacting the purified laminin with an acidic medium, preferably in the presence of a divalent cation.

[0117] As demonstrated by the inventors, the acid polymerization ability of the purified laminin obtained by other methods in the prior art, more precisely, the laminin obtained using an anion exchange chromatography step, is reduced or eliminated, and when in an acidic medium, they do not polymerize into polylaminin, which makes the polylaminin obtained by the extraction method and the purification method described herein another object of the present invention.

[0118] The sixth embodiment of the present invention relates to a kit which essentially comprises a first vial and a second vial, wherein the first vial contains a preparation containing purified laminin and the second vial contains an acidic preparation. These kits can be used for the immediate preparation of a polylaminin composition for treating central nervous system injuries (especially for treating spinal cord injuries). In the context of the present invention, the acidic preparation may comprise an osmotic pressure regulator, preferably sodium chloride, in an amount sufficient to achieve a physiological osmotic pressure range suitable for topical application to spinal cord injuries. In addition, the kit comprises a preparation having a divalent cation, which can be contained in the first vial, the second vial or even an additional third vial of the kit.

[0119] Within the scope of the present invention, the term "instant preparation" refers to the preparation of a mixture between the components of the first, second, and optionally third vials of the kit just prior to its therapeutic application. Preferably, the preparation is carried out in vitro. Thus, the kit may additionally include instructions for instant preparation of the poly-laminin composition for treating central nervous system injuries (especially for treating spinal cord injuries).

[0120] In clinical studies, it has been demonstrated that the kits of the present invention as well as poly-laminin can be used for the recovery of patients with spinal cord injuries. Therefore, the methods described herein are very important for public health, especially in addressing an injury for which there has been no treatment option until now.

[0121] Through the following examples, the superiority of the methods, uses, kits, and poly-laminin disclosed by the present invention will become clearer. The examples represent the preferred forms of carrying out the present invention and should not, therefore, limit the scope of the present invention.

[0122] Examples

[0123] Example 1: Obtaining a laminin-rich protein precipitate or extract from human placenta

[0124] The method for extracting laminin from human placenta according to the present invention is carried out through the following steps.

[0125] Human placentas were obtained from parturients through anonymous, gratuitous, and voluntary donation with the prior express consent of the parturients. They were selected according to strict eligibility criteria based on clinical history, genetic diseases, and infectious diseases. At the time of delivery, the donated placentas were placed in appropriately sealed primary and secondary plastic packages and then stored at -20 °C to maintain the integrity of the material until processing.

[0126] Using laminar flow, the material was thawed in water at room temperature, the outer membrane, remaining amnion, and umbilical cord were removed and discarded, and then the placental tissue was cut into cubes and washed with a refrigerated saline solution (NaCl 0.9% w / v) to eliminate clots and fluids. The chorion was not removed.

[0127] After adding 200 mL of ice-cold ultrapure water per 100 g of tissue, the clean placental tissue was homogenized in an industrial blender for 4 minutes. The fluids and blood released during homogenization were removed by centrifuging at 3000 × g for 5 minutes at 4 °C, the supernatant was discarded, and the centrifugation cycle was repeated twice to recover the solid material.

[0128] For laminin extraction, every 100 g of centrifuged solid tissue was resuspended in 200 mL of ice-cold extraction buffer (50 mM Tris, 1.0 M NaCl, 10 mM EDTA, pH 7.4). The suspension was slowly stirred with a magnetic stirrer for 8 to 16 hours at a temperature of 2 °C to 8 °C. Additionally, the liquid phase or supernatant containing soluble laminin was separated from the residual tissue by centrifugation (15,000×g, 30 minutes, 4 °C).

[0129] Finally, at 4 °C, laminin was precipitated from the supernatant recovered from the previous step using a 30% saturated salt concentration. Thus, 16.98 g of ammonium sulfate was slowly added per 100 mL of solution with slow stirring until completely dissolved, and then the solution was kept stirred for 2 hours at a temperature of 2 °C to 8 °C. After this period, high turbidity was observed due to the formation of protein precipitates in the suspension. The laminin-rich protein precipitate or extract was recovered by centrifugation (15,000×g, 30 minutes, 4 °C) and stored at -20 °C.

[0130] Example 2: Determination of the ideal saturation concentration of salt for laminin precipitation by ammonium sulfate salting-out

[0131] The ideal conditions for precipitating laminin with ammonium sulfate were determined by experimentation, i.e., at 4 °C, the saturation concentration of the salt was 20% to 80%.

[0132] At concentrations of 20% and 25%, the presence of laminin was detected in the supernatant fraction ( Figure 1 ), which is evidence of the incomplete precipitation of laminin from the solution and was not observed at concentrations of 30%, 40%, 60%, and 80%. In turn, the analysis of the precipitate fraction showed that the co-precipitation of impurities with laminin increased starting from a concentration of 40% ( Figure 2 ), which is undesirable as it reduces the quality of the laminin-rich protein extract, which will be the starting material for subsequent steps in the purification method. From the results, the researchers concluded that a 30% saturated concentration of ammonium sulfate is the ideal condition for the extraction method because there is a balance between the maximum precipitation of laminin (no laminin was detected in the supernatant) and the reduction of impurity co-precipitation, which is advantageous in subsequent purification steps as estimated from the total mass of the precipitate. However, this step of the method can be carried out under saturated conditions of 30% to 80% because laminin is completely precipitated, even though they constitute a smaller percentage of the extract. Saturated conditions between 20% and 30% can also be used, although the method has a lower yield due to the loss of laminin in the supernatant fraction.

[0133] Example 3: Optional filtration step before precipitation

[0134] Regarding the filters used, the removal of solid residues by filtration before salt precipitation was evaluated. Based on the results shown in Table 1, among the filters evaluated, depth filters were found to be the most suitable for reducing the turbidity of the suspension and improving the quality of the final protein extract.

[0135] Table 1: Filters tested in the purification step.

[0136]

[0137] Example 4: Purification of a laminin-rich protein extract to obtain purified laminin

[0138] The laminin-rich protein extract obtained as described in the previous examples was subjected to a purification method. It was demonstrated by SDS-PAGE that the purity of laminin in the material increased through the following purification steps, as Figure 3 shown. Other analytical methods performed are mentioned in the description of each step.

[0139] Example 4.1: Resuspension

[0140] In the same purification method, up to three protein extracts from different placentas were used together. As described below, the flasks containing the protein extracts were thawed at room temperature and diluted in resuspension buffer (20 mM Tris, 2 M urea, pH 7.5).

[0141] Resuspension buffer was added to the flasks containing the extracts, gently shaken to release any extract adhering to the flask walls, and then the flasks were washed with the buffer. This procedure was repeated for all flasks containing extracts, and the extract materials were collected in a single container of appropriate size and stirred with a magnetic stirrer. Under slow stirring, the conductivity was monitored with a conductivity meter, and resuspension buffer was slowly added to the solution until the measured conductivity reached the condition of <7 mS / cm. The solution was slowly stirred at room temperature for 15 minutes to ensure maximum dissolution of the extracts. After dissolution, samples were taken to evaluate the total protein concentration ( 280 ) by absorbance at 280 nm (A Figure 3 ).

[0142] Example 4.2: Filtration

[0143] The suspension obtained according to the procedure described in Example 4.1 was filtered to remove protein precipitates, cell debris, and residual tissue present in the solution as a preparation for the subsequent chromatography step.

[0144] Using a peristaltic pump, the suspension was pumped into a filtration capsule Merck-Millipore SHR OptiCap Capsule XL3 0.5 / 0.1μm, which has a 0.5μm pre-filter, a 0.1μm polyethersulfone (PES) filter and a 0.16m 2 filtration area. If clogging occurs during the method, the capsule is emptied and the material filtration is completed using a new capsule. After filtration, a clarified, particle-free solution is obtained. Samples were taken to evaluate the total protein concentration (A 280 ), molecular exclusion profile (SEC) and SDS-PAGE profile ( Figure 3 ).

[0145] Example 4.3: Cation Exchange Liquid Chromatography (CEX)

[0146] Cation exchange chromatography uses a resin functionalized with negatively charged groups that interact with positively charged solutes.

[0147] In this embodiment of the invention, a Millipore Vantage L Laboratory Column VL 32x250 column was used, which was packed with 41 mL of Cytiva MacroCap SP resin. The column was installed in a Cytiva Pure chromatograph. During this process, a mixture of buffer A and buffer B was used. Buffer A is a resuspension buffer consisting of 20 mM Tris and 2 M urea, pH 7.5. Buffer B is an elution buffer consisting of 20 mM Tris, 2 M urea and 1 M NaCl, pH 7.5. The ratios used in the mixture of buffer A and buffer B in each chromatographic step are described in Table 2 below, and the complete chromatographic method carried out according to this embodiment of the invention is shown in Figure 4 . The inventors determined the conductivity range in the method they programmed into the chromatograph, which mixes buffer A and buffer B with a gradually increasing salt concentration.

[0148] Table 2: Buffer solutions used in the CEX chromatographic method depending on the mixture to achieve the appropriate target conductivity (σ) at each step.

[0149]

[0150] After packing, the column was equilibrated with a mixture of buffer A and buffer B in the range of 7.0 mS / cm - 7.1 mS / cm. After equilibration, the steps of injecting the material into the column, washing and eluting laminin were carried out using an automated method.

[0151] Using a chromatograph sample pump, the resuspended and filtered extract was injected into the column at a flow rate of 90 cm / h (12 mL / min). Injection was continued until the air sensor associated with the sample pump detected that the contents had been completely injected into the column, and then the sample pump and tubing were automatically emptied with the injection of the resuspension buffer. Next, the column was washed with 10 column volumes (CV) of a mixture of buffer A and B with a conductivity of 7.0 mS / cm - 7.1 mS / cm at a flow rate of 90 cm / h to elute impurities with low interaction with the resin. Elution was monitored by absorbance at 280 nm, and a stable baseline indicated the elution of the above impurities, as Figure 5 shown.

[0152] Subsequently, laminin was eluted from the resin. The ratio between buffer A and B was changed to achieve a conductivity of 14.0 mS / cm to 14.1 mS / cm. An increase in absorbance at 280 nm monitored by the chromatograph indicated the start of protein elution, as Figure 5 shown.

[0153] The fractions collected ( Figure 5 ) contained an initial 0.85 CV (35 mL) elution peak, including the major fraction laminin, with a reduced amount of protein impurities. Therefore, this fraction proceeded to the next purification step. In the remaining elution peaks, laminin was still detected but at a lower concentration, and a large amount of related protein impurities were also detected, so it was considered unsuitable for further purification. Therefore, the fractions collected were characterized according to the total protein concentration (A 280 ) and SDS-PAGE profile ( Figure 3 ) and then stored in a refrigerator at a temperature of 2 °C to 8 °C.

[0154] Finally, after eluting the laminin-rich fraction of interest, the remaining impurities in the column were removed by changing the ratio of buffer A and B to achieve a conductivity of 48.0 mS / cm - 48.1 mS / cm. Next, the column was washed with 1 M sodium hydroxide and water and stored in 20% ethanol.

[0155] Example 4.4: Concentration

[0156] In the concentration step, the material eluted from the cation exchange was concentrated using 2 to 6 Merck-Millipore Amicon Ultra-15 concentrators (with a cut-off value of 30 kDa or 50 kDa). The target volume was calculated using the equation:

[0157] V 目标 =(C 洗脱 *V 洗脱 ) / 1.35

[0158] where V目标 = Target volume (mL) at the end of the concentration step, C 洗脱 = Total protein concentration (UA) of the eluted material in the CEX step in terms of A 280 and V 洗脱 = Volume (mL) of the eluted material in the CEX step.

[0159] Fill, balance the concentrator and centrifuge at 3000 × g for 10 minutes at 4 °C. After 10 minutes, pipette to stir the solution in the concentrator (to avoid aggregation at the bottom of the concentrator) and measure its volume. Repeat this cycle until the target volume is reached. Then combine the concentrated solution and measure its concentration again by A 280 Store the concentrated eluted material in a refrigerator at 2 °C to 8 °C.

[0160] During the development of the present invention, the inventors observed that when the purification method starts only from the protein extract of one placenta, a concentration step is required, but after scale-up, for example, from the extract of three placentas, the concentration step is not necessary. It was observed that when the total protein concentration analyzed by A 280 reaches a value equal to or greater than 1.35 AU, the concentration process of the material is too slow, resulting in protein aggregation in the sample. However, after scale-up, the eluted material from the CEX step has a total protein concentration analyzed by A 280 and a value higher than 1.35 AU, so the concentration step becomes unnecessary.

[0161] Example 4.5: Size Exclusion Liquid Chromatography (SEC)

[0162] In this embodiment of the present invention, purification is carried out using Cytiva Superose 6 prep grade resin, whose nominal separation range is from 5 kDa to 5000 kDa and is suitable for the separation of high molecular weight molecules such as laminin. The chromatography column used is HiLoad 16 / 600, 60 cm high, and the total resin volume is 120 mL.

[0163] After packing, equilibrate the column in PBS (phosphate buffered saline), then inject 5 mL (4.17% CV) of the eluted material (concentrated or not) from the CEX step into the column at a constant flow rate of 1 mL / min (29.8 cm / h). After injection, use PBS as the mobile phase for chromatography and keep the flow rate at 1 mL / min. Start collecting fractions at 46.5 mL (0.3875 CV) after the start of injection. The first 9.00 mL fraction collected contains the start and main part of the laminin peak. Immediately afterwards, a second fraction of 3.46 mL was also collected. The complete chromatography method carried out according to this embodiment of the present invention is shown in Figure 6 in.

[0164] The second fraction contains the final part of the laminin peak and possible impurities of protein nature, and its proportion may vary from batch to batch. To evaluate the quality of the material, the first and second fractions were analyzed by size exclusion chromatography (Cytiva Superose 6 Increase 10 / 300gl column), and their chromatograms were compared, as Figure 7 shown for two different batches in Figure 7 A). When the two fractions presented similar elution curves ( Figure 7 A), i.e., with consistent retention times and no secondary peaks, both fractions were used to compose the purified laminin pool for final processing, as this demonstrated that there were few impurities in the second fraction. Otherwise (

[0165] B), the purified laminin pool for final processing and subsequent steps was composed only of the first fraction. Figure 8 In this embodiment of the invention, the injection volume was limited to 5 mL, and multiple chromatographic runs were required to process all the material from the previous purification step. Typically, for a cation exchange eluate that has not undergone a concentration step, seven runs are required for complete processing. To accelerate the method, the inventors developed a chromatography in loop form (

[0166] ), which injects the material at a pre-calculated time so as to compress multiple runs into one run. Using this method, the waiting time for completing each run and the residence time of the dead volume (the time between injection and the start of elution of the first solute) were eliminated. A person skilled in the art can adapt the sample injection to the most convenient column, resin, and chromatographic method based on the knowledge of the prior art. 280 Finally, the fractions selected for the laminin pool from multiple runs were combined and characterized according to the theoretical concentration calculated from the A

[0167] Example 4.6: Concentration

[0168] After the size exclusion chromatography (SEC) step, the laminin pool was already in its final formulation (PBS), but it was still necessary to correct its concentration so that the A 280 value was greater than 0.25 UA. To achieve this, an equation was used to calculate the target volume:

[0169] V 目标 =(C 池 *V 池 ) / 0.32

[0170] where V 目标 = the target volume (mL) at the end of the concentration step, C 池= The total protein concentration (UA) represented by A of the laminin library eluted in the SEC step, and V 280 represents the total protein concentration (UA), and V 池 = The volume (mL) of the laminin library eluted in the SEC step.

[0171] The concentration process is similar to the process described above. Use two to six Merck - Millipore AmiconUltra - 15 concentrators with a cut - off value of 30 kDa or 50 kDa. Fill, balance the concentrators and centrifuge at 3000×g for 5 minutes at 4°C. After 5 minutes, pipette and agitate the solution in the concentrator and measure its volume. Repeat this cycle until the target volume is reached. Next, measure A 280 , and correct the concentration to the range of 0.30 AU to 0.34 AU by another centrifugation cycle or dilution with PBS. Then store the concentrated laminin pool in a refrigerator at 2°C to 8°C.

[0172] Example 4.7: Filtration, fractionation and storage

[0173] In a laminar flow, use a sterile Millipore Millex GP Filter Unit 0.22μm filter to filter the concentrated laminin pool. Next, fractionate the filtered pool in a sterile polypropylene tube and record its final volume. One of the samples is used for the final concentration analysis and final yield calculation by A 280 . Freeze this batch and store it in a refrigerator at - 80°C.

[0174] Example 5: Anion - exchange liquid chromatography (AEX)

[0175] Test this chromatographic step as an intermediate purification step for laminin. The purpose of this step is to remove urea from the solution and improve the material purity, thus improving its quality for injection in the final purification step. Develop on Cytiva MacroCap Q resin by directly injecting the material obtained from CEX chromatography.

[0176] Perform the AEX step on a MacroCap Q 3 mL column. First, inject the material eluted from the CEX step, then wash the column with a conductivity of 14 mS / cm to remove protein impurities with low interaction with the resin and remove urea. Wash the column with a buffer with a conductivity of 20 mS / cm. Elute laminin with a buffer with a conductivity of 28 mS / cm and collect the corresponding fractions. Finally, elute the remaining impurities with high interaction with the resin with 1M NaCl.

[0177] In terms of purification, the introduction of the AEX chromatographic step is successful ( Figure 9)。For the test batch, the relative purity of the material after AEX increased from 25% to 63%, and it was satisfactory to recover 81% of laminin from the sample. However, when analyzing the finished product batch, it was observed that laminin decreased or lost its acid polymerization ability, making its therapeutic use infeasible. Through two orthogonal techniques, DLS (Dynamic Light Scattering) and Pulldown, the loss of polymerization ability was observed ( Figure 10 ). Another three batches produced by introducing the AEX step showed the same decrease in acid polymerization ability. For this reason, the use of the AEX step was abandoned.

Claims

1. A method for extracting laminin from human placenta to obtain a protein extract rich in laminin, characterized in that, Comprising the following steps: (i) Treating the placenta to obtain clean placental tissue; (ii) Homogenizing the placental tissue and separating the homogenized tissue and the resulting fluid; (iii) Extracting laminin from the placental tissue; (iv) Optionally, filtering; and (v) Selectively precipitating laminin in the solution obtained in step (iii) or step (iv) by salting out to obtain the laminin-rich protein extract; wherein, in step (i), it is not necessary to remove the chorion from the placental tissue.

2. The method according to claim 1, wherein The method is carried out on fresh, refrigerated or frozen placentas.

3. The method according to claim 2, wherein The method is carried out on placentas frozen at -20°C and includes rapid or slow thawing of the placentas.

4. The method according to claim 3, characterized in that Preferably, rapid thawing of the placentas is carried out at room temperature.

5. The method according to claim 1, wherein step (i) is characterized by removing unwanted components and washing the placental tissue.

6. The method according to claim 5, wherein The washing is carried out with a physiologically acceptable liquid at low temperature, preferably with a cooled saline solution.

7. The method according to claim 1, wherein step (ii) is characterized in that the homogenization is carried out in an industrial stirrer with a physiologically acceptable liquid, preferably cooled ultrapure water.

8. The method according to claim 1, wherein step (ii) is characterized in that the homogenized tissue and the resulting fluid are separated by centrifugation.

9. The method according to claim 1, wherein step (iii) is characterized in that the extraction step is carried out in an extraction buffer containing Tris, NaCl and EDTA. Preferably, the buffer consists of 50 mM Tris, 1.0 M NaCl, 10 mM EDTA and has a pH of 7.

4.

10. The method according to claim 9, characterized in that, The ratio of the volume of the buffer to the mass of the placental tissue is 2:

1.

11. The method according to claim 9, characterized in that, The extraction is carried out with slow stirring.

12. The method according to claim 1, wherein step (iv) is characterized in that the filtration is carried out through a depth filter.

13. The method according to claim 1, wherein step (v) is characterized in that the precipitation of laminin is carried out with ammonium sulfate having a saturation concentration between 20% and 80%, preferably 30%.

14. A method for purifying laminin from a protein extract rich in laminin, characterized in that, Comprising the following steps: (a) Dissolving the laminin-rich protein extract in a resuspension buffer in the presence of a chaotropic agent; (b) Cation exchange chromatography; and (c) Size exclusion chromatography to obtain purified laminin.

15. The method according to claim 14, characterized in that The method additionally includes final treatment of the purified laminin.

16. The method according to claim 14, characterized in that, The method is carried out on a laminin-rich protein extract obtained from human placentas.

17. The method according to claim 16, wherein The laminin-rich protein extract is obtained by the method according to claims 1 to 13.

18. The method according to claim 14, wherein step (a) is characterized in that the chaotropic agent is urea.

19. The method according to claim 14, wherein step (a) is characterized in that the resuspension buffer is 20 mM tris containing 2 M urea and having a pH of 7.

5.

20. The method according to claim 14, wherein step (a) is characterized in that the buffer solution is added under slow stirring and the stirring is maintained until the protein extract is maximally dissolved. Preferably, the stirring is maintained for 15 minutes.

21. The method according to claim 14, wherein step (b) is characterized in that it comprises preparing the material obtained in step (a), preferably by filtration.

22. The method according to claim 21, characterized in that, The filtration is carried out on a filter of 0.5 μm or less, preferably on a 0.1 μm filter.

23. The method according to claims 21 and 22, characterized in that, The filtration is carried out using a combination of a pre-filter and a filter, preferably using a 0.5 μm pre-filter and a 0.1 μm filter.

24. The method according to claim 14, wherein step (b) is characterized in that the cation exchange chromatography is carried out using a cation exchange resin suitable for separating large biomolecules, preferably a resin having a large pore size in the basic matrix.

25. The method according to claim 24, wherein The cation exchange chromatography is carried out using a resin composed of a hydrophilic porous polymer or copolymer.

26. The method according to claims 24 and 25, characterized in that, The cation exchange chromatography is carried out using a resin which is a cross-linked copolymer of allyl dextran and N,N-methylenebisacrylamide, or a cross-linked agarose matrix or a stable reinforced cellulose membrane, preferably a resin which is a cross-linked copolymer of allyl dextran and N,N-methylenebisacrylamide.

27. The method according to claim 14, wherein step (b) is characterized in that the cation exchange chromatography is carried out using a sulfopropyl (SP) or methanesulfonate (S) group, preferably a sulfopropyl (SP) functionalized resin.

28. The method according to claim 14, wherein step (b) is characterized in that the laminin fraction is eluted with an ionic strength gradient, preferably by increasing the NaCl concentration.

29. The method according to claim 14, wherein step (b) is characterized in that the cation exchange chromatography is preferably carried out using a resin functionalized with sulfopropyl (SP), wherein, The material introduced into the column has a conductivity of 7 mS / cm, and the laminin fraction is eluted by adding NaCl as the conductivity of the eluent increases to a range of 14.0 mS / cm to 14.1 mS / cm.

30. The method according to claim 14, wherein step (c) is characterized in that chromatography is carried out using a size exclusion resin suitable for separating large biomolecules.

31. The method according to claim 30, wherein Size exclusion chromatography is carried out using a resin which is a hydrophilic porous polymer or copolymer.

32. The method according to claims 30 and 31, characterized in that, Size exclusion chromatography is carried out using a resin which is a highly cross-linked agarose matrix.

33. The method according to claims 30, 31, and 32, characterized in that, The resin has pores with a nominal separation range of 5 kDa to 5000 kDa.

34. The method according to claim 15, wherein The final treatment of the purified laminin includes one or more processes selected from concentration, filtration and fractionation.

35. The method according to claim 34, wherein The concentration process is carried out using a concentrator, preferably a concentrator having a cut-off value of 30 kDa or 50 kDa.

36. The method according to claim 35, characterized in that, Carry out the concentration process until an A value greater than 0.25 AU is reached. 280 value.

37. The method according to claim 34, wherein The filtration process is carried out using a sterilizing filter, preferably using a 0.2 μm filter.

38. A method for purifying laminin from a laminin-rich protein extract, characterized in that, It includes a cation exchange chromatography step, wherein the step is the only ion exchange chromatography step in the method.

39. A method for polymerizing laminin, characterized in that, The purified laminin is contacted with an acidic preparation to obtain a composition, wherein the purified laminin is obtained by the method defined in claims 14 to 38.

40. The method according to claim 39, wherein The composition has a pH in the range of 4.0 and 5.5, preferably between 4.2 and 4.

4.

41. The method according to claim 39, wherein It is carried out in the presence of an osmotic pressure regulator, preferably sodium chloride.

42. The method according to claim 41, wherein The osmotic pressure regulator is used in an amount sufficient to achieve physiological osmotic pressure, preferably in the range of 270 mOsmol / kg to 330 mOsmol / kg.

43. The method according to claim 39, wherein In the presence of divalent cations, preferably Ca 2+ This is carried out in the presence of 2+ .

44. The method according to claim 39, wherein The acidic preparation contains 30 mM acetic acid, 1.2 mM calcium chloride, and 144.87 mM sodium chloride. Use of laminin, characterized in that, The laminin is used to prepare a poly-laminin pharmaceutical composition for treating central nervous system injuries, mainly spinal cord injuries, wherein the laminin is purified by the method defined in claims 14 to 38.

46. Poly-laminin, characterized in that, The poly-laminin is obtained by acid polymerization of the purified laminin obtained by the purification method defined in claims 14 to 38.

47. A kit for the immediate preparation of a poly-laminin pharmaceutical composition for the treatment of spinal cord injury and central nervous system injury, characterized in that, The kit includes a first vial containing the purified laminin obtained by the purification method defined in claims 14 to 38 and a second vial containing the acidic preparation.

48. The kit according to claim 47, wherein, The acidic preparation additionally contains an osmotic pressure regulator, preferably sodium chloride.

49. The kit according to claim 47, wherein, The kit additionally includes divalent cations, preferably Ca 2+ preparations.

50. The kit according to claim 47, wherein The kit additionally includes instructions on the immediate preparation of the poly-laminin pharmaceutical composition and its therapeutic applications.

51. The kit according to claim 47, characterized in that, The acidic preparation contains 30 mM acetic acid, 1.2 mM calcium chloride, and 144.87 mM sodium chloride.

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