Liquid dosage form pharmaceutical composition containing plasmid DNA

Abstract

The present invention relates to a liquid dosage form pharmaceutical composition containing plasmid DNA. The liquid dosage form containing plasmid DNA according to the present invention exhibits excellent efficacy in terms of storage stability of the plasmid DNA, which is a pharma- ceutical active ingredient, and can be usefully used as a pharmaceutical composition with excellent product stability and safety.

Description

Technical Field

[0001] This patent application claims priority to Korean Patent Application No. 10-2022-0036970, filed with the Korean Intellectual Property Office on March 24, 2022, the disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a liquid pharmaceutical composition containing plasmid DNA.

[0003] (Blank)

Background Art

[0004] There is substantial clinical evidence that gene therapy involving the direct in vivo transfer of nucleic acid constructs (so-called "naked" DNA or RNA constructs) that are not packaged in viruses or virus-like particles may be effective in treating a variety of diseases. For example, direct intramuscular injection of a DNA plasmid construct expressing two isoforms of the human HGF protein (i.e., pCK-HGF-X7, also known as "VM202") has been proven effective in treating neuropathic pain. In a phase II clinical trial, injection of VM202 into the calf muscle of patients with diabetic peripheral neuropathy resulted in a significant reduction in pain, and this was sufficient to provide symptom relief that improved quality of life for three months when administered for two days at two-week intervals. Kessler et al., Annals Clin. Transl. Neurology 2(5):465-478 (2015). The same DNA plasmid construct described above has also been shown to be effective in treating patients with amyotrophic lateral sclerosis (ALS). In a Phase I clinical trial, nearly half of ALS patients remained stable or improved after VM202 administration, and it was observed that 47%, 50%, and 24% of subjects at 1, 2, and 3 months, respectively, did not experience a decrease in or an improvement in their ALSFRS-R (Amyotrophic Lateral Sclerosis Functional Rating Scale-revised) scores, which indicate the physical function of ALS patients. This is a better result than previously observed in control groups. Robert L. Sufit et al., Amyotrophic Lateral Sclerosis and Frontoemporal Degeneration 18:269-278 (2017).

[0005] However, the physical and chemical stability of such DNA or RNA constructs is known to be affected by the final formulation and dosage form, as well as storage conditions. Typically, pharmaceutical dosage forms are supplied in lyophilized form for storage stability, but they need to be reconstituted using sterile water for injection before being administered to patients. If the reconstitution process is not strictly followed, the drug may not be uniformly reconstituted, leading to drug loss or contamination during administration. Therefore, providing drugs in liquid form rather than lyophilized form can improve safety and competitiveness. Consequently, there is a need to develop liquid dosage forms of naked-DNA-based drugs that offer superior stability and safety.

[0006] (blank) [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention aims to provide a liquid dosage form containing plasmid DNA.

[0008] (blank) [Means for solving the problem]

[0009] According to one aspect of the present invention, the present invention provides a liquid dosage form containing plasmid DNA.

[0010] In this specification, the term "liquid formulation" means a pharmaceutical composition in liquid form as described herein, comprising plasmid DNA and at least one pharmaceutically acceptable carrier or excipient.

[0011] Since the liquid dosage form of the present invention relates to a general-purpose liquid dosage form of plasmid DNA that can be used regardless of the type of plasmid DNA, the object of the liquid dosage form of the present invention is not limited to a liquid dosage form for plasmid DNA (e.g., VM202) used as an embodiment of the present invention.

[0012] In one embodiment of the present invention, the plasmid DNA encodes human HGF or a variant thereof.

[0013] In one embodiment of the present invention, the plasmid DNA may be VM202, but is not limited thereto.

[0014] In one embodiment of the present invention, the plasmid DNA further comprises a promoter. Within the plasmid DNA, the promoter is operatively bound to a nucleic acid molecule encoding a protein. In this specification, the term “operatively bound” means a functional binding of a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) to another nucleic acid sequence, thereby the regulatory sequence regulating the transcription and / or decoding of the other nucleic acid sequence.

[0015] For example, when the vector of the present invention is an expression vector and the host is a eukaryotic cell, promoters derived from the genome of mammalian cells (e.g., metallothione promoter, beta-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., late adenovirus promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Roussarcoma virus (RSV) promoter) may be used, and generally have a polyadenylated sequence as a transcription termination sequence.

[0016] On the other hand, the expression vector of the present invention may include a selectable marker gene and / or reporter gene as a selective label for evaluating the presence or absence of transformation of the expression vector and protein expression.

[0017] Selectable marker genes include antibiotic resistance genes commonly used in this industry, such as resistance genes to ampicillin, gentamicin, cabenicillin, chloramphenicol, streptomycin, kanamycin, genethicin, neomycin, and tetracycline. Reporter genes include, but are not limited to, genes for luciferase, beta-galactosidase, chloramphenicol acetyltransferase, or green fluorescent protein.

[0018] Methods for introducing and expressing the recombinant vector of the present invention into cells are well known in the relevant art. The vector may be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by methods known in the art. For example, the vector may be delivered into host cells by physical, chemical, or biological means. The physical means include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. The chemical means include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The biological means include, but are not limited to, the use of DNA or RNA vectors, such as the lentiviruses and retroviruses mentioned above.

[0019] In this specification, the term "VM202" refers to the plasmid DNA also known as pCK-HGF-X7, which contains the pCK vector (SEQ ID NO: 6) and HGF-X7 cloned into the pCK vector (SEQ ID NO: 13). VM202 was deposited with the Korean Center for Microbial Conservation (KCCM) on March 12, 2002, under the Budapest Convention, with deposit number KCCM-10361.

[0020] As used in this application, the term "isoforms of hepatocyte growth factor (HGF)" refers to polypeptides having at least 80% the same amino acid sequence as HGF polypeptides that occur naturally from animals. The term includes polypeptides having at least 80% the same amino acid sequence as any full-length wild-type HGF polypeptide, and includes polypeptides having at least 80% the same amino acid sequence as naturally occurring HGF allele variants, splice variants, or deletion variants. In this invention, HGF isoforms include two or more isoforms selected from the group consisting of full-length HGF (flHGF), deleted variant HGF (dHGF), NK1, NK2, and NK4. According to a more specific embodiment of this invention, the HGF isoforms used in the method described in this application include flHGF (SEQ ID NO: 1) and dHGF (SEQ ID NO: 2).

[0021] The terms "human flHGF," "flHGF," and "fHGF" are used interchangeably in this application to refer to the human HGF protein, which is composed of amino acids 1-728. The amino acid sequence of flHGF is provided in SEQ ID NO: 1.

[0022] The terms "human dHGF" and "dHGF" are used interchangeably in this application as they mean a deleted variant of the HGF protein generated by alternative splicing of the human HGF gene. Specifically, "human dHGF" or "dHGF" means a human HGF protein with five amino acids (F, L, P, S, and S) deleted in the first kringle domain of the alpha chain from the full-length HGF sequence. Human dHGF is 723 amino acids long. The amino acid sequence of human dHGF is provided in SEQ ID NO: 2.

[0023] In one embodiment of the present invention, the plasmid DNA encodes SDF-1, IGF-1, c-Met, its fragments / isoforms / variants.

[0024] The term SDF-1 (stromal cell-derived factor 1) of the present invention is also known as C-X-C motif chemokine 12 (CXCL12), which is a chemokine protein encoded by the CXCL12 gene. SDF-1 is expressed from many cells and tissues and is distinguished into SDF-1 alpha and SDF-1 beta. SDF-1 is known to play an important role in angiogenesis, wound healing, nerve development, etc.

[0025] An exemplary amino acid sequence of human SDF-1 alpha is provided in SEQ ID NO: 21.

[0026] An exemplary nucleotide sequence encoding the human SDF-1 alpha is provided in SEQ ID NO: 17.

[0027] The term IGF-1 (Insulin like growth factor 1) in the present invention is also called somatomedin C and is a hormone having a molecular structure similar to insulin. IGF-1 may play an important role in promoting cell growth during the growth and development in the early childhood. IGF-1 is encoded by the IGF1 gene and is known to stimulate the growth of various types of cells, suppress cell death, and increase the protein production of cells.

[0028] An exemplary amino acid sequence of the Ea isoform of human IGF-1 is provided in SEQ ID NO: 19. An exemplary amino acid sequence of the Ec isoform of human IGF-1 is provided in SEQ ID NO: 20.

[0029] The term c-Met protein in the present invention is a protein also called tyrosine-protein kinase Met or hepatocyte growth factor receptor (HGFR). C-Met is encoded by the MET gene and is a kinase receptor essential for embryonic development, tissue formation, cell division, and wound healing having tyrosine kinase activity. In one embodiment of the present invention, the plasmid DNA may be pCK-SDF-1a (WO2016 / 048105A1), pTx-IGF-1X10 (WO2020 / 016655A2), pCMV3-cMet-flag (Sino Biological Inc., China), or a combination thereof, but is not limited thereto.

[0030] In one embodiment of the present invention, the pTx is a plasmid vector derived from pCK. pTx was generated by two sequential mutageneses of pCK. It was generated by removing unnecessary sequences between the Kanamycin resistance gene and ColE1 of pCK and optimizing the length of the HCMV intron sequence.

[0031] In one embodiment of the present invention, pCMV3 is a vector for mammalian protein expression induced by a CMV promoter.

[0032] (blank)

[0033] In one embodiment of the present invention, the liquid dosage form comprises plasmid DNA as the active ingredient of the pharmaceutical composition. The plasmid DNA may comprise genetic material for gene therapy. Specifically, the plasmid DNA may be capable of correcting the function of a defective gene or transcript, or it may encode polypeptides, sense or antisense oligonucleotides, or RNA (coding or non-coding; e.g., siRNA, shRNA, micro-RNA, and their antisense counterparts (e.g., antagoMi R)). Any dosage form containing plasmid DNA known in the art for use in gene therapy falls within the scope of the present invention.

[0034] In one embodiment of the present invention, the liquid dosage form contains plasmid DNA at a concentration of 0.01 to 5 mg / mL. More specifically, concentrations of 0.01 to 5 mg / mL, 0.01 to 3 mg / mL, 0.01 to 2 mg / mL, 0.01 to 1 mg / mL, 0.01 to 0.75 mg / mL, 0.01 to 0.6 mg / mL, 0.01 to 0.5 mg / mL, 0.1 to 5 mg / mL, 0.1 to 3 mg / mL, 0.1 to 2 mg / mL, 0.1 to 1 mg / mL, 0.1 to 0.75 mg / mL, 0.1 to 0.6 mg / mL, 0.1 to 0.5 mg / mL, and 0.2 to 5 mg Plasmid DNA may be included in concentrations of 0.2-3 mg / ml, 0.2-2 mg / ml, 0.2-1 mg / ml, 0.2-0.75 mg / ml, 0.2-0.6 mg / ml, 0.2-0.5 mg / ml, 0.3-5 mg / ml, 0.3-3 mg / ml, 0.3-2 mg / ml, 0.3-1 mg / ml, 0.3-0.75 mg / ml, 0.3-0.6 mg / ml, 0.3-0.5 mg / ml, or 0.5 mg / ml.

[0035] The plasmid DNA consists of 2,000 to 15,000 base pairs, 2,000 to 10,000 base pairs, 2,000 to 9,000 base pairs, 2,000 to 8,000 base pairs, 2,000 to 7,000 base pairs, 2,000 to 6,000 base pairs, 2,000 to 5,000 base pairs, 3,000 to 15,000 base pairs, 3,000 to 10,000 base pairs, and 3,000 to 9,000 base pairs. The plasmid DNA may be a polynucleotide having a length of 0 base pairs, 3,000 to 8,000 base pairs, 3,000 to 7,000 base pairs, 3,000 to 6,000 base pairs, 3,000 to 5,000 base pairs, 4,000 to 8,000 base pairs, 4,000 to 7,500 base pairs, 4,000 to 6,000 base pairs, 6,000 to 9,000 base pairs, or 7,000 to 8,000 base pairs. The plasmid DNA may be a polynucleotide having a length within the range provided in this application.

[0036] The liquid dosage form according to the present invention is for improving the stability of plasmid DNA. The stability of the plasmid DNA may be determined based on methods known in the art. In particular, the stability may be determined based on the conformation of the plasmid DNA, for example, whether they exist in a relatively stable supercoiled form or in relatively unstable open circle and linear forms. The conformation of the plasmid DNA may be determined by capillary electrophoresis or HPLC (High-Pressure Liquid Chromatography) of a sample containing plasmid DNA. The content of supercoiled DNA compared to open circle and linear forms may be measured under various conditions.

[0037] In one embodiment of the present invention, at least 90% of the plasmid DNA is supercoiled. In some embodiments, at least 92.5% of the plasmid DNA in the liquid composition is supercoiled, but is not limited to this. In some embodiments, at least 95% of the plasmid DNA in the liquid composition is supercoiled. In some embodiments, at least 97% of the plasmid DNA in the liquid composition is supercoiled. In some embodiments, at least 98% of the plasmid DNA in the liquid composition is supercoiled.

[0038] In one embodiment of the present invention, the liquid composition further comprises a buffer to maintain the pH of the pharmaceutical composition. The buffer may contain buffer compounds known in the art, such as TAPS, Bicine, Tris, Tricin, TAPSO, HEPES, TES, MPOS, PIPES, Cacodylate, or MES. The buffer may contain citric acid, monopotassium phosphate, boric acid, or diethyl barbituric acid. The buffer may be PBS, HEPES, TRIS, or TRIS / EDTA buffer. The buffer may be other phosphate buffers. Phosphate buffers may contain a mixture of monobasic dihydrogen phosphate and dibasic monohydrogen phosphate.

[0039] The buffer may be a potassium phosphate buffer. The potassium phosphate buffer may contain potassium phosphate at concentrations of approximately 30-50 mM, approximately 35-50 mM, approximately 40-50 mM, approximately 45-50 mM, approximately 30-45 mM, approximately 35-45 mM, approximately 40-45 mM, approximately 30-40 mM, approximately 35-40 mM, or approximately 40 mM.

[0040] The buffer contained in the liquid composition may have a pH of about 7 to 9. In some embodiments, the pH is about 7 to 9, about 7.5 to 9, about 8 to 9, about 7 to 8.5, about 7.5 to 8.5, about 8 to 8.5, about 7 to 8, about 7.5 to 8, or about 8.

[0041] In one embodiment of the present invention, the liquid dosage form is approximately 30-50 mM potassium phosphate with a pH of approximately 7.5-8.5.

[0042] In one embodiment of the present invention, the liquid dosage form further comprises a salt. The salt may be NaCl. The NaCl is present in amounts of approximately 0.5-1.5%, approximately 0.5-1.4%, approximately 0.5-1.3%, approximately 0.5-1.2%, approximately 0.5-1.1%, approximately 0.5-1.0%, approximately 0.5-0.9%, approximately 0.6-1.5%, approximately 0.6-1.4%, approximately 0.6-1.3%, approximately 0.6-1.2%, approximately 0.6-1.1%, approximately 0.6-1.0%, approximately 0.6-0.9%, and approximately 0. It is contained in concentrations of 7-1.5%, approximately 0.7-1.4%, approximately 0.7-1.3%, approximately 0.7-1.2%, approximately 0.7-1.1%, approximately 0.7-1.0%, approximately 0.7-0.9%, approximately 0.8-1.5%, approximately 0.8-1.4%, approximately 0.8-1.3%, approximately 0.8-1.2%, approximately 0.8-1.1%, approximately 0.8-1.0%, approximately 0.8-0.9%, or 0.9%.

[0043] In one embodiment of the present invention, the liquid dosage form contains approximately 40 mM potassium phosphate.

[0044] In one embodiment of the present invention, the liquid dosage form contains about 0.9% NaCl.

[0045] In one embodiment of the present invention, the pH of the liquid dosage form is approximately 8.0.

[0046] In one embodiment of the present invention, the liquid dosage form may contain about 40 mM potassium phosphate, about 0.9% NaCl, and have a pH of about 8.0. In this specification, the liquid dosage form according to the above embodiment of the present invention will be named Dosage Form A.

[0047] In one embodiment of the present invention, the plasmid in the liquid dosage form maintains a concentration of 80-100 SC% (supercoiled %), 85-100 SC%, 90-100 SC%, or 95-100 SC% after storage at 2-8°C for 3 months, 4 months, 5 months, or 6 months.

[0048] In one embodiment of the present invention, the plasmid in the liquid dosage form maintains concentrations of 80-100 SC%, 81-100 SC%, 82-100 SC%, 83-100 SC%, and 84-100 SC% even after storage at 15-30°C for at least 3, 4, 5, or 6 months. The aforementioned storage temperatures are 15-30°C, 16-30°C, 17-30°C, 18-30°C, 19-30°C, 20-30°C, 21-30°C, 22-30°C, 23-30°C, 24-30°C, 25-30°C, 15-28°C, 16-28°C, 17-28°C, 18-28°C, 19-28°C, 20-28°C, 21-28°C, 22-28°C, 23-28°C, 24-28°C, 25-28°C, 15-27°C, 1 The temperature range is 6-27°C, 17-27°C, 18-27°C, 19-27°C, 20-27°C, 21-27°C, 22-27°C, 23-27°C, 24-27°C, 25-27°C, 15-26°C, 16-26°C, 17-26°C, 18-26°C, 19-26°C, 20-26°C, 21-26°C, 22-26°C, 23-26°C, 24-26°C, 25-26°C, or 25°C, but is not limited thereto. The liquid dosage form according to the present invention contains an aqueous solution containing a stabilizing amount of sugar to improve the stability of plasmid DNA. The sugar may be sucrose, trehalose, mannitol, or sorbitol.

[0049] Therefore, the liquid dosage form of the present invention further comprises sucrose, trehalose, mannitol, sorbitol, leucine, or a combination thereof.

[0050] In one embodiment of the present invention, the liquid dosage form further comprises about 1-2% sucrose, about 0.5-2% trehalose, about 0.01-1% sorbitol, about 0.05-0.5% leucine, or a combination thereof. In another embodiment of the present invention, the liquid dosage form comprises about 1.5% sucrose, about 0.5-1.5% trehalose, about 0.05-0.25% sorbitol, about 0.1-0.5% leucine, or a combination thereof.

[0051] (blank)

[0052] In another embodiment of the present invention, the liquid dosage form of the present invention further contains about 0.15 to 1.5% mannitol in addition to the liquid dosage form of the above-described embodiment.

[0053] In one embodiment of the present invention, the liquid dosage form contains mannitol in amounts of approximately 0.15-1.5%, approximately 0.15-1.25%, approximately 0.15-1.0%, approximately 0.15-0.75%, approximately 0.15-0.5%, approximately 0.15-0.25%, approximately 0.25-1.5%, approximately 0.25-1.25%, approximately 0.25-1.0%, approximately 0.25-0.75%, and approximately 0. It contains, but is not limited to, concentrations of 25-0.5%, approximately 0.3-1.25%, approximately 0.3-1.0%, approximately 0.3-0.75%, approximately 0.3-0.5%, approximately 0.5-1.25%, approximately 0.5-1.0%, approximately 0.5-0.75%, approximately 1.5%, approximately 1.25%, approximately 1.0%, approximately 0.75%, approximately 0.5%, or approximately 0.25%.

[0054] In this specification, the liquid dosage form according to one aspect of the present invention described above is named Dosage Form B.

[0055] In one embodiment of the present invention, the plasmid DNA in the liquid dosage form maintains 90-100 SC% even after storage at 2-8°C for at least 3, 4, 5, or 6 months.

[0056] In one embodiment of the present invention, the plasmid DNA in the liquid dosage form maintains an SC% of 60-100%, 70-100%, 75-100%, 76-100%, 77-100%, 78-100%, 79-100%, or 80-100% even after storage at 15-30°C for at least 3, 4, 5, or 6 months. The aforementioned storage temperatures are 15-30°C, 16-30°C, 17-30°C, 18-30°C, 19-30°C, 20-30°C, 21-30°C, 22-30°C, 23-30°C, 24-30°C, 25-30°C, 15-28°C, 16-28°C, 17-28°C, 18-28°C, 19-28°C, 20-28°C, 21-28°C, 22-28°C, 23-28°C, 24-28°C, 25-28°C, 15-27°C, 16-27°C, 17-27°C, 18-27°C, 19-27°C, 20-27°C, 2 1-27°C, 22-27°C, 23-27°C, 24-27°C, 25-27°C, 15-26°C, 16-26°C, 17-26°C, 18-26°C, 19-26°C, 20-26°C, 21-26°C, 22-26°C, 23-26°C, 24-26°C, 25-26°C, 15-25°C, 16-25°C, 17-25°C, 18-25°C, 19-25°C, 20-25°C, 21-25°C, 22-25°C, 23-25°C, 24-25°C, or 25°C, but not limited to these.

[0057] In yet another embodiment of the present invention, the liquid dosage form of the present invention further comprises the liquid dosage form of the above embodiment plus approximately 0.001-0.3% mannitol, approximately 0.05-0.5% sorbitol, approximately 0.05-0.5% sucrose, approximately 0.15-3% trehalose, approximately 0.01-1% leucine, or a combination thereof.

[0058] In this specification, the liquid dosage form according to one aspect of the present invention described above is named Dosage Form C.

[0059] In one embodiment of the present invention, the liquid dosage form contains mannitol in amounts of approximately 0.001-0.3%, approximately 0.005-0.3%, approximately 0.01-0.3%, approximately 0.02-0.3%, approximately 0.03-0.3%, approximately 0.04-0.3%, approximately 0.05-0.3%, approximately 0.001-0.25%, approximately 0.005-0.25%, approximately 0.01-0.25%, approximately 0.02-0.25%, approximately 0.03-0.25%, approximately 0.04-0.25%, approximately 0.05-0.25%, approximately 0.001-0.2%, and approximately 0.005 ~0.2%, approximately 0.01~0.2%, approximately 0.02~0.2%, approximately 0.03~0.2%, approximately 0.04~0.2%, approximately 0.05~0.2%, approximately 0.001~0.15%, approximately 0.005~0.15%, approximately 0.01~0.15%, approximately 0.02~0.15%, approximately 0.03~0.15%, approximately 0.04~0.15%, approximately 0.05~0.15%, approximately 0.001~0.1%, approximately 0.005~0.1%, approximately 0.01~0.1%, approximately 0.02~0.1%, approximately 0.03~0.1%, approximately 0.04~0 0.1%, approximately 0.05-0.1%, approximately 0.001-0.075%, approximately 0.005-0.075%, approximately 0.01-0.075%, approximately 0.02-0.075%, approximately 0.03-0.075%, approximately 0.04-0.075%, approximately 0.05-0.075%, approximately 0.001-0.07%, approximately 0.005-0.07%, approximately 0.01-0.07%, approximately 0.02-0.07%, approximately 0.03-0.07%, approximately 0.04-0.07%, approximately 0.05-0.07%, approximately 0.001-0.06%, approximately 0.0 It may be included in concentrations of 0.5-0.06%, approximately 0.01-0.06%, approximately 0.02-0.06%, approximately 0.03-0.06%, approximately 0.04-0.06%, approximately 0.05-0.06%, approximately 0.001-0.05%, approximately 0.005-0.05%, approximately 0.01-0.05%, approximately 0.02-0.05%, approximately 0.03-0.05%, approximately 0.04-0.05%, approximately 0.01%, approximately 0.03%, approximately 0.05%, approximately 0.07%, approximately 0.075%, or approximately 0.1%, but is not limited to these.

[0060] In one embodiment of the present invention, the liquid dosage form contains sorbitol in concentrations of approximately 0.05-0.5%, approximately 0.075-0.5%, approximately 0.1-0.5%, approximately 0.125-0.5%, approximately 0.15-0.5%, approximately 0.2-0.5%, approximately 0.25-0.5%, approximately 0.3-0.5%, approximately 0.35-0.5%, approximately 0.4-0.5%, approximately 0.05%, 0.075%, approximately 0.1%, or approximately 0.15%.

[0061] In one embodiment of the present invention, the liquid dosage form contains sucrose in concentrations of approximately 0.05% to 0.5%, approximately 0.075% to 0.5%, approximately 0.1% to 0.5%, approximately 0.125% to 0.5%, approximately 0.15% to 0.5%, approximately 0.2% to 0.5%, approximately 0.3% to 0.5%, approximately 0.35% to 0.5%, approximately 0.4% to 0.5%, approximately 0.05%, 0.075%, approximately 0.1%, 0.15%, approximately 0.2%, or approximately 0.3%.

[0062] In one embodiment of the present invention, the liquid dosage form contains trehalose in amounts of approximately 0.15-3%, approximately 0.15-2.5%, approximately 0.15-2%, approximately 0.15-1.75%, approximately 0.15-1.5%, approximately 0.15-1.25%, approximately 0.15-1%, approximately 0.15-0.8%, approximately 0.15-0.75%, approximately 0.15-0.7%, approximately 0.15-0.6%, approximately 0.15-0.5%, approximately 0.25-3%, and approximately 0. 0.25~2.5%, approximately 0.25~2%, approximately 0.25~1.75%, approximately 0.25~1.5%, approximately 0.25~1.25%, approximately 0.25~1%, approximately 0.25~0.8%, approximately 0.25~0.75%, approximately 0.25~0.7%, approximately 0.25~0.6%, approximately 0.25~0.5%, approximately 0.5~3%, approximately 0.5~2.5%, approximately 0.5~2%, approximately 0.5~1.75%, approximately 0.5~1.5%, approximately 0.5 ~1.25%, approximately 0.5~1%, approximately 0.5~0.8%, approximately 0.5~0.75%, approximately 0.5~0.7%, approximately 0.5~0.6%, approximately 0.75~3%, approximately 0.75~2.5%, approximately 0.75~2%, approximately 0.75~1.75%, approximately 0.75~1.5%, approximately 0.75~1.25%, approximately 0.75~1%, approximately 0.75~0.8%, approximately 1~3%, approximately 1~2.5%, approximately 1~2%, approximately 1~1.75% It contains, but is not limited to, concentrations of approximately 1-1.5%, 1-1.25%, 1.25-3%, 1.25-2.5%, 1.25-2%, 1.25-1.75%, 1.25-1.5%, 1.5-3%, 1.5-2.5%, 1.5-2%, 1.5-1.75%, 0.5%, 1.0%, 1.25%, 1.5%, 2%, 2.5%, or 3%.

[0063] In one embodiment of the present invention, the liquid dosage form contains leucine in amounts of approximately 0.01-1%, approximately 0.01-0.9%, approximately 0.01-0.8%, approximately 0.01-0.7%, approximately 0.01-0.6%, approximately 0.01-0.5%, approximately 0.01-0.4%, approximately 0.01-0.3%, approximately 0.01-0.2%, approximately 0.01-0.1%, approximately 0.03-1%, approximately 0.03-0.9%, approximately 0.03-0.8%, approximately 0.03-0.7%, approximately 0.03-0.6%, and approximately 0.03 ~0.5%, approximately 0.03~0.4%, approximately 0.03~0.3%, approximately 0.03~0.2%, approximately 0.03~0.1%, approximately 0.05~1%, approximately 0.05~0.9%, approximately 0.05~0.8%, approximately 0.05~0.7%, approximately 0.05~0.6%, approximately 0.05~0.5%, approximately 0.05~0.4%, approximately 0.05~0.3%, approximately 0.05~0.2%, approximately 0.05~0.1%, approximately 0.075~1%, approximately 0.075~0.9%, approximately 0.075~0.8%, Approximately 0.075-0.7%, approximately 0.075-0.6%, approximately 0.075-0.5%, approximately 0.075-0.4%, approximately 0.075-0.3%, approximately 0.075-0.2%, approximately 0.075-0.1%, approximately 0.08-1%, approximately 0.08-0.9%, approximately 0.08-0.8%, approximately 0.08-0.7%, approximately 0.08-0.6%, approximately 0.08-0.5%, approximately 0.08-0.4%, approximately 0.08-0.3%, approximately 0.08-0.2%, approximately 0.08-0.1%, It contains, but is not limited to, concentrations of approximately 0.09-1%, 0.09-0.9%, 0.09-0.8%, 0.09-0.7%, 0.09-0.6%, 0.09-0.5%, 0.09-0.4%, 0.09-0.3%, 0.09-0.2%, 0.09-0.1%, 0.01%, 0.03%, 0.05%, 0.075%, 0.08%, 0.09%, 0.1%, 0.2%, or 0.3%.

[0064] In one embodiment of the present invention, the liquid dosage form further comprises about 0.05% mannitol, about 0.15% sorbitol, about 0.3% sucrose, about 1.5% trehalose, about 0.1% leucine, or a combination thereof.

[0065] In one embodiment of the present invention, the plasmid DNA in the liquid dosage form maintains an SC% of 90-100, 91-100, 92-100, 93-100, or 94-100 even after storage at 2-8°C for at least 3, 4, 5, 6, 7, 8, or 9 months.

[0066] In one embodiment of the present invention, the plasmid DNA in the liquid dosage form maintains SC% of 80-100, 81-100, 82-100, 83-100, 84-100, and 85-100 even after storage at 15-30°C for at least 3, 4, 5, 6, 7, 8, or 9 months. The aforementioned storage temperatures are 15-30°C, 16-30°C, 17-30°C, 18-30°C, 19-30°C, 20-30°C, 21-30°C, 22-30°C, 23-30°C, 24-30°C, 25-30°C, 15-28°C, 16-28°C, 17-28°C, 18-28°C, 19-28°C, 20-28°C, 21-28°C, 22-28°C, 23-28°C, 24-28°C, 25-28°C, 15-27°C, 16-27°C, 17-27°C, 18-27°C, 19-27°C, 20-27°C, 2 1-27°C, 22-27°C, 23-27°C, 24-27°C, 25-27°C, 15-26°C, 16-26°C, 17-26°C, 18-26°C, 19-26°C, 20-26°C, 21-26°C, 22-26°C, 23-26°C, 24-26°C, 25-26°C, 15-25°C, 16-25°C, 17-25°C, 18-25°C, 19-25°C, 20-25°C, 21-25°C, 22-25°C, 23-25°C, 24-25°C, or 25°C, but not limited to these.

[0067] In yet another embodiment of the present invention, a liquid dosage form of plasmid DNA further comprises about 0.001-0.3% mannitol, about 0.05-0.5% sorbitol, about 0.05-0.5% sucrose, about 0.15-3% trehalose, about 0.045-0.055% leucine, or a combination thereof.

[0068] In one embodiment of the present invention, the liquid dosage form contains mannitol in amounts of approximately 0.001-0.3%, approximately 0.005-0.3%, approximately 0.01-0.3%, approximately 0.02-0.3%, approximately 0.03-0.3%, approximately 0.04-0.3%, approximately 0.05-0.3%, approximately 0.001-0.25%, approximately 0.005-0.25%, approximately 0.01-0.25%, approximately 0.02-0.25%, approximately 0.03-0.25%, approximately 0.04-0.25%, approximately 0.05-0.25%, approximately 0.001-0.2%, and approximately 0.005 ~0.2%, approximately 0.01~0.2%, approximately 0.02~0.2%, approximately 0.03~0.2%, approximately 0.04~0.2%, approximately 0.05~0.2%, approximately 0.001~0.15%, approximately 0.005~0.15%, approximately 0.01~0.15%, approximately 0.02~0.15%, approximately 0.03~0.15%, approximately 0.04~0.15%, approximately 0.05~0.15%, approximately 0.001~0.1%, approximately 0.005~0.1%, approximately 0.01~0.1%, approximately 0.02~0.1%, approximately 0.03~0.1%, approximately 0.04~0 0.1%, approximately 0.05-0.1%, approximately 0.001-0.075%, approximately 0.005-0.075%, approximately 0.01-0.075%, approximately 0.02-0.075%, approximately 0.03-0.075%, approximately 0.04-0.075%, approximately 0.05-0.075%, approximately 0.001-0.07%, approximately 0.005-0.07%, approximately 0.01-0.07%, approximately 0.02-0.07%, approximately 0.03-0.07%, approximately 0.04-0.07%, approximately 0.05-0.07%, approximately 0.001-0.06%, approximately 0.0 It may be included in concentrations of 0.5-0.06%, approximately 0.01-0.06%, approximately 0.02-0.06%, approximately 0.03-0.06%, approximately 0.04-0.06%, approximately 0.05-0.06%, approximately 0.001-0.05%, approximately 0.005-0.05%, approximately 0.01-0.05%, approximately 0.02-0.05%, approximately 0.03-0.05%, approximately 0.04-0.05%, approximately 0.01%, approximately 0.03%, approximately 0.05%, approximately 0.07%, approximately 0.075%, or approximately 0.1%, but is not limited to these.

[0069] In one embodiment of the present invention, the liquid dosage form is approximately 30-39 mM, approximately 31-39 mM, approximately 32-39 mM, approximately 33-39 mM, approximately 34-39 mM, approximately 34-39 mM, approximately 35-39 mM, approximately 36-39 mM, approximately 37-39 mM, approximately 30-38.5 mM, approximately 31-38.5 mM, approximately 32-38.5 mM, Approx. 33~38.5mM, approx. 34~38.5mM, approx. 34~38.5mM, approx. 35~38.5mM, approx. 36~38.5mM, approx. 37~38.5mM, approx. 30~38mM, approx. 31~38mM, approx. 32~38mM, approx. 33~38mM, approx. 34~38mM, approx. 34~38mM, approx. 35~38mM, approx. 36~38 mM, approximately 37~38mM, approximately 30~37.5mM, approximately 31~37.5mM, approximately 32~37.5mM, approximately 33~37.5mM, approximately 34~37.5mM, Approx. 34~37.5mM, Approx. 35~37.5mM, Approx. 36~37.5mM, Approx. 37~37.5mM, Approx. 30~37.3mM, Approx. 31~37.3mM, Approx. 3 It contains potassium phosphate at concentrations of 2-37.3 mM, approximately 33-37.3 mM, approximately 34-37.3 mM, approximately 34-37.3 mM, approximately 35-37.3 mM, approximately 36-37.3 mM, approximately 37-37.3 mM, approximately 36 mM, approximately 37 mM, approximately 37.3 mM, approximately 37.5 mM, approximately 38 mM, or approximately 39 mM.

[0070] In one embodiment of the present invention, the liquid dosage form contains NaCl in amounts of approximately 0.8-1.5%, approximately 0.85-1.5%, approximately 0.9-1.5%, approximately 0.95-1.5%, approximately 0.97-1.5%, approximately 0.975-1.5%, approximately 0.98-1.5%, approximately 0.8-1.25%, approximately 0.85-1.25%, approximately 0.9-1.25%, approximately 0.97-1.25%, approximately 0.975-1.25%, approximately 0.98-1.25%, approximately 0.8-1.2%, approximately 0.85-1.2%, approximately 0.9-1.2%, approximately 0.95-1.2%, approximately 0.97- It contains, but is not limited to, concentrations of 1.2%, approximately 0.975-1.2%, approximately 0.98-1.2%, approximately 0.8-1.1%, approximately 0.85-1.1%, approximately 0.9-1.1%, approximately 0.95-1.1%, approximately 0.97-1.1%, approximately 0.975-1.1%, approximately 0.98-1.1%, approximately 0.8-1.0%, approximately 0.85-1.0%, approximately 0.9-1.0%, approximately 0.95-1.0%, approximately 0.97-1.0%, approximately 0.975-1.0%, approximately 0.98-1.0%, approximately 0.8%, approximately 0.9%, approximately 0.95%, or approximately 1.0%.

[0071] In one embodiment of the present invention, the liquid dosage form comprises about 37 mM potassium phosphate, about 1.0% NaCl, about 0.05% mannitol, about 0.15% sorbitol, about 0.3% sucrose, about 1.5% trehalose, about 0.05% leucine, or a combination thereof, and has a pH of about 8.0.

[0072] In this specification, the liquid dosage form according to one aspect of the present invention described above is named Dosage Form D.

[0073] In one embodiment of the present invention, the plasmid DNA in the liquid dosage form maintains SC% of 90-100, 91-100, 92-100, 93-100, 94-100, 95-100, 95-100, and 96-100 even after storage at 2-8°C for at least 3, 4, 5, 6, 7, 8, or 9 months.

[0074] In one embodiment of the present invention, the plasmid DNA in the liquid dosage form maintains an SC% of 80-100, 81-100, 82-100, 83-100, 84-100, 85-100, 86-100, 87-100, 88-100, or 89-100 even after storage at 15-30°C for at least 3, 4, 5, 6, 7, 8, or 9 months. The aforementioned storage temperatures are 15-30°C, 16-30°C, 17-30°C, 18-30°C, 19-30°C, 20-30°C, 21-30°C, 22-30°C, 23-30°C, 24-30°C, 25-30°C, 15-28°C, 16-28°C, 17-28°C, 18-28°C, 19-28°C, 20-28°C, 21-28°C, 22-28°C, 23-28°C, 24-28°C, 25-28°C, 15-27°C, 16-27°C, 17-27°C, 18-27°C, 19-27°C, 20-27°C, 2 1-27°C, 22-27°C, 23-27°C, 24-27°C, 25-27°C, 15-26°C, 16-26°C, 17-26°C, 18-26°C, 19-26°C, 20-26°C, 21-26°C, 22-26°C, 23-26°C, 24-26°C, 25-26°C, 15-25°C, 16-25°C, 17-25°C, 18-25°C, 19-25°C, 20-25°C, 21-25°C, 22-25°C, 23-25°C, 24-25°C, or 25°C, but not limited to these.

[0075] In one embodiment of the present invention, the liquid dosage form (Dosage Form A to Dosage Form D) according to one aspect of the present invention suppresses changes in the SC% (supercoiled %) of plasmid DNA.

[0076] In specific embodiments of the present invention, the SC% (supercoiled %) of plasmid DNA contained in the liquid dosage form composition maintains a measured value of 80-100%, 85-100%, 90-100%, or 95-100% even after storage at 2-8°C for at least 3, 4, 5, or 6 months, based on the initial measured value. For example, if the initial measured value of the SC% of plasmid DNA contained in the liquid dosage form composition of the present invention is 90 SC%, the SC% of plasmid DNA measured again from the liquid dosage form after 6 months at 2-8°C may be 72-90 SC. The "initial measured value" refers to the SC% of plasmid DNA measured immediately after adding plasmid DNA to the liquid dosage form of the present invention.

[0077] In another embodiment of the present invention, the SC% (supercoiled %) of plasmid DNA contained in the liquid dosage form composition maintains a measured value of 70-100%, 75-100%, 80-100%, 81-100%, 82-100%, 83-100%, 84-100%, or 85-100% even after storage at 15-30°C for at least 3, 4, 5, or 6 months, based on the initial measured value.

[0078] For example, if the initial measured SC% of plasmid DNA contained in the liquid dosage form composition of the present invention is 90 SC%, the SC% of plasmid DNA measured again after 6 months at 30°C may be between 63 SC% and 90 SC%.

[0079] The aforementioned storage temperatures are 15-30°C, 16-30°C, 17-30°C, 18-30°C, 19-30°C, 20-30°C, 21-30°C, 22-30°C, 23-30°C, 24-30°C, 25-30°C, 15-28°C, 16-28°C, 17-28°C, 18-28°C, 19-28°C, 20-28°C, 21-28°C, 22-28°C, 23-28°C, 24-28°C, 25-28°C, 15-27°C, 16-27°C, 17-27°C, 18-27°C, 19-27°C, 20-27°C, 2 1-27°C, 22-27°C, 23-27°C, 24-27°C, 25-27°C, 15-26°C, 16-26°C, 17-26°C, 18-26°C, 19-26°C, 20-26°C, 21-26°C, 22-26°C, 23-26°C, 24-26°C, 25-26°C, 15-25°C, 16-25°C, 17-25°C, 18-25°C, 19-25°C, 20-25°C, 21-25°C, 22-25°C, 23-25°C, 24-25°C, or 25°C, but not limited to these.

[0080] In yet another embodiment of the present invention, the liquid dosage form is manufactured in unit dose form as a vial, ampoule, bottle, or pre-filled syringe.

[0081] A liquid dosage form according to one aspect of the present invention may be administered to a mammalian subject to treat various diseases. The liquid dosage form of the present invention may be delivered by various means, for example, orally or parenteral routes, such as intravenous, intramuscular, intraendocardial, intramyocardial, intrapericardial, intraventricular, intraarticular, intradermal, intracerebral, intrarenal, intrahepatic, and intrasplenic. It may be administered intralymphatic, subcutaneous, intraabdominal, intratesticular, intraovarian, intrauterine, sternal, intratratracheal, intrathoracic, intradural, intraspinal, intramedullary, intramural, intrascorionic, and intra-arterial injection or infusion, or topically via rectal, intranasal, inhalation, or intraocular. In specific manifestations, the method of transmission is intramuscular, intramyocardial, intravenous, intracerebral, or intrarenal.

[0082] In one embodiment of the present invention, the nucleic acid construct is administered by injection of a liquid pharmaceutical composition. In a specific embodiment, the polynucleotide construct is administered by intramuscular injection. Typically, the polynucleotide construct is administered by intramuscular injection near the site of effect. In some embodiments, the polynucleotide construct is administered into the muscles of the limbs, heart, or other body part of the target.

[0083] In some embodiments, the construct is injected subcutaneously or intradermally. In some embodiments, the polynucleotide construct is administered by intravascular transport. In certain embodiments, the construct is injected by retrograde intravenous injection.

[0084] The typical daily dose of the liquid dosage form composition of the present invention must be determined based on various relevant factors, including the condition being treated, the chosen route of administration, the patient's age, sex, and weight, and the severity of the patient's symptoms, and it should be understood that it can be administered as a single dose or in divided doses. The polynucleotide construct is administered in a therapeutically effective dose.

[0085] In some embodiments of the method described herein, the polynucleotide construct is administered in a total dose of 1 μg to 200 mg, 1 mg to 200 mg, 1 mg to 100 mg, 1 mg to 50 mg, 1 mg to 20 mg, 5 mg to 10 mg, 16 mg, 8 mg, or 4 mg.

[0086] In typical implementations, the total dose is divided into multiple individual injection doses. In some implementations, the total dose is divided into multiple identical injection doses. In some implementations, the total dose is divided into unequal injection doses.

[0087] In various divided dose implementations, the total dose is administered to 4, 8, 16, 24, or 32 different injection sites.

[0088] In some embodiments, the injectable dose is 0.1 to 5 mg. In specific embodiments, the injectable dose is 0.1 mg, 0.15 mg, 0.2 mg, 0.25 mg, 0.3 mg, 0.35 mg, 0.4 mg, 0.45 mg, or 0.5 mg.

[0089] The total dose may be administered in one visit or two or more visits.

[0090] In typical examples of divided dose implementation, all multiple injectable doses are administered within one hour of each other. In some examples, all multiple injectable doses are administered within 1.5, 2, 2.5, or 3 hours of each other.

[0091] In various implementations of this method, whether administered as a single, unified dose (unitary dose) or divided into multiple injectable doses, the total dose of the polynucleotide construct is administered to the subject only once.

[0092] In some embodiments, the administration of a total dose of a polynucleotide construct to multiple injection sites over one, two, three, or four visits may constitute a single cycle. In particular, the administration of 32 mg, 16 mg, 8 mg, or 4 mg of a polynucleotide construct to multiple injection sites over two visits may constitute a single cycle. The two visits may be spaced 3, 5, 7, 14, 21, or 28 days apart.

[0093] In some embodiments, the period may be repeated. The period may be repeated two, three, four, five, six, or more times.

[0094] In some embodiments, the cycle may be repeated for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more after the previous cycle.

[0095] In some cases, the total dose administered in a subsequent cycle is the same as the total dose administered in the previous cycle. In some cases, the total dose administered in a subsequent cycle is different from the total dose administered in the previous cycle.

[0096] Currently, in a preferred example, the nucleic acid construct is administered in a dose of 8 mg per affected limb, equally divided into multiple intramuscular injections and multiple visits, with each of the multiple injections in any single visit being administered at a different injection site. In a specific embodiment, the nucleic acid construct is administered in a dose of 8 mg per affected limb, equally divided into a first dose of 4 mg per limb on day 0 and a second dose of 4 mg per limb on day 14, where each of the first and second doses is equally divided into multiple injection doses.

[0097] The actual dose, rate, and time course of administration will vary depending on the nature and severity of the disease being treated. In typical cases, the polynucleotide construct is administered in a dose effective in reducing the symptoms of the disease, such as pain. In some cases, the dose is effective in reducing symptoms within one week of administration. In some cases, the dose is effective in reducing symptoms within two, three, or four weeks after administration.

[0098] Plasmid DNA may be administered simultaneously or sequentially, alone or in combination with other plasmid DNA, depending on the condition being treated.

[0099] In some embodiments, the pharmaceutical composition in liquid dosage form contains plasmid DNA encoding human HGF. The pharmaceutical composition in liquid dosage form may be administered to treat a variety of diseases, for example, diseases previously established to be treatable by the administration of plasmid DNA. Plasmid DNA may encode a therapeutic gene such as human HGF. These diseases include, but are not limited to, ischemic or hepatic disease, coronary artery disease (CAD), amyotrophic lateral sclerosis (ALS), peripheral artery disease (diabetic ulcer), and diabetic peripheral neuropathy (DPN), or neuropathy due to disease, injury, infection, or vitamin deficiency. For example, neurological disorders may be induced by diabetes, vitamin deficiency, autoimmune disease, genetic or hereditary disorder, amyloidosis, uremia, toxins or poisons, trauma or injury, tumors, or idiopathic. The references provided herein are included herein by reference in their entirety.

[0100] (blank) [Effects of the Invention]

[0101] The liquid dosage form containing plasmid DNA according to the present invention exhibits excellent efficacy in terms of the storage stability of plasmid DNA, which is the pharmaceutical active ingredient, and can therefore be usefully used as a pharmaceutical composition with excellent product stability and safety.

[0102] (blank) [Brief explanation of the drawing]

[0103] [Figure 1] This figure shows the results of testing the long-term stability of liquid dosage form A of the present invention.

[0104] [Figure 2A] This figure shows the results of a statistical analysis of the influence of each factor in order to derive liquid dosage forms B and C of the present invention. (*KP=Potassium Phosphate, N=NaCl, MT=Mannitol, SR=Sucrose, TH=Trehalose, LC=Leucine) [Figure 2B] This figure shows the results of a statistical analysis of the influence of each factor in order to derive liquid dosage forms B and C of the present invention. (*KP=Potassium Phosphate, N=NaCl, MT=Mannitol, SR=Sucrose, TH=Trehalose, LC=Leucine)

[0105] [Figure 3A] This figure shows the composition derived from statistical analysis to derive the optimal liquid dosage form of the present invention. [Figure 3B] This figure shows the composition derived from statistical analysis to derive the optimal liquid dosage form of the present invention.

[0106] [Figure 4] This figure shows the results of statistical analysis to derive the optimal liquid dosage form for improving the long-term stability of plasmid DNA in the present invention. [Figure 5] This figure shows the results of statistical analysis to derive the optimal liquid dosage form for improving the long-term stability of plasmid DNA in the present invention.

[0107] [Figure 6] This figure compares the stability of plasmid DNA when the liquid dosage form of the present invention is manufactured as a vial and a pre-filled syringe, in order to confirm whether there are any differences in storage stability depending on the unit dose form.

[0108] (blank) [Modes for carrying out the invention]

[0109] (blank) [Examples]

[0110] (blank)

[0111] Throughout this specification, unless otherwise specified, the percentages "%" used to indicate the concentration of a particular substance refer to (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid.

[0112] (blank)

[0113] [Table 1] [Table 1]

[0114] ●Example 1. Development of Plasmid DNA Liquid Dosage Form A

[0115] The inventors screened Plasmid DNA Liquid Dosage Form A of the present invention a total of five times. The component preparation, BDP production, vial filling, sampling, and SC% analysis used in all five screenings were identical. In this example, vials were washed according to the vial washing protocol up to 7 days before BDP production and stored in a 70°C dry oven before use. Rubber stoppers were sterilized using a vendor (West-DAIKYO SEIKO) and used immediately after opening for testing.

[0116] (blank)

[0117] ● BDP Manufacturing (Bulk Drug Product Formulation)

[0118] The 1X and 2X buffers used in this example were prepared up to 7 days before the start of production of each final BDP (Bulk Drug Product). The 2X buffer was prepared without NaCl, with a buffer strength of X2, mannitol, and sucrose concentration. (However, if the NaCl concentration of the final formulation was 0.9%, the 2X buffer should also be 0.9% NaCl; if it was 1.1%, the 2X buffer should be 1.3% NaCl.)

[0119] In the case of 1X buffer, the composition was identical to that of the final dosage form.

[0120] This test involved the production of small-scale buffer (100 mL), and taking into account the manufacturing tolerances of actual commercial production processes, the 1X and 2X buffers were produced with a manufacturing tolerance range of ~2%.

[0121] The API used in this test was a VM202 DS sample (lot no. 88084) produced by Company A, which was thawed for 20-24 hours by transferring it from a -70°C deep freezer to a 2-8°C refrigerator before use.

[0122] The thawed DS was mixed with 2X and 1X buffers to produce BDP with a final pDNA concentration of 0.48–0.52 mg / mL (target 0.5 mg / mL).

[0123] The BDP manufacturing process and sampling & analysis procedures are as shown in Table 2 below.

[0124] [Table 2] BDP (Bulk Drug Product) Manufacturing and Sampling Procedures [Table 2]

[0125] The list of dosage forms used in the first to fifth screenings is shown in Tables 3 to 7 below.

[0126] [Table 3] List of dosage forms used in the first screening [Table 3]

[0127] [Table 4] List of dosage forms used in the second screening [Table 4]

[0128] [Table 5] List of dosage forms used in the third screening [Table 5]

[0129] List of dosage forms used in the fourth screening [Table 6] [Table 6]

[0130] Since the fourth screening was conducted in two separate experiments, P40 ctrl was prepared and analyzed twice.

[0131] [Table 7] List of dosage forms used in 5 screenings (long-term stability experiment) [Table 7]

[0132] ●Screening analysis method

[0133] High temperatures accelerate pDNA SC% degradation. In this example, liquid DP vials were stored in a 70°C oven, and samples were taken on Day 0, Day 1, Day 3, and Day 5 for SC% analysis (using HPLC). The analytical samples were stored in a -70°C deep freezer immediately after sampling, and thawed in a 2-8°C cold room one hour before HPLC analysis.

[0134] To reduce sample analysis time and costs, samples with a pDNA SC% of less than 10% were not analyzed in subsequent samples.

[0135] (blank)

[0136] ● Long-term stability study

[0137] To confirm the long-term stability of the liquid dosage form, the inventors set up conditions of 2-8°C, 25°C, and 37°C to check stability under various temperature conditions and analyzed the SC% at one-month intervals. The 2-8°C samples were stored statically in an HX refrigerator, the 25°C samples were stored statically at room temperature, and the 37°C samples were stored statically in a 37°C incubator.

[0138] To maintain consistent sampling conditions, only a maximum of four samples were taken from one vial (5 ml filled), and the sample was discarded after the final pH measurement.

[0139] All other conditions were the same as those used in the existing 1st to 4th screening rounds.

[0140] (blank)

[0141] ●Screening analysis results

[0142] Example 1-1. First screening (buffering agent, pH screening)

[0143] The inventors attempted to determine the type of buffer and pH range that can stably maintain VM202pDNA as a raw material.

[0144] First, to confirm the effectiveness of various buffering agents, potassium phosphate, tris, histidine, and sodium citrate were used as buffering agents.

[0145] The final formulation list was selected by applying the possible pH range for each buffer. (However, the pH was set to not exceed 8.0, taking into consideration literature (Antoine Al-Achi, 2013).)

[0146] (blank)

[0147] The results of the first screening SC% (using HPLC) analysis are shown in Table 8 below.

[0148] [Table 8] First screening results (buffering agent, pH screening) [Table 8]

[0149] The SC% of VM202pDNA tended to be higher as the pH approached 8.0, and it was most stable when potassium phosphate was used as a buffer.

[0150] Specifically, in PC8.0 and PD8.0, the SC% was maintained at over 30% for 3 days, and in PC7.4 and PD7.4, the SC% on day 1 was maintained at over 60%, but the SC% on day 3 was only about 10%, confirming a decrease in SC% stability compared to PC8.0 and PD8.0.

[0151] Based on the results described above, analysis revealed that when using the same buffer, higher pH levels resulted in better stability of SC%.

[0152] Furthermore, we confirmed that higher buffer strength leads to longer-term maintenance of pH during manufacturing (see PC8.0, PD8.0, TC8.0, and TD8.0 results). Therefore, we confirmed that, with the same buffer, higher pH and buffer strength lead to greater SC% stability.

[0153] The type of buffer also affects the stability of SC%, and it was confirmed that VM202 pDNA exhibits high stability in potassium phosphate buffer and sodium citrate buffer.

[0154] In summary, the most suitable buffer for the VM202 liquid formulation was determined to be potassium phosphate buffer at pH 8.0.

[0155] (blank)

[0156] ●Examples 1-2. Second screening (excipient screening)

[0157] Based on the results of the first screening (potassium phosphate, pH 8.0), we attempted to explore the optimal excipient concentration range that contributes to pDNA stability by varying the concentrations of mannitol and sucrose, which are used as pDNA stabilizers.

[0158] (blank)

[0159] The basic composition was based on potassium phosphate, pH 8.0, which was identified as the optimal buffering agent in the first screening mentioned above.

[0160] We compared the stability of pDNA by varying the concentrations of mannitol and sucrose.

[0161] (blank)

[0162] The results of the second screening SC% (using HPLC) analysis are shown in Table 9 below.

[0163] [Table 9] Second screening results (excipient screening) [Table 9]

[0164] No significant difference was observed in the SC% of pDNA over 5 days in PMS0.1, PMS0.5, PMS1, and PMS2. Furthermore, while both PN45 and P20 / MS maintained an SC% of over 70% on day 1, the SC% on day 3 showed that PN45 exhibited more than 30% greater stability compared to P20 / MS.

[0165] Based on the above results, analysis confirmed that the SC% of VM202 (a non-GMP production sample from Company A) is unrelated to the concentrations of mannitol and sucrose. (See PMS0.1 and PMS2 results.)

[0166] Furthermore, since the Day 3 SC% of PN45 was approximately 30% higher than that of P20 / MS, it was concluded that buffer strength, compared to mannitol and sucrose content, had a greater impact on the SC% stability of pDNA. (See PN45 and P20 / MS pDNA SC% results.)

[0167] Therefore, in the third screening, it was determined that mannitol and sucrose could be removed from the formulation composition.

[0168] (blank)

[0169] ●Examples 1-3. Third screening (salt screening)

[0170] Based on reference literature indicating that when pDNA depurination occurs, salt neutralizes the negative charge of the phosphate group of pDNA, thereby stabilizing it, the inventors attempted to explore the optimal type and concentration range of salt that contributes to the pDNA SC% stability of VM202.

[0171] Based on the results of the first and second screenings described above, a basic composition of 40 mM potassium phosphate, 0.45% NaCl, and pH 8.0 was adopted.

[0172] Based on academic data indicating that salts such as NaCl and MgCl2 affect pDNA stability, the inventors conducted experiments using various concentrations of NaCl and MgCl2.

[0173] Finally, the salt concentration range was set considering the osmolality required for intramuscular injection, which is the injectable form of VM202. (Ideally 280-360 mOsm / kg, preferably <600 mOsm / kg)

[0174] The results of the SC% (using HPLC) analysis from the third screening are shown in Table 10 below.

[0175] [Table 10] Third screening results (salt screening) [Table 10]

[0176] We confirmed that the SC% stability of VM202 pDNA increased with increasing NaCl concentration. (See PN45 and PN9 results.)

[0177] Furthermore, we confirmed that the SC% stability of pDNA significantly increases when the NaCl concentration is 0.9%.

[0178] In the case of MgCl2, an increase in SC% of approximately 10% was observed when NaCl was present at 0.45%. However, it was confirmed that as time passed, MgCl2 reacted with water and precipitated as Mg(OH)2.

[0179] We confirmed that the maximum amount of MgCl2 that can be dissolved in 40 mM potassium phosphate and 0.9% NaCl is 0.01%, and that any amount greater than that precipitates.

[0180] MgCl2 precipitated on day 5 in PN9M1 and PN9M5. (HPLC analysis completed)

[0181] Based on the results above, we confirmed that SC% stability increases significantly as the concentration of NaCl increases. (See PN45 and PN9 results.)

[0182] Furthermore, MgCl2 showed signs of precipitating as Mg(OH)2 in aqueous solution at concentrations above a certain level (PN45M conditions). This is because the conditions used in this round were at a high pH of 8.0, and therefore, high (OH) - It was determined that Mg(OH)2 was formed due to the ion concentration. Therefore, it was confirmed that at salt concentrations above PN45M, Mg(OH)2 cannot improve the SC% safety of pDNA.

[0183] In summary, when the salt was added alone with 0.9% NaCl, the SC% stability of the pDNA was highest (SC% on Day 5: 44%). Based on the results up to the third screening, the optimal composition for the liquid dosage form of VM202 pDNA (Company A) was determined to be 40 mM potassium phosphate and 0.9% NaCl.

[0184] (blank)

[0185] ●Example 1-4. 4th Screening Round 4 Screening (Double Buffer Screening)

[0186] The inventors investigated whether pDNA SC% was more stable when using two types of buffers, potassium phosphate and tris (double buffer), compared to when using only potassium phosphate as a buffer. Based on the finding that pDNA was more stable with increasing pH in the first to third screenings, they attempted to determine whether pDNA would be further stabilized after adjusting the pH upward using two types of buffers (double buffer).

[0187] (blank)

[0188] First, two types of buffers were prepared by mixing the phosphate buffer and Tris buffer used previously in different ratios, and the pDNA was prepared in liquid dosage form.

[0189] Based on the results of the first screening, which showed that pDNA was more stable at higher pH levels, we attempted to test the pDNA stability by preparing a mixture of phosphate and Tris at a pH higher than 8.0.

[0190] The NaCl concentration was fixed at 0.9% based on the results of the third screening.

[0191] (blank)

[0192] The results of the SC% (using HPLC) analysis from the fourth screening are shown in Table 11 below.

[0193] [Table 11] Results of the 4th screening (double buffer screening) [Table 11]

[0194] In experiments where the pH was fixed at 8.0 and the phosphate and Tris ratios were varied, it was confirmed that pDNA stability increased with higher phosphate ratios. (See P / T 3:1 and P / T 1:3 results.) In experiments where the phosphate and Tris ratios were fixed and the pH was varied, it was confirmed that pDNA stability increased with higher pH.

[0195] Ultimately, when using double buffer, pDNA stability at a phosphate-to-Tris ratio of 1:1 and pH 8.7 showed similar results to that at 40 mM phosphate and pH 8.0.

[0196] (blank)

[0197] From the fourth screening, we confirmed that the higher the phosphate ratio, the longer the stability of pDNA SC% was maintained.

[0198] We confirmed that, when the ratio of phosphate to Tris is the same, pDNA becomes more stable at higher pH levels.

[0199] Therefore, in the case of the double-buffer formulation, we confirmed that the stability of pDNA increases as the ratio of phosphate buffer increases and as the pH of the formulation increases.

[0200] However, we confirmed that the SC% stability of P40 and P / T8.7 were similar. This means that the effect of double buffering is not significant in terms of pDNA SC% stability compared to existing single-buffer formulations. Also, when using double buffering, 40 mM of each buffer strength is used, which increases the osmolality.

[0201] Therefore, based on the above results, it is determined that the existing single buffer, 40 mM phosphate, and pH 8.0 are the most suitable.

[0202] (blank)

[0203] ●Examples 1-5. 5th Screening (Long-term Stability of the Final Candidate Group)

[0204] From the first to fourth screenings, we attempted to confirm the long-term stability of the selected 40 mM phosphate, 0.9% NaCl, pH 8.0 formulation and additional lead candidates at various temperatures.

[0205] (blank)

[0206] To confirm the saturation point of the buffer and salt, the selected conditions of 40 mM phosphate, 0.9% NaCl, and pH 8.0 were supplemented with conditions of 60 mM phosphate, 0.9% NaCl / 100 mM phosphate, and 1.1% NaCl, and long-term stability tests were conducted.

[0207] First, we sampled and examined the results at different time periods under three conditions: DP storage temperature of 2-8°C, accelerated conditions of 25°C, and stress conditions of 37°C.

[0208] To maintain consistent sampling conditions, only a maximum of four samples were taken from one vial (5 ml filled), and any remaining sample was discarded after the final pH measurement.

[0209] The plan for the long-term stability test is described in Table 12 below.

[0210] [Table 12] Long-term stability test plan [Table 12]

[0211] The SC% (using HPLC) analysis results from the five screenings are shown in Table 13 and Figure 1 below.

[0212] [Table 13] Fifth screening (long-term stability of the final candidate group) SC% results [Table 13]

[0213] - N / A means not analyzed.

[0214] We confirmed that the compositions of P40, P60, and P100 all had similar SC% after storage under their respective temperature conditions.

[0215] In the case of P40, after storage at 25°C for 6 months, it was confirmed that the SC% was 80% or higher (exceeding the VM202DP Acceptance Criteria).

[0216] As a result, as previously predicted, we determined that this composition is stable for more than 18 months when stored at 2-8°C, and we confirmed that when stored at 2-8°C for 6 months, there was no difference of more than 1% in SC% compared to T0.

[0217] We confirmed that the SC% 80% could be maintained for approximately two months under the harsh (stress) condition of 37°C.

[0218] (blank)

[0219] From the above results, it was confirmed that the SC% analysis results were similar under all conditions: P40, P60, and P100. Therefore, it was determined that the phosphate buffer strength and the VM202 pDNA SC% stability with NaCl were maintained at 40 mM, 0.9%, and pH 8.0, and that NaCl or pH stability did not have any further effect on SC%.

[0220] Therefore, based on the above results, the P40 dosage form (pH 8.0, 40 mM phosphate, 0.9% NaCl) is determined to be the most suitable final dosage form for VM202 DP.

[0221] (blank)

[0222] ●Conclusion of Example 1

[0223] Based on the results of a total of four screenings and one 6-month stability test, we successfully developed a VM202 liquid dosage form with the target stability (25°C, 3-month storage, SC% 80% or higher).

[0224] This example confirmed that not only pH but also the type of buffer affects the stability of VM202 pDNA. Furthermore, in the case of MgCl2, which was expected to maintain DNA stability more effectively than NaCl based on academic data, it precipitated while forming Mg(OH)2 and did not contribute to DNA stability. This is because, contrary to academic data, the VM202 formulation had a relatively high pH of 8.0, and high (OH) - This is determined to be because Mg(OH)2 is formed as an ion.

[0225] Furthermore, a 6-month stability test confirmed that VM202 pDNA remained stable (SC% 83%) even after 6 months of storage at 25°C in a phosphate buffer (40mM), NaCl (0.9%), and pH 8.0 composition.

[0226] The final derived composition will be further verified for storage stability at 2-8°C through long-term stability testing (18 months) if necessary. Based on the final derived composition, the range of compositions applicable to future commercial production will be determined.

[0227] (blank)

[0228] In this Example 1, the final composition derived from the fifth screening was as shown in Table 14 below.

[0229] [Table 14] Final derived composition [Table 14]

[0230] The final derived composition (40 mM potassium phosphate, 0.9% NaCl, pH 8.0) showed stability exceeding the DP spec standard for 3 months under 25°C storage conditions (89.5% SC), and this dosage form is judged to be stable for more than 18 months under 2-8°C storage conditions. Based on the final derived composition, we plan to determine the range of compositions applicable to commercial production in the future.

[0231] (blank)

[0232] (blank)

[0233] ●Example 2: Development of Plasmid DNA Liquid Dosage Forms B and C

[0234] When the composition of the liquid dosage form derived from Example 1 was applied to VM202 DS produced by Company C, Company W, and Helixmith, it was confirmed that the SC% stability differed from that of VM202 DS produced by Company A. Therefore, this experiment was conducted to extend the limitations of the liquid dosage form, which had been restricted to VM202 DS produced by Company A. The liquid dosage form established in Example 1 and applied to Company A's VM202 DS was named Liquid Dosage Form A (Formulation A).

[0235] (blank)

[0236] Similar to the previous example, the stability of VM202DP was evaluated using the SC% ratio, which is one of the factors that determine the quality of VM202.

[0237] Assuming that the basic characteristics of VM202 DS are identical, this embodiment was screened based on the liquid dosage form A composition selected in Example 1.

[0238] (blank)

[0239] Based on the screening results of liquid dosage form A, liquid dosage form B (Formulation B) was selected, and liquid dosage form C (Formulation C) with the optimal concentration of each excipient was selected through DoE.

[0240] The stability target for this screening was to maintain the DP Spec (SC% 85%) standard for at least three months under storage conditions of 20-25°C. This target was set based on research showing that the degradation of pDNA SC% accelerates as the storage temperature increases. It was assumed that if a sample is stable for three months at 20-25°C, it will be stable for at least 18 months at the DP storage temperature of 2-8°C.

[0241] Furthermore, to shorten the development period, the pDNA degradation rate was accelerated by exposing the pDNA to 70°C for 5 days (accelerated conditions) before analyzing the SC%. For long-term stability testing, samples were taken every 2 weeks (every month for 2-8°C) after storage at 2-8°C, 20-25°C, and 37°C, and the SC% was measured for 3 months. (The 2-8°C test was planned for 3 years.)

[0242] (blank)

[0243] The four screenings conducted in this example were carried out in the same manner as in Example 1, following the process of component preparation, BDP production, vial packaging (vial filling), sampling, and SC% analysis.

[0244] The vials used in the test were washed according to the vial washing protocol up to two days before BDP production, in a manner as close as possible to the actual production process, and stored in a 70°C dryer before use. The rubber stoppers were sterilized by Vendor (West-DAIKYO SEIKO) and used immediately after opening.

[0245] ● Manufacturing of dosage forms

[0246] The 1X buffer and 2X buffer used in this embodiment were manufactured up to 3 days before the start of each BDP production cycle.

[0247] For 2X buffers, the buffer strength was adjusted to twice the concentrations of mannitol, sucrose, sorbitol, trehalose, leucine, and arginine. (However, the NaCl concentration was calculated by substituting the final NaCl concentration (A) into the following formula: *(A × 2) - 0.9 = NaCl concentration of 2X buffer)

[0248] In the case of 1X buffer, the composition is identical to that of the final dosage form.

[0249] This example involved the production of a small-scale buffer (100 mL), and taking into account the manufacturing tolerances of actual commercial production processes, the 1X and 2X buffers were produced within a manufacturing tolerance range of approximately 2%.

[0250] The APIs used in this embodiment were VM202 DS samples produced by Company A (lot no. 88084), Company C (lot no. 2017#0042U), Helixmith's own VM202 DS, and Company W (lot no. DEV19-035, 049, 061, 068). These samples were thawed for 20-24 hours by transferring them from a -70°C deep freezer to a 2-8°C refrigerator before use.

[0251] The thawed DS was mixed with 2X and 1X buffers to produce BDP with a final pDNA concentration of 0.48–0.52 mg / mL (target 0.5 mg / mL).

[0252] The BDP manufacturing process and sampling and analysis procedures are as shown in Table 15 below.

[0253] (blank)

[0254] [Table 15] BDP Manufacturing and Sampling Procedure [Table 15]

[0255] The list of dosage forms used in the first to fourth screenings is shown in Tables 16 to 21 below.

[0256] [Table 16] First Press Screening List 1 (Excipient)

Table 16

[0257] In the previous example regarding the liquid dosage form A, the buffer optimal for liquid dosage form development was selected as potassium phosphate, so the buffer was fixed as potassium phosphate in this example.

[0258] (Blank)

[0259] [Table 17] First Press Screening List 2 (Arginine)

Table 17

[0260] [Table 18] First Press Screening List 3 (Range)

Table 18

[0261] [Table 19] Second Round Test Design (Design of Experiment, DoE)

Table 19

[0262] [Table 20] Third Round Long-Term Stability Test and Comparative Dosage Form List A

Table 20

[0263] [Table 21] Fourth Round Scale-Up Procedure Adjustment 请注意,原文中 疑似应为 ,我在翻译中保留了原文形式。你可根据实际情况进行调整。

Table 21

[0264] After BDP production, the pH was adjusted to 8.0, and after filtering using Supor EKV-Mini Kleenpak Capsules filter (Pall co., LTD), aliquots were made into vials.

[0265] (Blank)

[0266] ● Analysis

[0267] Since high temperature accelerates the degradation of pDNA SC%, in this test, after storing the liquid DP vials in a 70°C oven, samples were taken on Day 0 and Day 5, and SC% (using HPLC) was analyzed. (In the case of Round 3, sampling on Day 0, 1, 3, 5)

[0268] The analysis samples were stored in a -70°C deep freezer immediately after sampling, and after thawing in a 2 - 8°C refrigerator 1 hour before sample analysis, HPLC analysis was performed.

[0269] To reduce sample analysis time and cost, samples with a pDNA SC% of less than 10% were not analyzed for the samples sampled after that sample.

[0270] (Blank)

[0271] ● Long-term Stability Study

[0272] To confirm long-term stability under various temperature conditions, conditions of 2 - 8, 20 - 25, and 37°C were set, samples were taken at 2-week intervals (in the case of 2 - 8°C, monthly), and SC% was analyzed.

[0273] Samples at 2-8°C were stored statically in the HX refrigerator, samples at 25°C were stored statically at room temperature in laboratory 530, and samples at 37°C were stored statically in a 37°C incubator.

[0274] To maintain consistent sampling conditions, each vial (filled with 5 ml) was sampled only five times, with a maximum of 200 µl, before being discarded.

[0275] (blank)

[0276] ● Scale-up process adjustment study

[0277] The process was scaled up from a small-scale (~15 ml BDP) process to a large-scale (150 ml BDP) process, and the SC% stability of the pDNA was confirmed. The method for confirming SC% stability was the same as in Example 1.

[0278] To determine whether the presence or absence of a filter process affects the SC% stability of pDNA in each liquid dosage form A and C composition, experiments were conducted separately for BDP production with and without filter use.

[0279] (blank)

[0280] ●Example 2-1. First screening (excipients)

[0281] Considering that the efficacy of the liquid dosage form A composition established in Example 1 is limited to Company A's DS, we attempted to develop a composition with similar stability to liquid dosage form A + Company A's DS for VM202 DS produced by other CMOs such as Company C and Company W. To this end, we added various excipients and tried to identify the types and concentration ranges of excipients that can increase the SC% stability of VM202 pDNA produced at various production sites other than Company A.

[0282] (blank)

[0283] ● Test design

[0284] To confirm the effects of various excipients, mannitol, sucrose, sorbitol, trehalose, leucine, and arginine were treated at appropriate concentrations, and then significant ranges were examined.

[0285] The concentration ranges for each excipient were selected based on a review of FDA-approved component of biosimilars data (Reference) and literature (Rev Bras Otorrinolaringol, 2006;72(3):400-6).

[0286] (blank)

[0287] ●Results

[0288] The results of the osmolality and SC% (using HPLC) analysis of the first pre-screening samples 1-3 are shown in Tables 22-24 below.

[0289] [Table 22] First Pre-screening 1 (Excipient) Results [Table 22]

[0290] To account for the overlap in excipient concentrations across pre-screenings 1-3, osmolality was measured only in pre-screening 1.

[0291] The SC% of VM202 pDNA tended to be higher with increasing concentrations of sucrose and trehalose, or with increasing pH (see result A005), when mannitol was included (similar results for both 0.5% and 1.5%).

[0292] [Table 23] First Pre-screening 2 (Arginine) Results [Table 23]

[0293] In Pre-Screening 2, the NaCl concentration was set to 0.38%. The purpose of Pre-Screening 2 was to confirm whether the increase in SC% stability of A005, which was confirmed in Pre-Screening 1, was due to the increase in pH caused by arginine, or to the effect of arginine itself.

[0294] To confirm this, we divided the samples into two groups: one with an additional arginine (0.0025%) added to the existing excipient, and the other without. We then adjusted the pH of both groups to 8.0 (to eliminate the effects of pH increase) and attempted to confirm the effect of arginine itself on increasing SC%. The results showed no effect of arginine addition on increasing SC% stability.

[0295] (blank)

[0296] The increase in SC% stability of A005, confirmed from pre-screening 1, was due to an increase in pH, and it was confirmed that arginine itself had no effect. Therefore, arginine was excluded from subsequent dosage form compositions.

[0297] [Table 24] First Pre-Screening 3 (Range) Results [Table 24]

[0298] In the case of pre-screening 3, the test was conducted under pH 7.9 conditions to confirm stability under more severe conditions. Compared to the control group conditions (liquid dosage form A composition), M025 showed the highest level of increase in SC%, and the remaining excipients also showed a significant increase in SC% stability.

[0299] (blank)

[0300] ● Consideration

[0301] Among the added excipients, mannitol showed the highest increase in SC% and was found to be similar for mannitol concentrations ranging from 0.25% to 1.5%. Assuming that the effect of mannitol saturates at 0.25%, the mannitol concentration range for the DoE experiment was set to 0.05% to 0.25%.

[0302] Furthermore, we selected a liquid dosage form B (Formulation B) by adding only 0.25% mannitol to the existing liquid dosage form A (Formulation A) composition and attempted to compare it with the liquid dosage form C (Formulation C) composition developed using DoE.

[0303] The remaining excipients were also given a saturation point (maximum 1.5%) and their concentration ranges were set using the same principle. In the case of sorbitol and leucine, considering that the SC% increases with lower concentrations, the concentrations were set to the ranges of 0.05-0.25% and 0.1-0.5%, respectively.

[0304] Furthermore, we confirmed that pH buffering is more stably maintained with disaccharides such as sucrose and trehalose compared to monosaccharides such as mannitol and sorbitol.

[0305] In summary, the results above confirm the possibility of developing a new dosage form composition applicable to VM202 DS produced at other production sites such as Company C and Company W by adding an additional excipient to the existing dosage form A, which was previously limited to Company A's VM202 DS. Furthermore, we attempted to maximize the efficiency of the DoE experiment by setting the concentration range in which each excipient affects SC% stability.

[0306] (blank)

[0307] ●Example 2-2. Second Experiment Design (DoE)

[0308] Using the candidate excipients and their concentration ranges selected in the first pre-screening, we aim to select the optimal excipient composition for stabilizing VM202 pDNA using a DoE (Degree of Excipient) analysis.

[0309] (blank)

[0310] ● Test design

[0311] Based on the pDNA stability-enhancing effects of various excipients confirmed in the initial excipient screening, we compared pDNA stability by combining various concentration ranges to identify the effects of inter-excipient combinations and the major excipient factors.

[0312] (blank)

[0313] The DOE was designed randomly and performed in two iterations.

[0314] The excipients identified using DOE were as follows:

[0315] Mannitol, sucrose, sorbitol, trehalose, leucine

[0316] The results of the second DOE SC% (using HPLC) analysis are shown in Table 25 below.

[0317] [Table 25] Results of the second trial design [Table 25-1]

[0318] The above results, analyzed using statistical processing, are shown in Figures 2A and 2B. Figure 2A shows the results of an experiment conducted using a statistical design to confirm the effect of changes in seven factors (KP to LC) on the %SC of pDNA. When applying the statistical hypothesis validation criterion p=0.05, it was confirmed that all major factors except MT and TH directly affected %SC, and that pH*MT and TH*TH affected %SC as alternating and square factors. Specifically, it was determined that pDNA stability increased as the concentration of KP (potassium phosphate), N (NaCl), and pH increased within the selected range.

[0319] In the case of SR (Sucrose) and LC (Leucine), it was determined that pDNA stability increased as the concentration decreased within the selected range.

[0320] In the case of MT (Mannitol), due to its interaction with pH, ​​it was determined that pDNA stability is most increased when the MT concentration is low, especially when the pH value is high.

[0321] This allowed us to derive a model equation that embodies the influence of each factor on %SC, which is shown in Figure 2B.

[0322] Based on the above analysis, the derived optimal compositions are shown in Figures 3A and 3B.

[0323] (blank)

[0324] ● Consideration

[0325] As shown in Figure 3A, there are a total of seven factors influencing %SC: KP, N, pH, MT, SR, TH, and LC. Using these factors, the optimal model was derived and confirmed to be solution 1. Applying the model using solution 1 and statistically analyzing the spinning formula, it was estimated that the range in which the mean value of the %SC statistic is distributed is approximately 19.209 to 22.845 at a 95% confidence level. Furthermore, it was predicted that individual %SC values ​​will be distributed within the range of 17.451 to 26.603 at a 95% confidence level in the future.

[0326] Based on the results above, among the three compositions derived from the data, we selected the first composition (solution 1) with the highest SC%.

[0327] Furthermore, the inventors determined the concentration or value of each factor in a way that maximizes the y value, i.e., %SC, within the range of each factor (Figure 3B).

[0328] In the case of sorbitol, the DOE results showed that sorbitol had only a slight effect on the SC% stability of pDNA, and it was fixed at an intermediate value of 0.15% under DOE sorbitol conditions.

[0329] [Table 25-2]

[0330] ●Examples 2-3. Manufacturing of liquid dosage form C (Formulation C: potassium phosphate 40mM, NaCl 0.9%, pH 8.0, mannitol 0.05%, sorbitol 0.15%, sucrose 0.3%, trehalose 1.5%, leucine 0.1%) and comparison of dosage forms A, B, and C (long-term stability test at 70°C)

[0331] Long-term stability tests were conducted to confirm the stability of the liquid dosage form C composition under refrigerated conditions (2-8°C), room temperature storage conditions (20-25°C), and harsh conditions (37°C).

[0332] This study aimed to confirm the long-term pDNA stability of liquid dosage forms B and C, selected through the previous screening, and to confirm the pDNA stability of VM202 DS production sites in combination with liquid dosage forms A, B, and C.

[0333] (blank)

[0334] ● Test design

[0335] The first pre-screening confirmed that mannitol has the greatest pDNA stabilization effect as a single excipient.

[0336] We applied liquid dosage form B, which was obtained by adding only 0.25% mannitol to the existing liquid dosage form A, and liquid dosage form C, which was developed by DoE, to two lots of VM202 DS produced by HX and W Company, and compared them with the existing control condition, liquid dosage form A.

[0337] Long-term stability tests for liquid dosage forms A, B, and C at 70°C involved sampling on Days 0, 1, 3, and 5, and the SC% was confirmed.

[0338] (blank)

[0339] The test plan for the third long-term stability test is shown in Table 26.

[0340] [Table 26] Third long-term stability test plan (liquid dosage forms B and C) [Table 26]

[0341] - Time point T0.5 means 0.5 months (2 weeks).

[0342] ●Results

[0343] The SC% (using HPLC) analysis results from the third experiment are shown in Tables 27 and 28 below.

[0344] [Table 27] Comparison of dosage forms A, B, and C at 70°C (3rd time). [Table 27]

[0345] [Table 28] Third long-term stability test (dosage forms B and C) [Table 28]

[0346] - For day 1, 3, and 5 samples, and for samples taken at 2-8°C, only some samples were analyzed. - If analysis of the sample was not possible, it was indicated as N / A.

[0347] (blank)

[0348] As can be seen from Table 27, liquid dosage form C showed similar stability in HX, Company W, and ALD-produced DS at 70°C after 5 days (average SC 20.6%), while in the case of liquid dosage form B, Company A (SC 6.6%) confirmed that liquid dosage form A had generally lower SC% stability (9.8%).

[0349] This was similar to the long-term stability results in Table 28. In the case of liquid dosage form C, all lots of DS from HX and W companies showed an SC% of 60.0-70.5% at 37°C for 12 weeks, while liquid dosage form B showed a large difference between DS lots, ranging from 29.0-72.0%.

[0350] The long-term stability results at 25°C showed that liquid dosage form C maintained an average SC% of 79.4% over 6 months, while liquid dosage form B maintained an average SC% of 75.7%, confirming that VM202 pDNA was more stably maintained in liquid dosage form C.

[0351] (blank)

[0352] ● Consideration

[0353] Based on the above results, it is determined that liquid dosage form C, developed by DoE, exhibits less variation between DS lots compared to liquid dosage form B, and maintains VM202 pDNA SC% more stably under room temperature conditions (20-25°C).

[0354] Furthermore, in the case of liquid dosage form C, the target condition of 85% or higher SC% after storage at 25°C for 3 months, which was the objective of this test, is also met. (Average 86.7% after 3 months of storage)

[0355] Therefore, through the third screening, we successfully developed liquid dosage form C, which is applicable not only to VM202 DS produced by Company A, but also to VM202 DS produced by HX, W, and other companies.

[0356] Furthermore, considering that all of the above experiments were conducted on a small scale, we will attempt to confirm whether the results can be reproduced under scale-up conditions.

[0357] (blank)

[0358] (blank)

[0359] ●Example 2-4. Fourth Scale-Up Procedure Adjustment (Scale-Up A, C)

[0360] To confirm whether the liquid dosage form C composition developed in the first to third trials could be applied directly under scale-up conditions, and to confirm the SC% stability with and without a filter process, the following tests were conducted.

[0361] (blank)

[0362] ● Test design

[0363] The liquid dosage form procedure, previously performed on a 20 mL scale, was scaled up to a 300 mL scale.

[0364] To determine the effect of the filter process, 300 mL of manufactured BDP was divided into two 150 mL portions. One 150 mL was filtered, and the other 150 mL was filled into vials without filtration and subjected to a stress test.

[0365] The aforementioned process was applied to liquid dosage forms C and A, and the effects of scale-up and the presence or absence of a filter process were confirmed in each case.

[0366] For the fourth scale-up procedure adjustment, a harsh 70°C test was performed, with sampling on Days 0, 1, 3, and 5 to confirm the SC%.

[0367] (blank)

[0368] ●Results

[0369] The results of the SC% (using HPLC) analysis from the fourth screening are shown in Table 29 below.

[0370] [Table 29] 4th Scale-Up Procedure Adjustment [Table 29]

[0371] As can be seen in Table 29, in the case of liquid dosage form C, it was confirmed that the SC% remained constant regardless of whether a filter process was used. In contrast, in the case of liquid dosage form A, when a filter process was used, the day 3 SC% was 8.9%, which was a clear difference of more than 30% compared to when no filter process was used (40.6%).

[0372] (blank)

[0373] ● Consideration

[0374] In practice, filtering processes are essential in the production process due to issues of bioburden or sterility; therefore, the development of liquid dosage forms that can be filtered is necessary.

[0375] This test confirmed that liquid dosage form C maintains stable SC% even when a filter process is applied. (In contrast, liquid dosage form A was found to be unable to maintain SC% stability when a filter process is applied.)

[0376] Therefore, this embodiment successfully developed liquid dosage form C, which is a liquid dosage form that can be applied to actual production procedures.

[0377] (blank)

[0378] ●Conclusion of Example 2

[0379] Through a total of four screenings, we successfully developed a VM202 liquid dosage form that is applicable to VM202 DS produced at various production sites (Rounds 1-2), satisfies the target SC% stability of the test (Round 3), and is finally applicable to the actual production process (Round 4).

[0380] The long-term stability results for liquid dosage form C at 25°C (3rd test) showed that the average SC% at week 12 was 86.7%, exceeding the target of 85%.

[0381] Furthermore, this embodiment confirmed that the stability of VM202 pDNA varied depending on the manufacturing environment and lot, and that the presence or absence of a filter process also affected pDNA stability. Therefore, when introducing the liquid dosage form into actual production in the future, it is judged that the process by which VM202 DS was produced, the material of the filter introduced into production, and the process time should all be considered as factors that may affect the stability of the final liquid DP.

[0382] The final derived composition, liquid dosage form C, will be further tested for long-term stability (36 months) at 2-8°C if necessary to confirm its storage stability. Based on this final derived composition, the range of compositions applicable to future commercial production will be determined.

[0383] (blank)

[0384] The composition derived from the fourth test is shown in Table 30 below.

[0385] [Table 30] Final derived composition [Table 30]

[0386] Liquid dosage form C, the final derived composition constructed using DoE (40 mM potassium phosphate, 0.9% NaCl, pH 8.0, mannitol 0.05%, sucrose 0.3%, sorbitol 0.15%, trehalose 1.5%, leucine 0.1%), showed stability exceeding the DP spec standard for 3 months under storage conditions of 20-25°C (86.7% SC), and this dosage form is judged to be stable for 18 months or more under storage conditions of 2-8°C.

[0387] Based on the final derived composition, we plan to develop compositions that can be applied to commercial production in the future.

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[0389] ●Example 3: Development of plasmid DNA liquid dosage form D

[0390] Based on the previous Examples 1 and 2, we have confirmed that when the derived liquid dosage forms B and C were applied to VM202 DS produced by Company W and Helixmith, a liquid dosage form with a considerably higher level of stability compared to the lyophilized dosage form was developed.

[0391] In this example, tests were conducted to derive the most stable and optimal composition for VM202 using the RSM (Reaction Surface Design) method for liquid dosage form C composition. The optimal liquid dosage form derived by RSM was named liquid dosage form D.

[0392] (blank)

[0393] Similar to the previous example, the stability of VM202DP was evaluated using the SC% ratio, which is one of the factors that determine the quality of VM202.

[0394] This study, which involved improving the composition of liquid dosage form C, confirmed the main effects, interactions, and curvature effects between the factors of each composition. The test was conducted by deriving and applying the optimal composition through further improvements.

[0395] In the previous example, liquid dosage form C, in which each excipient had the optimal concentration, was selected using DoE, and the composition was further improved considering the interfactorial reactions confirmed from liquid dosage form C.

[0396] The stability target for this test is to maintain the DP Spec (SC% 85%) standard for at least 3 months under storage conditions of 20-25°C. This target was selected based on research showing that the degradation of pDNA SC% accelerates as the storage temperature increases, assuming that if stability is maintained for 3 months at 20-25°C, stability will be maintained for at least 18 months at the DP storage temperature of 2-8°C.

[0397] The improved liquid dosage form D was subjected to long-term stability testing with liquid dosage form C as the control group. Samples were collected every 4 weeks at 2-8°C, and every 2 weeks after storage at 20-25°C and 37°C, and the SC% was measured for 3 months. (The 2-8°C period was planned for 3 years.)

[0398] The order of the tests (workflow) was as shown in Table 31 below.

[0399] [Table 31] Test order [Table 31]

[0400] ● Exam Overview

[0401] The three screenings conducted in this study were performed using the same process: components preparation, BDP manufacturing, vial filling, sampling, and SC% analysis.

[0402] The vials used in this test were washed according to the vial washing protocol up to two days prior to BDP production, in a manner as close as possible to the actual production process, and were stored in a 70°C dryer before use. The rubber stoppers were sterilized by Vendor (West-DAIKYO SEIKO) and used immediately after opening.

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[0404] ●Manufacturing

[0405] The 1X and 2X buffers used in the tests were manufactured up to three days before the start of BDP production for each test run.

[0406] For 2X buffers, the buffer strength was adjusted to twice the concentrations of mannitol, sucrose, sorbitol, trehalose, leucine, and arginine. However, the NaCl concentration was calculated by substituting the final NaCl concentration (A) into the following formula: *(A × 2) - 0.9 = NaCl concentration of 2X buffer.

[0407] In the case of 1X buffer, the composition is identical to that of the final dosage form.

[0408] This test involved the production of small-scale buffers (100 mL), and taking into account the manufacturing tolerances of actual commercial production processes, the 1X and 2X buffers were produced within a manufacturing tolerance range of ~2%.

[0409] The APIs used in the test were VM202 DS (lots: 8th and 9th) produced by Company A, Company C, and Helixmith itself, and VM202 DS (Company W 1: 1912063, Company W 2: 1912073). They were thawed for 16-18 hours by transferring them from a -70°C deep freezer to a 2-8°C refrigerator before use.

[0410] The thawed DS was mixed with 2X and 1X buffers to produce BDP with a final pDNA concentration of 0.48–0.52 mg / mL (target 0.5 mg / mL).

[0411] The BDP manufacturing process and sampling and analysis procedures were as shown in Table 32 below.

[0412] [Table 32] BDP Manufacturing and Sampling Procedure [Table 32]

[0413] The list of RSM dosage forms used in the first screening is shown in Table 33 below.

[0414] [Table 33] First trial design (RSM) [Table 33]

[0415] The compositions of liquid dosage forms C and D used in the second long-term stability test were as shown in Table 34 below.

[0416] [Table 34] List of long-term stable dosage forms (second edition) [Table 34]

[0417] ●Analysis

[0418] High temperatures accelerate pDNA SC% degradation; therefore, liquid DP vials were stored in a 70°C oven, and samples were taken on Day 0 and Day 5 for SC% analysis (using HPLC). The analytical samples were stored in a -70°C deep freezer immediately after sampling, and thawed in a 2-8°C refrigerator one hour before HPLC analysis.

[0419] (blank)

[0420] ● Long-term stability test

[0421] To confirm long-term stability under various temperature conditions, samples were collected at 2-8°C, 20-25°C, and 37°C according to the sampling plan described in Table 36, and the SC% was analyzed.

[0422] Samples kept at 2-8°C were stored statically in the HX refrigerator, samples kept at 25°C statically at room temperature in laboratory 530, and samples kept at 37°C statically in a 37°C incubator.

[0423] To maintain consistent sampling conditions, only 10 samples were taken from each vial (5 ml filled), at a maximum of 100 µl each, before discarding.

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[0426] ● Test results and analysis

[0427] ●Example 3-1: First test design (RSM; reaction surface design)

[0428] Using formulation C, which was previously developed in our research, we designed a reaction surface to ensure the optimal composition in which each factor contributes to pDNA stability. This was done to confirm the optimal composition and combination that could be derived by examining the main effects, interactions, and curvature effects between various factors such as each excipient, buffer, pH, and salt.

[0429] (blank)

[0430] ● Test design

[0431] We confirmed the degree to which the equation for the final composition of liquid dosage form C, derived in Examples 1 and 2 above, and the impact on the stability between each factor.

[0432] Using the acquired data, the final composition was refined by considering the interactions between each factor that can be confirmed during the RSM design phase, thereby securing the optimal combination of factors applicable to pDNA stability, i.e., the best case composition.

[0433] The target of the optimal composition to be confirmed by the DOE is as follows:

[0434] : Potassium phosphate, sodium chloride, and leucine

[0435] The results of the SC% (using HPLC) analysis using the first trial design (RSM) are shown in Table 35 below.

[0436] [Table 35] Results of the first trial design (RSM) [Table 35]

[0437] ●Results

[0438] The above results, analyzed using statistical processing, are shown in Figures 4 and 5.

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[0440] ● Consideration

[0441] Figure 4 shows the effect of each factor used in pDNA preparation (e.g., KP, NaCl, pH, etc.) on %SC. Regarding Figure 4, it was found that the remaining factors other than SB all had a certain degree of influence on %SC. Of these, the factors with the strongest influence, considering the degree of visual evaluation, were potassium phosphate (KP), sodium chloride (N), and leucine (LC). Therefore, these three factors were used for dosage form optimization using RSM design, and the remaining excipients, whose optimal composition had already been ensured by the liquid dosage form C DOE in previous studies, were made to have the same composition as liquid dosage form C.

[0442] The optimal composition of the remaining excipients was selected based on the presence or absence of main effects and interactions between each factor.

[0443] Figure 5 shows the effect of changes in NaCl and KP concentrations on %SC after fixing leucine at 0.06%. As shown in Figure 5, it can be seen that %SC increases as the concentration of sodium chloride (NaCl,N) increases and as the concentration of potassium phosphate (KP) decreases. Specifically, when the leucine (LC) value is 0.06%, the optimal concentration ranges for potassium phosphate (KP) and sodium chloride (N) are confirmed to be 37.0~37.3 mM and 0.975~1.0%, respectively. Therefore, based on this, the upper limit for leucine was set to 0.055%, and the final concentration was set to 0.05% (leucine range: 0.045~0.055%).

[0444] The final derived liquid dosage form D had the following composition:

[0445] ●Liquid dosage form D: Potassium phosphate 37mM, NaCl 1.0%, pH 8.0, Mannitol 0.05%, Sorbitol 0.15%, Sucrose 0.3%, Trehalose 1.5%, Leucine 0.05%

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[0447] ●Second long-term stability test (liquid dosage form D)

[0448] ● Exam Overview

[0449] This test was conducted to confirm the long-term pDNA stability of liquid dosage form D composition, selected by the aforementioned DOE, and to confirm the long-term pDNA stability with liquid dosage form C.

[0450] (blank)

[0451] ● Test design

[0452] We improved the concentrations of potassium phosphate, sodium chloride, and leucine in the existing liquid dosage form C composition to obtain liquid dosage form D composition, and then applied this to compare it with liquid dosage form C as a control composition.

[0453] For liquid dosage forms C and D, samples were taken every 4 weeks at 2-8°C, and every 2 weeks after storage at 20-25°C and 37°C, and the SC% was measured for 3 months. (The 2-8°C period was planned over 2 years.)

[0454] The DS systems used were two HX DS systems and two W company DS systems, for a total of four systems used in the experiment.

[0455] The test plan for the second long-term stability test is described in Table 36 below.

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[0457] [Table 36] Second long-term stability study plan (dosage forms D and C) [Table 36]

[0458] -Time point T0.5 means 0.5 months (2 weeks).

[0459] ● Test results

[0460] The results of the SC% (using HPLC) analysis in the second test are shown in Table 37 below.

[0461] [Table 37] Results of the second long-term stability test (dosage forms D and C) [Table 37]

[0462] - Liquid dosage form D (HX1, 25°C) was discontinued in week 4, and liquid dosage form C (HX1, 25°C) was discontinued in week 6.

[0463] [Table 38] Results of the second long-term stability test (average %SC) [Table 38]

[0464] As can be seen in Table 38, liquid dosage form C showed an average SC stability of 84.1% at 20-25°C and 24 weeks, while liquid dosage form D showed an SC stability of 89.8% at 20-25°C and 24 weeks. Furthermore, liquid dosage form C showed an average SC stability of 66.4% at 37°C and 12 weeks, while liquid dosage form D showed an SC stability of 71.9% at 37°C and 12 weeks.

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[0466] ● Consideration

[0467] Based on the above results, analysis confirmed that liquid dosage form D, which is a composition improved from liquid dosage form C by RSM, exhibits a 5.9% increase in SC stability under room temperature conditions (20-25°C) compared to liquid dosage form C, and a 5.5% increase in SC stability under accelerated conditions of 37°C.

[0468] Furthermore, in the case of liquid dosage form C, the target condition of 85% or higher SC after 3 months of storage at 25°C was met (89.4% SC), and liquid dosage form D was confirmed to exhibit an even higher SC stability of 92.8%, ensuring an improved final composition. (Average %) after 3 months of storage.

[0469] Therefore, we successfully developed liquid dosage form D, which is the optimal composition that can maintain pDNA stability far better than liquid dosage form C.

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[0471] ●Conclusion of Example 3

[0472] This Example 3 confirmed that liquid dosage form D, the final composition derived from the pDNA stability aspect, is far more stable in maintaining pDNA under both accelerated conditions (25°C) and harsh conditions (37°C) compared to liquid dosage form C, which was confirmed in a previous study.

[0473] In fact, while liquid dosage form C showed 89.4% (% SC) at 25°C for 3 months, exceeding the VM202 DP spec standard of 85%, liquid dosage form D, an improved version of C, showed 92.8% at 25°C for 3 months. Therefore, we developed a dosage form that can more stably maintain the stability of pDNA in a liquid state.

[0474] Since it was confirmed that liquid dosage form C, the original composition, was applicable to VM202 DS produced at various production sites, satisfied the target SC% stability for this test, and was applicable to actual production processes, it was confirmed that liquid dosage form D, which was developed by improving upon this composition, exhibited the same effect while showing even greater stability.

[0475] This combination was confirmed to be the optimal one for enhancing the safety of pDNA through synergistic effects between the liquid dosage form compositions, and it is presumed that it can be maintained more stably for up to two years or more under refrigerated conditions, which is the DP storage temperature.

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[0478] ●Example 4: Long-term stability test of pre-filled syringe dosage form

[0479] The inventors conducted tests on the long-term stability of liquid dosage forms C and D developed in Examples 1 to 3 when manufactured as pre-filled syringe dosage forms.

[0480] Liquid dosage forms C and D, each containing 0.5 mg / ml of plasmid DNA, were prepared in vials and pre-filled syringes, respectively. The SC% was measured over a 3-month period by sampling at 2–8°C every 4 weeks. The results are shown in Table 39 and Figure 6.

[0481] [Table 39] [Table 39]

[0482] As shown in Table 39 and Figure 6, both vials and pre-filled syringes maintained an SC% of over 95% even after storage at 2-8°C for more than 6 months (24 weeks). Therefore, it was found that there is no difference in storage stability even when manufactured in the form of pre-filled syringes.

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[0484] ●Example 5: Stability testing of dosage forms C and D using various plasmids

[0485] Experiments were conducted to confirm whether other plasmids besides VM202 exhibited the same stability as liquid dosage forms C and D developed in Examples 2 and 3. The plasmids used were pCK-SDF-1a (WO2016 / 048105A1), pTx-IGF-1X10 (WO2020 / 016655A2), and pCMV3-cMet-flag (Sino Biological Inc., China). The experimental method was the same as in the existing examples. Liquid dosage forms C and D of the three plasmids were stored at 2-8°C and 25°C for 4 weeks, respectively, and the SC% was measured.

[0486] The results are shown in Table 40 below.

[0487] [Table 40] [Table 40]

[0488] As shown in Table 40, all three plasmids exhibited high stability in liquid dosage forms C or D. In particular, pCK-SDF-1a showed a significant decrease in stability even with brief exposure to its initial composition of 0.9% NaCl (SC% plummeted to 80%), but it was found to be highly stable in dosage forms C or D for 4 weeks without a significant decrease in SC%. This demonstrates that liquid dosage forms C or D, especially liquid dosage form D, are capable of stably maintaining even highly unstable plasmid DNA.

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[0490] ●Example 6: Long-term stability test of pre-filled syringe formulations using various plasmids

[0491] The stability of the three plasmids used in Example 5—pCK-SDF-1a (WO2016 / 048105A1), pTx-IGF-1X10 (WO2020 / 016655A2), and pCMV3-cMet-flag (Sino Biological Inc., China)—was measured in SC% after being prepared as pre-filled syringes using liquid dosage form D and stored for 4 weeks.

[0492] The results are shown in Table 41.

[0493] [Table 41] [Table 41]

[0494] As shown in Table 41, similar to the results in Example 5, all three plasmids were confirmed to be very stable even when stored for 4 weeks as a pre-filled syringe formulation (D formulation). Exemplary embodiments of the present invention are described below. <1> A liquid dosage form of plasmid DNA containing 30-50 mM potassium phosphate, 0.5-1.5% NaCl, and plasmid DNA, with a pH of 7.5-8.5. <2> The aforementioned liquid dosage form contains 40 mM potassium phosphate. <1> Liquid dosage form of plasmid DNA as described above. <3> The aforementioned liquid dosage form contains 0.9% NaCl. <1> Liquid dosage form of plasmid DNA as described above. <4> The pH of the aforementioned liquid dosage form is 8.0. <1> Liquid dosage form of plasmid DNA as described above. <5> The plasmid DNA in the aforementioned liquid dosage form maintains 80-100 SC% (supercoiled %) even after storage at 20-30°C for 6 months. <1> Liquid dosage form of plasmid DNA as described above. <6> The liquid dosage form further comprises sucrose, trehalose, mannitol, sorbitol, leucine, or a combination thereof. <1> Liquid dosage form of plasmid DNA as described above. <7> The aforementioned liquid dosage form further comprises 1-2% sucrose, 0.5-2% trehalose, 0.01-1% sorbitol, 0.05-0.5% leucine, or a combination thereof. <1> Liquid dosage form of plasmid DNA as described above. <8> The plasmid DNA is contained at a concentration of 0.01 to 5 mg / mL. <1> Liquid dosage form of plasmid DNA as described above. <9> The aforementioned liquid dosage form further contains 0.15-1.5% mannitol. <1> Liquid dosage form of plasmid DNA as described above. <10> The plasmid DNA in the aforementioned liquid dosage form maintains 90-100 SC% even after storage at 2-8°C for at least 6 months. <9> Liquid dosage form of plasmid DNA as described above. <11> The plasmid DNA in the liquid dosage form maintains 60-100 SC% even after storage at 15-30°C for at least 3, 4, 5, or 6 months. <9> Liquid dosage form of plasmid DNA as described above. <12> The aforementioned liquid dosage form further comprises 0.001-0.3% mannitol, 0.05-0.5% sorbitol, 0.05-0.5% sucrose, 0.15-3% trehalose, 0.01-1% leucine, or a combination thereof. <1> Liquid dosage form of plasmid DNA as described above. <13> The plasmid DNA in the liquid dosage form maintains 90-100 SC% even after storage at 2-8°C for at least 3, 4, 5, 6, 7, 8, or 9 months. <12> Liquid dosage form of plasmid DNA as described above. <14> The plasmid DNA in the liquid dosage form maintains 80-100 SC% even after storage at 20-30°C for at least 3, 4, 5, 6, 7, 8, or 9 months. <12> Liquid dosage form of plasmid DNA as described above. <15> The aforementioned liquid dosage form further comprises 0.001-0.3% mannitol, 0.05-0.5% sorbitol, 0.05-0.5% sucrose, 0.15-3% trehalose, 0.045-0.055% leucine, or a combination thereof. <1> Liquid dosage form of plasmid DNA as described above. <16> The aforementioned liquid dosage form contains 37-37.3 mM potassium phosphate. <15> Liquid dosage form of plasmid DNA as described above. <17> The aforementioned liquid dosage form contains 0.975-1.0% NaCl. <15> Liquid dosage form of plasmid DNA as described above. <18> The plasmid DNA in the liquid dosage form maintains 90-100 SC% even after storage at 2-8°C for at least 3, 4, 5, 6, 7, 8, or 9 months. <15> Liquid dosage form of plasmid DNA as described above. <19> The plasmid DNA in the liquid dosage form maintains 80-100 SC% even after storage at 20-30°C for at least 3, 4, 5, 6, 7, 8, or 9 months. <15> Liquid dosage form of plasmid DNA as described above. <20> The aforementioned liquid dosage form is manufactured as a vial, ampoule, bottle, or pre-filled syringe. <1> ~ <19> A liquid dosage form of plasmid DNA as described in any of the following.

Claims

1. A pharmaceutical composition in liquid dosage form for gene therapy, comprising 35-45 mM potassium phosphate, 0.8-1.1% NaCl, 0.04-0.1% mannitol, 0.1-0.5% sorbitol, 0.25-0.5% sucrose, 1.25-2% trehalose, 0.03-0.1% leucine, and plasmid DNA, with a pH of 7.5-8.

5.

2. The pharmaceutical composition in liquid dosage form according to claim 1, wherein the composition contains 40 mM potassium phosphate.

3. The pharmaceutical composition in liquid dosage form according to claim 1, wherein the composition contains 0.9% NaCl.

4. The pharmaceutical composition in liquid dosage form according to claim 1, wherein the pH of the composition is 8.

0.

5. The pharmaceutical composition in liquid dosage form according to claim 1, wherein the plasmid DNA in the composition maintains 80-100 SC% (supercoiled %) even after storage at 20-30°C for 6 months.

6. A pharmaceutical composition in liquid dosage form according to claim 1, comprising the plasmid DNA at a concentration of 0.1 to 1 mg / mL.

7. The pharmaceutical composition in liquid dosage form according to claim 1, wherein the plasmid DNA in the composition maintains 90-100 SC% even after storage at 2-8°C for at least 6 months.

8. The pharmaceutical composition in liquid dosage form according to claim 1, wherein the plasmid DNA in the composition maintains 60-100 SC% even after storage at 15-30°C for at least 3 months, 4 months, 5 months, or 6 months.

9. The pharmaceutical composition in liquid dosage form according to claim 1, wherein the plasmid DNA in the composition maintains 90-100 SC% even after storage at 2-8°C for at least 3, 4, 5, 6, 7, 8, or 9 months.

10. The pharmaceutical composition in liquid dosage form according to claim 1, wherein the plasmid DNA in the composition maintains 80-100 SC% even after storage at 20-30°C for at least 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, or 9 months.

11. The pharmaceutical composition in liquid dosage form according to claim 1, wherein the composition comprises 37 to 37.3 mM potassium phosphate.

12. The pharmaceutical composition in liquid dosage form according to claim 1, wherein the composition comprises 0.975 to 1.0% NaCl.

13. The pharmaceutical composition in liquid dosage form according to any one of claims 1 to 12, wherein the composition is prepared as a vial, ampoule, bottle, or pre-filled syringe.

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