Carrier for long-term storage of biopharmaceuticals and manufacturing method thereof

Abstract

The present invention relates to a carrier for the long-term storage of biopharmaceuticals and a manufacturing method thereof, and specifically, to a carrier using freeze-drying of inorganic nanoparticles for the preservation and distribution of biopharmaceuticals comprising proteins or nucleic acids as active ingredients, and a manufacturing method thereof.

Description

Delivery vehicle for long-term storage of biological drugs and method for manufacturing the same

[0001] The present invention relates to a delivery vehicle for the long-term storage of biological pharmaceuticals and a method for manufacturing the same. Specifically, it relates to a delivery vehicle utilizing freeze-drying of inorganic nanoparticles for the preservation and distribution of biological pharmaceuticals containing proteins or nucleic acids as active ingredients, and a method for manufacturing the same.

[0002] Biological drugs, or biopharmaceuticals, refer to medicines based on living cells, tissues, microorganisms, or biologically derived materials that are utilized for the prevention, diagnosis, or treatment of diseases. They exert therapeutic effects primarily by utilizing proteins, antibodies, genes, or cells to regulate biological responses or activate the immune system. Types include protein-based therapeutics (e.g., monoclonal antibodies, plasma-derived proteins), vaccines, gene therapies, and cell therapies, and they are applied to a wide range of diseases, including autoimmune diseases, cancer, infectious diseases, genetic disorders, and rare diseases. In particular, biological drugs enable personalized treatment for diseases and play an important role in providing treatment options for conditions that are difficult to resolve with conventional synthetic drugs through highly specific and effective mechanisms of action.

[0003] Recently developed biological drugs have disadvantages that make stability, delivery, and storage difficult and challenging due to their increasing complexity. For example, mRNA vaccines face difficulties such as requiring low-temperature (frozen) distribution due to their low stability. The following are materials used for the transport of biological drugs.

[0004] - Lipid Nanoparticles (LNP): Used in RNA vaccines and the like for the protection and intracellular delivery of mRNA, aiding in stability and effective delivery.

[0005] - Polymer Nanoparticles (PNP): Used to enhance the controlled release and stability of drugs, various polymer materials with biocompatibility and biodegradability are utilized.

[0006] - Porous Silica Nanoparticles (PSN): Enable drug adsorption and controlled release through a large surface area, and are used to encapsulate various biological molecules.

[0007] - Gold Nanoparticles: Their surfaces can be functionalized, making them suitable for drug delivery or diagnostic purposes; they are primarily applied to the delivery of anticancer drugs.

[0008] - Polymeric Nanocapsules: These are carriers composed of biocompatible and biodegradable polymers that enable the protection and controlled release of drugs.

[0009] Recently, freeze-drying processes have also been utilized for the long-term preservation of biopharmaceuticals. However, even in this case, problems arise, such as reduced efficacy due to changes in vaccine antigen structure and surface charge, functional degradation caused by the instability of protein degradation pathways, phase separation and aggregation of lipid nanoparticles (LNPs), changes in the particle size and shape of polymer nanoparticles (PNPs), and effects resulting from stress on the capsid proteins and lipid membranes of viral particles.

[0010] Accordingly, the inventors have completed the present invention regarding a method for manufacturing a biopharmaceutical delivery system comprising a freezing formulation using silica nanoparticles with improved preservation of biopharmaceuticals such as genes, proteins, and vaccines, and a delivery system manufactured therefrom.

[0011] Accordingly, the object of the present invention is to provide a method for manufacturing a biopharmaceutical delivery system comprising: 1) loading a biopharmaceutical substance onto porous inorganic nanoparticles through electrostatic bonding; 2) removing unbound biopharmaceutical substance through centrifugation; and 3) adding a cryopreservative to the porous inorganic nanoparticles and freeze-drying them.

[0012] Another objective of the present invention is to provide a biopharmaceutical delivery system manufactured by the method described above.

[0013] Another object of the present invention is to provide a cryopreservation composition for preparing a porous inorganic nanoparticle freeze-dried formulation comprising a biopharmaceutical, comprising one or more sugars selected from sucrose, trehalose, D-mannitol, and dextran, and one or more buffers selected from HEPES, Tris, or PBS.

[0014] To achieve the above objectives, the present invention provides a method for manufacturing a biopharmaceutical delivery system comprising: 1) loading a biopharmaceutical substance onto porous inorganic nanoparticles through electrostatic bonding; 2) removing unbound biopharmaceutical substances through centrifugation; and 3) adding a cryopreservative to the porous inorganic nanoparticles and freeze-drying them.

[0015] To achieve another objective of the present invention, the present invention provides a biopharmaceutical delivery system manufactured by the above-described method.

[0016] To achieve another objective of the present invention, the present invention provides a cryopreservation composition for preparing a porous inorganic nanoparticle freeze-dried formulation comprising a biopharmaceutical, comprising one or more sugars selected from sucrose, trehalose, D-mannitol, and dextran, and one or more buffers selected from HEPES, Tris, or PBS.

[0017]

[0018] The present invention will be described in detail below.

[0019] As one aspect of the present invention, the present invention relates to a method for manufacturing a biopharmaceutical delivery system comprising: 1) loading a biopharmaceutical substance onto porous inorganic nanoparticles through electrostatic bonding; 2) removing unbound biopharmaceutical substance through centrifugation; and 3) adding a cryopreservative to the porous inorganic nanoparticles and freeze-drying.

[0020] In the present invention, the biological drug is a drug developed using living cells, tissues, microorganisms, or materials derived therefrom, and is primarily based on proteins, antibodies, genes, cells, etc. Such drugs are used for the prevention, diagnosis, and treatment of diseases; they are particularly effective for diseases requiring personalized treatment or the regulation of complex biological responses, and exhibit more complex and specific actions than compound-based drugs.

[0021] In the present invention, for the purpose of long-term preservation and easy transport of the above-mentioned biological pharmaceuticals, a formulation was prepared by loading biological pharmaceutical substances, such as proteins, antibodies, genes, and cells constituting the biological pharmaceuticals, onto porous inorganic nanoparticles and freeze-drying them.

[0022] In the present invention, 'porous inorganic nanoparticles' are nanometer-sized particles composed of inorganic materials having a porous structure, and are utilized for purposes such as drug delivery, catalysis, and adsorption due to their large surface area and numerous nano-sized pores within. Types may include porous silica nanoparticles (PSN), porous metal oxide nanoparticles such as titanium oxide or aluminum oxide, porous carbon nanoparticles, etc., but are not limited to these types. In one embodiment of the present invention, a biopharmaceutical substance was loaded using porous silica nanoparticles.

[0023] The porous inorganic nanoparticles of the present invention may have a diameter in the nm range, but are not limited thereto, a diameter of 10 to 500 nm, 100 to 400 nm, preferably 200 to 300 nm.

[0024] First, the biopharmaceutical substance of the present invention is bound to the porous inorganic nanoparticles. The method of binding is not limited and may be bound through covalent bonding, electrical interaction, coordination bonding, hydrophobic interaction, etc. In one embodiment of the present invention, the biopharmaceutical substance was loaded onto porous nanosilica particles through binding via an electrostatic reaction. In this process, after binding the biopharmaceutical substance to the porous inorganic nanoparticles, a process of removing the unbound biopharmaceutical substance through centrifugation may be additionally included.

[0025] The porous inorganic nanoparticles loaded with a biopharmaceutical substance are mixed with a cryopreservative and freeze-dried. The freeze-drying process is repeated. In this specification, the term "temperature" refers to the temperature inside the freeze-drying chamber (i.e., the internal temperature of the freeze-drying chamber, "internal temperature"). Likewise, the term "pressure" refers to the pressure inside the freeze-drying chamber (i.e., the internal pressure of the freeze-drying chamber, "internal pressure").

[0026] In one embodiment, porous inorganic nanoparticles loaded with a biopharmaceutical substance are mixed with a cryopreservative and, after pre-freezing at about -80°C, a lyophilized formulation is obtained in a lyophilized chamber. The lyophilized chamber is cooled to a temperature in the range of about -70°C to about -85°C to produce a frozen formulation and maintained for a period of 12 to 48 hours, preferably 24 hours. Cooling occurs at a rate of about 0.5°C / min and maintenance is carried out for a period of 12 to 48 hours, preferably about 24 hours.

[0027] A cryoprotectant may be included during the freeze-drying process described above. A cryoprotectant is a substance used to protect cells, proteins, nanoparticles, or other biological materials from damage during the freezing process, and it serves to prevent the formation of ice crystals, destruction of cell membranes, protein denaturation, and aggregation of nanoparticles that occur as the material freezes. The cryoprotectant may include low molecular weight cryoprotectants such as glycerol or DMSO, high molecular weight cryoprotectants containing sugars such as trehalose, sucrose, dextran, and mannitol, or substances such as polyvinylpyrrolidone (PVP) or glucose. In one embodiment of the present invention, one or more sugars selected from sucrose, trehalose, D-mannitol, and dextran were included, and although not limited thereto, sucrose and dextran were mixed in a certain weight ratio and used.

[0028] In addition, during freeze-drying, a buffer may be additionally included to maintain pH, osmotic pressure, etc., in addition to the above-mentioned cryopreservative. The above buffer is not limited to any specific type and various known buffers may be selected and used. Suitable buffers may be selected from PBS (Phosphate Buffered Saline), Tris-HCl (Tris-Hydrochloride), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), or Citrate Buffer, and preferably, one of HEPES, Tris-HCl, or PBS may be selected.

[0029] The biopharmaceutical delivery system produced by the manufacturing method of the present invention has the effect of enabling long-term preservation at room temperature. In one embodiment, lipid nanoparticles (LNP), lipofectamine (Lipo), and inorganic nanoparticles (NB) 2 The delivery effect of the biopharmaceutical substance was confirmed through ). It was confirmed that in the solution state, chemical properties were denatured and activity was lost even during short-term storage, but in the case of the freeze-dried formulation of the present invention, it was confirmed that the transfection efficiency was maintained at 20% or higher even when hydrated after being stored at room temperature for 30, 60, and 90 days following freeze-drying.

[0030] In another aspect, the present invention relates to a biopharmaceutical delivery vehicle manufactured by the above method. The biopharmaceutical delivery vehicle manufactured by the above manufacturing method has the effect of being able to be stored at room temperature for a long period of time, preferably for 90 days or more, preferably for 60 days or more, and more preferably for 30 days or more.

[0031] In another aspect, the present invention relates to a cryopreservation composition for preparing a porous inorganic nanoparticle freeze-dried formulation, which is one of a biopharmaceutical delivery vehicle comprising a biopharmaceutical, comprising one or more sugars selected from sucrose, trehalose, D-mannitol, and dextran; and one or more buffers selected from HEPES, Tris-HCl, or PBS.

[0032]

[0033] The present invention relates to a method for manufacturing a biopharmaceutical delivery vehicle in the form of a freeze-dried formulation comprising porous inorganic nanoparticles loaded with a biopharmaceutical drug. The delivery vehicle manufactured according to the method of the present invention has the effect of facilitating the long-term preservation and distribution of a biopharmaceutical drug containing a biopharmaceutical substance, such as a protein, antibody, or nucleic acid, as an active ingredient.

[0034] Figure 1 is a diagram comparing a TEM image (Figure 1a) of a porous silica nanoparticle freeze-dried formulation containing the biopharmaceutical of the present invention and the characteristics of porous silica nanoparticles before and after freeze-drying (Figures 1b to 1e).

[0035] FIGS. 2a to 2c show the results of experiments conducted to select the optimal combination of cryopreservative and buffer in the process of manufacturing a porous silica nanoparticle freeze-dried formulation containing the biopharmaceutical of the present invention.

[0036] Figures 3a to 3c show the dispersion stability and cell viability confirmed by analyzing the size and surface charge of each particle synthesized using a cryopreservation agent under optimized conditions.

[0037] Figures 4a to 4d show the results of comparing the activity maintenance effect of biopharmaceuticals contained in freeze-dried formulations of lipid nanoparticles (LNP) and porous inorganic nanoparticles (PSN), respectively, through GFP gene expression.

[0038] Figures 5a and 5b are experimental results confirming the long-term storage efficiency of a porous silica nanoparticle freeze-dried formulation containing a biopharmaceutical in cells.

[0039] Figures 6a to 6d show the results of analyzing the in vivo tumor-targeted gene editing and GFP knockout efficiency using freeze-dried porous inorganic nanoparticles.

[0040]

[0041] Hereinafter, embodiments are described in detail to specifically explain the present specification. However, the embodiments according to the present specification may be modified in various different forms, and the scope of the present specification is not to be interpreted as being limited to the embodiments described below. The embodiments of the present specification are provided to more completely explain the present specification to those with average knowledge in the art.

[0042]

[0043] Experimental Example 1. Preparation of a lyophilized formulation of porous inorganic nanoparticles containing a biopharmaceutical

[0044] Porous silica nanoparticles with a size of 200 to 300 nm were loaded with Cas9-RNP, a biopharmaceutical substance, by electrostatically reacting them for 1 hour. Then, a cryopreservative mixture of sucrose, trehalose, D-mannitol, and dextran was added in a ratio of 0.005 to 0.2 w / w (%), and a freeze-drying process was carried out. At this time, PBS (P2101-100, GenDEPOT), Tris-HCl (648315, Calbiochem), and HEPES (CA011-010, GenDEPOT) were used as buffers.

[0045] It was confirmed through TEM and SEM images that porous silica nanoparticles freeze-dried under the above conditions were loaded onto the freeze-dried particles (Fig. 1a). The pore size and surface area of ​​the particles were measured as shown in Fig. 1b. In addition, the loading content of Cas9-RNP on the silica nanoparticles during freeze-drying was confirmed as shown in Fig. 1c. The zeta potential and dynamic changes of the silica inorganic nanoparticles before and after freeze-drying were confirmed as shown in Figs. 1D and 1E.

[0046] Experimental Example 2. Comparison of a lyophilized formulation of porous inorganic nanoparticles containing a biopharmaceutical and lipid nanoparticles (LNP).

[0047] For screening each cryopreservation agent, enzyme activity evaluations were compared using a TMB-based colorimetric assay before and after freeze-drying of lipid nanoparticles (LNP) and porous inorganic nanoparticles (PSN) using HRP enzymes.

[0048] To select the cryopreservative with the highest loading retention rate, cryopreservation windows for each element were screened at a ratio of 0.005 to 0.2 w / w%, and the ratio with the highest loading retention rate was selected. (Fig. 2a)

[0049] The substances exhibiting the highest retention ratios were compared across various combinations to select the highest ratio, and an optimized combination of cryopreservatives was chosen. (Fig. 2b)

[0050] In addition, regarding the buffers used during the freeze-drying process, it was confirmed that the activity of the biopharmaceutical substances was maintained in the order of HEPES > Tris > PBS. (Fig. 2c)

[0051] The dispersion stability was confirmed by analyzing the size and surface charge of each particle synthesized using the previously optimized cryopreservative (Fig. 3a). It was confirmed that PSN maintained higher activity than LNP even after freeze-drying using the previously optimized cryopreservative. This was attributed to the fact that HRP is well supported and protected within the porous structure of silica nanoparticles, exhibiting better stability under the optimized cryopreservative conditions. In other words, as shown in Fig. 3b, PSN demonstrated a higher ability to maintain activity after freeze-drying compared to LNP.

[0052] In addition, to investigate the effect of cryopreservatives on cell viability, experiments were conducted using Lipofectamin 2000 (Lipo), lipid nanoparticles (LNP), and porous silica nanoparticles (PSN). As a result, cell viability decreased with increasing concentration for Lipo and LNP, whereas PSN nanoparticles did not affect cell viability even at high concentrations. (Fig. 3c)

[0053]

[0054] Experimental Example 3. Comparison of gene editing efficiency using a lyophilized formulation of porous inorganic nanoparticles containing a biopharmaceutical and lipid nanoparticles (LNPs)

[0055] GFP-HeLa cells (4x10⁶⁶⁶) in a 24-well plate 4) was dispensed and treated with particles in a serum-free culture medium, and after 4 hours, the medium was replaced with a complete culture medium and incubated for 48 hours.

[0056] Specifically, 1 μg of Cas9 protein and 0.5 μg of sgRNA were reacted at room temperature for 5 minutes, then 30 μg of nanoparticles were added and reacted at room temperature for 1 hour. RNP was loaded onto the nanoparticles by electrostatic force, and after centrifugation at 8000 rpm for 10 minutes, the pellet was redispersed in 5% sucrose, 2.5% dextran, and 20 mM HEPES buffer. After pre-freezing at approximately -80°C, the mixture was freeze-dried in a freeze-dryer for 24 hours to obtain a freeze-dried preparation.

[0057] After freeze-drying, GFP-HeLa cells were treated with the particles, and after 4 hours, the medium was replaced with complete medium and cultured for 48 hours. Nuclei were stained with DAPI (P36966, Invitrogen), and fluorescence images were obtained using confocal analysis, confirming that the activity of the nanoparticles was maintained after freeze-drying (Fig. 4a). Gene editing efficiency was analyzed using flow cytometry (FACS) (Fig. 4b), and the indel ratio was evaluated via genomic DNA sequencing using Illumina NGS (Fig. 4c). Consequently, while the efficiency of LNP and Lipo decreased significantly after freeze-drying, the freeze-dried nanoparticles maintained an efficiency of over 40%. Western blot (WB) analysis quantitatively confirmed the GFP protein bands in GFP-HeLa cell knockout by PSN nanoparticles before and after freeze-drying, compared to LNP and Lipo (Fig. 4d).

[0058]

[0059] Experimental Example 4. Confirmation of long-term preservation efficiency of a porous inorganic nanoparticle freeze-dried formulation containing a biopharmaceutical.

[0060] For the composition in the form of a dried powder obtained by the optimized freeze-drying method in Experimental Examples 1 and 2 above, long-term storage at room temperature was performed to evaluate the stability and solubility of the formulation.

[0061] Similar to previous experiments, the long-term storage efficiency of Lipo and LNP containing PSN was compared in GFP-HeLa. LNP and Lipo lost all activity within a short period in solution and did not exhibit activity even when conducted under the previously optimized freeze-drying conditions. In the case of freeze-dried PSN, it was confirmed that it maintained a transfection efficiency of over 20% for more than 90 days (Fig. 5). There are limitations to maintaining the structural stability and biological activity of organic-based materials, particularly after long-term storage. In contrast, freeze-dried porous silica nanoparticles demonstrated long-term efficacy in powder form; this formulation may be particularly advantageous in the context of infectious diseases where rapid response is critical and demonstrates potential as a scalable and reliable platform for long-term storage and emergency preparedness.

[0062]

[0063] Experimental Example 5. Analysis of In Vivo Tumor-Targeted Gene Editing and GFP Knockout Efficiency Using Injectable Lyophilized Inorganic Nanoparticles

[0064] GFP-HeLa cells (5X10 6A tumor xenograft mouse model was constructed using cells, and tumors were induced in Balb / c nude mice (5w, male) via subcutaneous injection. When the tumor reached 100 mm³, the mice were divided into three groups, and for each group, intratumoral injections were performed using PBS, Lipid Nanoparticles (LNP), Lipofectamine 2000 (Lipo), and Porous Inorganic Nanoparticles (PSN) as samples before and after lyophilization. The total injection dose was 40 μL, and the ratio of Cas9, gRNA, and PSN was 2:1:73 (w / w), with LNP at 5 mg / mL, Lipofectamine at 0.25 mg / mL, and PSN at 18.25 mg / mL.

[0065] Three days after injection, tumor tissue was collected, and the fluorescence intensity of the GFP-expressing cancer tissue was measured using the IVIS Spectrum In Vivo Imaging System. This confirmed that PSN significantly reduced GFP expression compared to the control group, based on the fluorescence intensity within the tumor (Fig. 6b).

[0066] In addition, GFP expression efficiency was compared using a flow cytometry analyzer (FACS) to measure GFP knockout efficiency in cancer tissue, and it was confirmed that GFP-negative cells increased significantly in PSN before and after freeze-drying (Fig. 6c).

[0067] In addition, the tumor sections were analyzed using CLSM imaging to confirm that the in vitro experimental results were consistent with the in vivo results, suggesting that the freeze-dried nanoparticles possessed excellent genome editing capabilities in vivo (Fig. 6d).

[0068]

[0069] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of the claims should be interpreted as being included in the invention.

[0070]

[0071] In one embodiment, the present invention relates to a method for manufacturing a biopharmaceutical delivery system comprising: 1) loading a biopharmaceutical substance onto porous inorganic nanoparticles through electrostatic bonding; 2) removing unbound biopharmaceutical substance through centrifugation; and 3) adding a cryopreservative to the porous inorganic nanoparticles and freeze-drying them.

[0072] As an example, the porous inorganic nanoparticles may be selected from the group comprising porous silica nanoparticles (PSN), porous metal oxide nanoparticles such as titanium oxide or aluminum oxide, and porous carbon nanoparticles.

[0073] As an example, the above cryopreservative may comprise one or more sugars selected from the group comprising sucrose, trehalose, D-mannitol, and dextran, either alone or in combination.

[0074] As an example, when performing the freeze-drying above, a buffer selected from PBS (Phosphate Buffered Saline), Tris-HCl (Tris-Hydrochloride), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), or Citrate Buffer (citric acid buffer) may be additionally included.

[0075] The biopharmaceutical substance that can be delivered through the delivery vehicle of the present invention may be selected from the group comprising proteins, antibodies, genes, nucleic acid sequences, or cells.

[0076] As an example, a method for manufacturing a biopharmaceutical delivery system, wherein the porous inorganic nanoparticles have a diameter of 10 to 500 nm.

[0077] As an example, the freeze-drying may be carried out at a temperature of about -70°C to about -85°C for 12 to 48 hours.

[0078] In another aspect, the present invention relates to a biopharmaceutical delivery vehicle produced by the above-described manufacturing method. The biopharmaceutical delivery vehicle can be stored for a long period of time at room temperature for 90 days or more.

[0079] In another aspect, the present invention relates to a cryopreservative composition for preparing a porous inorganic nanoparticle freeze-dried formulation for biopharmaceutical delivery, comprising one or more sugars selected from sucrose, trehalose, D-mannitol, and dextran, and one or more buffers selected from HEPES, Tris, or PBS.

Claims

1. 1) A step of loading a biopharmaceutical substance onto porous inorganic nanoparticles via electrostatic bonding; 2) a step of removing unbound biopharmaceutical substances through centrifugation; and 3) A method for manufacturing a biopharmaceutical delivery system comprising the step of adding a cryopreservative to the porous inorganic nanoparticles and freeze-drying them.

2. In Paragraph 1, A method for manufacturing a biopharmaceutical delivery system, wherein the above-mentioned porous inorganic nanoparticles are selected from the group comprising porous silica nanoparticles (PSN), porous metal oxide nanoparticles such as titanium oxide or aluminum oxide, and porous carbon nanoparticles.

3. In Paragraph 1, A method for manufacturing a biopharmaceutical delivery agent, wherein the above cryopreservative comprises one or more sugars selected from the group comprising sucrose, trehalose, D-mannitol, and dextran, either alone or in combination.

4. In Paragraph 1, A method for manufacturing a biopharmaceutical delivery system, wherein, when performing step 3) above, a buffer selected from PBS (Phosphate Buffered Saline), Tris-HCl (Tris-Hydrochloride), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MES (2-(N-morpholino)ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), or Citrate Buffer (citric acid buffer) is additionally included.

5. In Paragraph 1, A method for manufacturing a biopharmaceutical delivery vehicle, wherein the biopharmaceutical substance is selected from the group comprising proteins, antibodies, genes, nucleic acid sequences, or cells.

6. In Paragraph 1, A method for manufacturing a biopharmaceutical delivery system, wherein the porous inorganic nanoparticles have a diameter of 10 to 500 nm.

7. In Paragraph 1, A method for manufacturing a biopharmaceutical delivery vehicle, wherein the freeze-drying is carried out at a temperature of about -70°C to about -85°C for 12 to 48 hours.

8. A biopharmaceutical delivery system produced by the manufacturing method of paragraph 1.

9. In Paragraph 8, The above-mentioned biological drug delivery system is a biological drug delivery system capable of long-term storage at room temperature for 90 days or more.

10. A cryopreservative composition for preparing a porous inorganic nanoparticle freeze-dried formulation for biopharmaceutical delivery, comprising one or more sugars selected from sucrose, trehalose, D-mannitol, and dextran, and one or more buffers selected from HEPES, Tris, or PBS.

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