Liquid 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 dosage pharmaceutical composition comprising plasmid DNA.

[0003] (blank) [Background technology]

[0004] There is substantial clinical evidence that gene therapy involving the direct in vivo delivery of nucleic acid constructs that are not packaged in viruses or virus-like particles (so-called "naked" DNA or RNA constructs) can 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 called "VM202") has been shown to be effective in treating neuropathic pain. In a Phase II clinical trial, injection of VM202 into the gastrocnemius muscle of patients with diabetic peripheral neuropathy was found to significantly reduce pain, and this was sufficient to provide symptomatic relief with improved quality of life for three months with treatment on two days every two weeks. Kessler et al., Annals Clin. Transl. Neurology 2(5):465-478(2015). The same DNA plasmid construct was also found to be effective in treating amyotrophic lateral sclerosis (ALS) patients. In a Phase I clinical trial, nearly half of ALS patients maintained a stable or improved condition after VM202 administration, with 47%, 50%, and 24% of subjects experiencing no decrease or improvement in the ALSFRS-R (Amyotrophic Lateral Sclerosis Functional Rating Scale-revised) score at 1 month, 2 months, and 3 months, respectively, which is a better result than observed in the historical control group. Robert L. Sufit et al., Amyotrophic Lateral Sclerosis and Frontoemporal Degeneration18:269-278(2017).

[0005] However, it is known that the physical and chemical stability of such DNA or RNA construct materials is affected by the final formulation and dosage form, storage conditions, etc. Generally, pharmaceutical dosage forms are provided in a lyophilized form for storage stability, but they need to be reconstituted with water for injection before being administered to patients. If the manual is not followed precisely during the reconstitution of such drugs, the drugs cannot be uniformly reconstituted, resulting in drug loss or contamination during administration. Therefore, if the drugs are provided in a liquid dosage form rather than a lyophilized form, safety and product competitiveness can be improved. Therefore, there is a need to develop a liquid dosage form of naked-DNA-based drugs with excellent stability and safety.

[0006] (blank) Summary of the Invention [Problem to be solved by the invention]

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

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

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

[0010] As used herein, the term "liquid formulation" refers to a pharmaceutical composition in liquid form, comprising plasmid DNA and at least one pharma- ceutically acceptable carrier or excipient, as described herein.

[0011] The liquid dosage form of the present invention is a general-purpose liquid dosage form of plasmid DNA that can be used regardless of the type of plasmid DNA, and therefore the liquid dosage form of the present invention is not limited to a liquid dosage form for the 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, but is not limited to, VM202.

[0014] In one embodiment of the present invention, the plasmid DNA further comprises a promoter. In the plasmid DNA, the promoter is operably linked to a nucleic acid molecule encoding a protein. As used herein, the term "operably linked" refers to the functional association of a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcription regulator binding sites) with another nucleic acid sequence, whereby the control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0015] For example, when the vector of the present invention is an expression vector and a eukaryotic cell is used as a host, a promoter derived from the genome of a mammalian cell (e.g., metallothionine promoter, beta-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late 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 Rous sarcoma virus (RSV) promoter) may be used, which generally has a polyadenylation sequence as a transcription termination sequence.

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

[0017] Selectable marker genes include antibiotic resistance genes commonly used in the art, such as resistance genes to ampicillin, gentamicin, cavenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline, etc. Reporter genes include, but are not limited to, genes such as luciferase, beta-galactosidase, chloramphenicol acetyltransferase, or green fluorescent protein.

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

[0019] As used herein, the term "VM202" refers to a plasmid DNA comprising pCK vector (SEQ ID NO: 6) and HGF-X7 (SEQ ID NO: 13) cloned into the pCK vector, also called pCK-HGF-X7. VM202 was deposited under the Budapest Treaty with the Korea Center for Microorganisms (KCCM) on March 12, 2002 under the deposit number KCCM-10361.

[0020] The term "isoforms of hepatocyte growth factor (HGF)" as used herein refers to a polypeptide having an amino acid sequence at least 80% identical to the amino acid sequence of an HGF polypeptide naturally occurring in an animal. The term includes a polypeptide having an amino acid sequence at least 80% identical to any full-length wild-type HGF polypeptide, and includes a polypeptide having an amino acid sequence at least 80% identical to a naturally occurring HGF allelic variant, splice variant, or deletion variant. In the present invention, the 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 the present invention, the HGF isoforms used in the methods described herein include flHGF (SEQ ID NO: 1) and dHGF (SEQ ID NO: 2).

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

[0022] The terms "human dHGF" and "dHGF" are used interchangeably herein to mean a deletion variant of the HGF protein generated by alternative splicing of the human HGF gene. Specifically, "human dHGF" or "dHGF" means a human HGF protein in which five amino acids (F, L, P, S, and S) are 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, or fragments / variants / mutants thereof.

[0024] The term SDF-1 (stromal cell-derived factor 1) as used herein is also known as C-X-C motif chemokine 12 (CXCL12), and is a chemokine protein encoded by the CXCL12 gene. SDF-1 is expressed in many cells and tissues and is divided into SDF-1 alpha and SDF-1 beta. SDF-1 is known to play an important role in angiogenesis, wound healing, neurodevelopment, 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 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 with a molecular structure similar to that of insulin. IGF-1 may play an important role in promoting cell proliferation during early childhood development. IGF-1 is encoded by the IGF1 gene and is known to stimulate the growth of various types of cells, inhibit cell death, and increase cellular protein production.

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

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

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

[0032] (blank)

[0033] In one embodiment of the present invention, the liquid dosage form includes plasmid DNA as an active ingredient of the pharmaceutical composition. The plasmid DNA may include genetic material for gene therapy. Specifically, the plasmid DNA may correct the function of a defective gene or transcript, or may code for a polypeptide, a sense or antisense oligonucleotide, or an RNA (coding or non-coding; e.g., siRNA, shRNA, micro-RNA, and their antisense counterparts (e.g., antagoMi R)). Any plasmid DNA-containing dosage form known in the art for use in gene therapy is within the scope of the present invention.

[0034] In one embodiment of the present invention, the liquid formulation contains plasmid DNA at a concentration of 0.01-5 mg / mL. More specifically, the liquid formulation contains plasmid DNA at a concentration of 0.01-5 mg / mL, 0.01-3 mg / mL, 0.01-2 mg / mL, 0.01-1 mg / mL, 0.01-0.75 mg / mL, 0.01-0.6 mg / mL, 0.01-0.5 mg / mL, 0.1-5 mg / mL, 0.1-3 mg / mL, 0.1-2 mg / mL, 0.1-1 mg / mL, 0.1-0.75 mg / mL, 0.1-0.6 mg / mL, 0.1-0.5 mg / mL, 0.2-5 mg / mL, 0.4-5 mg / mL, 0.5-5 mg / mL, 0.5-5 mg / mL, 0.6-5 mg / mL, 0.7 ... The plasmid DNA may be contained at a concentration of 0.3 to 5 mg / ml, 0.3 to 3 mg / ml, 0.3 to 2 mg / ml, 0.3 to 1 mg / ml, 0.3 to 0.75 mg / ml, 0.3 to 0.6 mg / ml, 0.2 to 0.5 mg / ml, 0.3 to 5 mg / ml, 0.3 to 3 mg / ml, 0.3 to 2 mg / ml, 0.3 to 1 mg / ml, 0.3 to 0.75 mg / ml, 0.3 to 0.6 mg / ml, 0.3 to 0.5 mg / ml, or 0.5 mg / ml.

[0035] The plasmid DNA may have a length 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, 3,000 to 9,000 base pairs, The plasmid DNA may be a polynucleotide having a length within the ranges provided herein.

[0036] The liquid dosage form of 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 are present in a relatively stable supercoiled form or in a relatively less stable open circle and linear form. The conformation of the plasmid DNA may be determined by capillary electrophoresis or HPLC (High-Pressure Liquid Chromatography) of a sample containing the plasmid DNA. The content of supercoiled DNA compared to the open circle and linear forms may be measured under various conditions.

[0037] In one embodiment of the invention, at least 90% of the plasmid DNA is supercoiled. In some non-limiting embodiments, at least 92.5% of the plasmid DNA in the liquid composition is supercoiled. 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 include buffer compounds known in the art, such as TAPS, bicine, tris, tricine, 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 include 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 a concentration of about 30 to 50 mM, about 35 to 50 mM, about 40 to 50 mM, about 45 to 50 mM, about 30 to 45 mM, about 35 to 45 mM, about 40 to 45 mM, about 30 to 40 mM, about 35 to 40 mM, or about 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 formulation has a potassium phosphate concentration of about 30-50 mM, pH of about 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 may be present in an amount of about 0.5-1.5%, about 0.5-1.4%, about 0.5-1.3%, about 0.5-1.2%, about 0.5-1.1%, about 0.5-1.0%, about 0.5-0.9%, about 0.6-1.5%, about 0.6-1.4%, about 0.6-1.3%, about 0.6-1.2%, about 0.6-1.1%, about 0.6-1.0%, about 0.6-0.9%, about 0. The compound is present in a concentration of about 7-1.5%, about 0.7-1.4%, about 0.7-1.3%, about 0.7-1.2%, about 0.7-1.1%, about 0.7-1.0%, about 0.7-0.9%, about 0.8-1.5%, about 0.8-1.4%, about 0.8-1.3%, about 0.8-1.2%, about 0.8-1.1%, about 0.8-1.0%, about 0.8-0.9%, or 0.9%.

[0043] In one embodiment of the invention, the liquid dosage form comprises about 40 mM potassium phosphate.

[0044] In one embodiment of the 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 about 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 one embodiment of the present invention described above is designated as dosage form A.

[0047] In one embodiment of the present invention, the plasmid in the liquid formulation maintains 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 formulation maintains 80-100SC%, 81-100SC%, 82-100SC%, 83-100SC%, or 84-100SC% after storage at 15-30°C for at least 3 months, 4 months, 5 months, or 6 months. The storage temperature is 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 may be, but is not limited to, 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. The liquid formulation according to the present invention includes an aqueous solution containing a stabilizing amount of sugar to improve the stability of the plasmid DNA. The sugar may be sucrose, trehalose, mannitol, or sorbitol.

[0049] Thus, the liquid dosage forms of the present invention may further comprise sucrose, trehalose, mannitol, sorbitol, leucine, or combinations 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 one embodiment of the present invention, the liquid dosage form further 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 a liquid dosage form of plasmid DNA according to another embodiment of the present invention, the liquid dosage form of the present invention further contains about 0.15 to 1.5% of mannitol in addition to the liquid dosage form according to the above-mentioned embodiment.

[0053] In one embodiment of the present invention, the liquid dosage form contains mannitol at about 0.15-1.5%, about 0.15-1.25%, about 0.15-1.0%, about 0.15-0.75, about 0.15-0.5%, about 0.15-0.25%, about 0.25-1.5%, about 0.25-1.25%, about 0.25-1.0%, about 0.25-0.75, about 0. Concentrations include, but are not limited to, about 25-0.5%, about 0.3-1.25%, about 0.3-1.0%, about 0.3-0.75, about 0.3-0.5%, about 0.5-1.25%, about 0.5-1.0%, about 0.5-0.75%, about 1.5%, about 1.25%, about 1.0%, about 0.75%, about 0.5%, or about 0.25%.

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

[0055] In one embodiment of the present invention, the plasmid DNA in the liquid dosage form maintains 90-100% SC% 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 60-100, 70-100, 75-100, 76-100, 77-100, 78-100, 79-100, or 80-100 SC% after storage at 15-30°C for at least 3 months, 4 months, 5 months, or 6 months. The storage temperature is 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 The temperature may be, but is not limited to, 1 to 27°C, 22 to 27°C, 23 to 27°C, 24 to 27°C, 25 to 27°C, 15 to 26°C, 16 to 26°C, 17 to 26°C, 18 to 26°C, 19 to 26°C, 20 to 26°C, 21 to 26°C, 22 to 26°C, 23 to 26°C, 24 to 26°C, 25 to 26°C, 15 to 25°C, 16 to 25°C, 17 to 25°C, 18 to 25°C, 19 to 25°C, 20 to 25°C, 21 to 25°C, 22 to 25°C, 23 to 25°C, 24 to 25°C, or 25°C.

[0057] In a liquid dosage form of plasmid DNA according to still another embodiment of the present invention, the liquid dosage form of the present invention further comprises about 0.001 to 0.3% mannitol, about 0.05 to 0.5% sorbitol, about 0.05 to 0.5% sucrose, about 0.15 to 3% trehalose, about 0.01 to 1% leucine, or a combination thereof, in addition to the liquid dosage form according to the embodiment described above.

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

[0059] In one embodiment of the present invention, the liquid dosage form contains mannitol at about 0.001-0.3%, about 0.005-0.3%, about 0.01-0.3%, about 0.02-0.3%, about 0.03-0.3%, about 0.04-0.3%, about 0.05-0.3%, about 0.001-0.25%, about 0.005-0.25%, about 0.01-0.25%, about 0.02-0.25%, about 0.03-0.25%, about 0.04-0.25%, about 0.05-0.25%, about 0.001-0.2%, about 0.005 ~0.2%, approx. 0.01-0.2%, approx. 0.02-0.2%, approx. 0.03-0.2%, approx. 0.04-0.2%, approx. 0.05-0.2%, approx. 0.001-0.15%, approx. 0.005-0.15%, approx. 0.01-0.15%, approx. 0.02-0.15%, approx. 0.03-0.15%, approx. 0.04-0.15%, approx. 0.05-0.15%, approx. 0.001-0.1%, approx. 0.005-0.1%, approx. 0.01-0.1%, approx. 0.02-0.1%, approx. 0.03-0.1%, approx. 0.04-0 .1%, approx. 0.05-0.1%, approx. 0.001-0.075%, approx. 0.005-0.075%, approx. 0.01-0.075%, approx. 0.02-0.075%, approx. 0.03-0.075%, approx. 0.04-0.075%, approx. 0.05-0.075%, approx. 0.001-0.07%, approx. 0.005-0.07%, approx. 0.01-0.07%, approx. 0.02-0.07%, approx. 0.03-0.07%, approx. 0.04-0.07%, approx. 0.05-0.07%, approx. 0.001-0.06%, approx. The concentration of the sucrose ionizing agent may be, but is not limited to, about 0.05-0.06%, about 0.01-0.06%, about 0.02-0.06%, about 0.03-0.06%, about 0.04-0.06%, about 0.05-0.06%, about 0.001-0.05%, about 0.005-0.05%, about 0.01-0.05%, about 0.02-0.05%, about 0.03-0.05%, about 0.04-0.05%, about 0.01%, about 0.03%, about 0.05%, about 0.07%, about 0.075%, or about 0.1%.

[0060] In one embodiment of the present invention, the liquid dosage form contains sorbitol at a concentration of, but not limited to, about 0.05-0.5%, about 0.075-0.5%, about 0.1-0.5%, about 0.125-0.5%, about 0.15-0.5%, about 0.2-0.5%, about 0.25-0.5%, about 0.3-0.5%, about 0.35-0.5%, about 0.4-0.5%, about 0.05%, 0.075%, about 0.1%, or about 0.15%.

[0061] In one embodiment of the present invention, the liquid dosage form contains sucrose at a concentration of about 0.05%-0.5%, about 0.075-0.5%, about 0.1-0.5%, about 0.125-0.5%, about 0.15-0.5%, about 0.2-0.5%, about 0.25-0.5%, about 0.3-0.5%, about 0.35-0.5%, about 0.4-0.5%, about 0.05%, 0.075%, about 0.1%, 0.15%, about 0.2%, or about 0.3%, but is not limited to these.

[0062] In one embodiment of the present invention, the liquid dosage form contains trehalose at about 0.15-3%, about 0.15-2.5%, about 0.15-2%, about 0.15-1.75%, about 0.15-1.5%, about 0.15-1.25%, about 0.15-1%, about 0.15-0.8%, about 0.15-0.75%, about 0.15-0.7%, about 0.15-0.6%, about 0.15-0.5%, about 0.25-3%, about 0. .25-2.5%, approx. 0.25-2%, approx. 0.25-1.75%, approx. 0.25-1.5%, approx. 0.25-1.25%, approx. 0.25-1%, approx. 0.25-0.8%, approx. 0.25-0.75%, approx. 0.25-0.7%, approx. 0.25-0.6%, approx. 0.25-0.5%, approx. 0.5-3%, approx. 0.5-2.5%, approx. 0.5-2%, approx. 0.5-1.75%, approx. 0.5-1.5%, approx. 0.5 ~1.25%, approx. 0.5-1%, approx. 0.5-0.8%, approx. 0.5-0.75%, approx. 0.5-0.7%, approx. 0.5-0.6%, approx. 0.75-3%, approx. 0.75-2.5%, approx. 0.75-2%, approx. 0.75-1.75%, approx. 0.75-1.5%, approx. 0.75-1.25%, approx. 0.75-1%, approx. 0.75-0.8%, approx. 1-3%, approx. 1-2.5%, approx. 1-2%, approx. 1-1.75% , about 1-1.5%, about 1-1.25%, about 1.25-3%, about 1.25-2.5%, about 1.25-2%, about 1.25-1.75%, about 1.25-1.5%, about 1.5-3%, about 1.5-2.5%, about 1.5-2%, about 1.5-1.75%, 0.5%, about 1.0%, about 1.25%, about 1.5%, about 2%, about 2.5%, or about 3%.

[0063] In one embodiment of the present invention, the liquid dosage form contains leucine at about 0.01-1%, about 0.01-0.9%, about 0.01-0.8%, about 0.01-0.7%, about 0.01-0.6%, about 0.01-0.5%, about 0.01-0.4%, about 0.01-0.3%, about 0.01-0.2%, about 0.01-0.1%, about 0.03-1%, about 0.03-0.9%, about 0.03-0.8%, about 0.03-0.7%, about 0.03-0.6%, about 0.03 ~0.5%, about 0.03~0.4%, about 0.03~0.3%, about 0.03~0.2%, about 0.03~0.1%, about 0.05~1%, about 0.05~0.9%, about 0.05~0.8%, about 0.05~0.7%, about 0.05~0.6%, about 0.05~0.5%, about 0.05~0.4%, about 0.05~0.3%, about 0.05~0.2%, about 0.05~0.1%, about 0.075~1%, about 0.075~0.9%, about 0.075~0.8%, About 0.075~0.7%, About 0.075~0.6%, About 0.075~0.5%, About 0.075~0.4%, About 0.075~0.3%, About 0.075~0.2%, About 0.075~0.1%, About 0.08~1%, About 0.08~0.9%, About 0.08~0.8%, About 0.08~0.7%, About 0.08~0.6%, About 0.08~0.5%, About 0.08~0.4%, About 0.08~0.3%, About 0.08~0.2%, About 0.08~0.1%, Concentrations include, but are not limited to, about 0.09-1%, about 0.09-0.9%, about 0.09-0.8%, about 0.09-0.7%, about 0.09-0.6%, about 0.09-0.5%, about 0.09-0.4%, about 0.09-0.3%, about 0.09-0.2%, about 0.09-0.1%, about 0.01%, about 0.03%, about 0.05%, about 0.075%, about 0.08%, about 0.09%, about 0.1%, about 0.2%, or about 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 after storage at 2-8°C for at least 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, or 9 months.

[0066] 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, or 85-100 after storage at 15-30°C for at least 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, or 9 months. The storage temperature is 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 The temperature may be, but is not limited to, 1 to 27°C, 22 to 27°C, 23 to 27°C, 24 to 27°C, 25 to 27°C, 15 to 26°C, 16 to 26°C, 17 to 26°C, 18 to 26°C, 19 to 26°C, 20 to 26°C, 21 to 26°C, 22 to 26°C, 23 to 26°C, 24 to 26°C, 25 to 26°C, 15 to 25°C, 16 to 25°C, 17 to 25°C, 18 to 25°C, 19 to 25°C, 20 to 25°C, 21 to 25°C, 22 to 25°C, 23 to 25°C, 24 to 25°C, or 25°C.

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

[0068] In one embodiment of the present invention, the liquid dosage form contains mannitol at about 0.001-0.3%, about 0.005-0.3%, about 0.01-0.3%, about 0.02-0.3%, about 0.03-0.3%, about 0.04-0.3%, about 0.05-0.3%, about 0.001-0.25%, about 0.005-0.25%, about 0.01-0.25%, about 0.02-0.25%, about 0.03-0.25%, about 0.04-0.25%, about 0.05-0.25%, about 0.001-0.2%, about 0.005 ~0.2%, approx. 0.01-0.2%, approx. 0.02-0.2%, approx. 0.03-0.2%, approx. 0.04-0.2%, approx. 0.05-0.2%, approx. 0.001-0.15%, approx. 0.005-0.15%, approx. 0.01-0.15%, approx. 0.02-0.15%, approx. 0.03-0.15%, approx. 0.04-0.15%, approx. 0.05-0.15%, approx. 0.001-0.1%, approx. 0.005-0.1%, approx. 0.01-0.1%, approx. 0.02-0.1%, approx. 0.03-0.1%, approx. 0.04-0 .1%, approx. 0.05-0.1%, approx. 0.001-0.075%, approx. 0.005-0.075%, approx. 0.01-0.075%, approx. 0.02-0.075%, approx. 0.03-0.075%, approx. 0.04-0.075%, approx. 0.05-0.075%, approx. 0.001-0.07%, approx. 0.005-0.07%, approx. 0.01-0.07%, approx. 0.02-0.07%, approx. 0.03-0.07%, approx. 0.04-0.07%, approx. 0.05-0.07%, approx. 0.001-0.06%, approx. The concentration of the sucrose ionizing agent may be, but is not limited to, about 0.05-0.06%, about 0.01-0.06%, about 0.02-0.06%, about 0.03-0.06%, about 0.04-0.06%, about 0.05-0.06%, about 0.001-0.05%, about 0.005-0.05%, about 0.01-0.05%, about 0.02-0.05%, about 0.03-0.05%, about 0.04-0.05%, about 0.01%, about 0.03%, about 0.05%, about 0.07%, about 0.075%, or about 0.1%.

[0069] In one embodiment of the present invention, the liquid dosage form may have a concentration of about 30-39 mM, about 31-39 mM, about 32-39 mM, about 33-39 mM, about 34-39 mM, about 34-39 mM, about 35-39 mM, about 36-39 mM, about 37-39 mM, about 30-38.5 mM, about 31-38.5 mM, about 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 a concentration of 2 to 37.3 mM, about 33 to 37.3 mM, about 34 to 37.3 mM, about 34 to 37.3 mM, about 35 to 37.3 mM, about 36 to 37.3 mM, about 37 to 37.3 mM, about 36 mM, about 37 mM, about 37.3 mM, about 37.5 mM, about 38 mM, or about 39 mM.

[0070] In one embodiment of the present invention, the liquid dosage form contains about 0.8-1.5%, about 0.85-1.5%, about 0.9-1.5%, about 0.95-1.5%, about 0.97-1.5%, about 0.975-1.5%, about 0.98-1.5%, about 0.8-1.25%, about 0.85-1.25%, about 0.9-1.25%, about 0.95-1.25%, about 0.97-1.25%, about 0.975-1.25%, about 0.98-1.25%, about 0.8-1.2%, about 0.85-1.2%, about 0.9-1.2%, about 0.95-1.2%, about 0.97-1.25%, about 0.975-1.25%, about 0.98-1.25%, about 0.8-1.25%, about 0.85-1.2%, about 0.9-1.2%, about 0.95-1.2%, about 0.97-1.25 ...8-1.25%, about 0.8-1.2 The concentration of the sucrose ionizing agent may be, but is not limited to, about 1.2%, about 0.975 to 1.2%, about 0.98 to 1.2%, about 0.8 to 1.1%, about 0.85 to 1.1%, about 0.9 to 1.1%, about 0.95 to 1.1%, about 0.97 to 1.1%, about 0.975 to 1.1%, about 0.98 to 1.1%, about 0.8 to 1.0%, about 0.85 to 1.0%, about 0.9 to 1.0%, about 0.95 to 1.0%, about 0.97 to 1.0%, about 0.975 to 1.0%, about 0.98 to 1.0%, about 0.8%, about 0.9%, about 0.95%, or about 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 designated as dosage form D.

[0073] 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, 94-100, 95-100, 95-100, or 96-100 after storage at 2-8°C for at least 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 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 after storage at 15-30°C for at least 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, or 9 months. The storage temperature is 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 The temperature may be, but is not limited to, 1 to 27°C, 22 to 27°C, 23 to 27°C, 24 to 27°C, 25 to 27°C, 15 to 26°C, 16 to 26°C, 17 to 26°C, 18 to 26°C, 19 to 26°C, 20 to 26°C, 21 to 26°C, 22 to 26°C, 23 to 26°C, 24 to 26°C, 25 to 26°C, 15 to 25°C, 16 to 25°C, 17 to 25°C, 18 to 25°C, 19 to 25°C, 20 to 25°C, 21 to 25°C, 22 to 25°C, 23 to 25°C, 24 to 25°C, or 25°C.

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

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

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

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

[0079] The storage temperature is 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 The temperature may be, but is not limited to, 1 to 27°C, 22 to 27°C, 23 to 27°C, 24 to 27°C, 25 to 27°C, 15 to 26°C, 16 to 26°C, 17 to 26°C, 18 to 26°C, 19 to 26°C, 20 to 26°C, 21 to 26°C, 22 to 26°C, 23 to 26°C, 24 to 26°C, 25 to 26°C, 15 to 25°C, 16 to 25°C, 17 to 25°C, 18 to 25°C, 19 to 25°C, 20 to 25°C, 21 to 25°C, 22 to 25°C, 23 to 25°C, 24 to 25°C, or 25°C.

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

[0081] The liquid dosage forms according to one aspect of the present invention may be administered to a mammalian subject to treat a variety of diseases. The liquid dosage forms of the present invention may be administered by a variety of delivery methods, for example, orally or parenterally, including intravenous, intramuscular, intraendocardial, intramyocardial, intrapericardial, intraventricular, intraarticular, intradermal, intracerebral, intrarenal, intrahepatic, intrasplenic, etc. The compositions may be administered intraperitoneally, intralymphatic, subcutaneously, intraabdominal, intratesticular, intraovarian, intrauterine, sternal, intratracheal, intrathoracic, intradural, intraspinal, intramedullary, intramural, intrachorionic and intraarterial injection or infusion, or topically via rectal, intranasal, inhalation or intraocular routes. In certain embodiments, the method of delivery 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 affected site. In some embodiments, the polynucleotide construct is administered into the muscles of the limbs, heart, or other body parts of the subject.

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

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

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

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

[0087] In various divided dose embodiments, the total dose is administered into 4, 8, 16, 24 or 32 separate injection sites.

[0088] In some embodiments, the injection dose is 0.1-5 mg, hi certain embodiments, the injection 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 administered dose may be administered in one visit or over two or more visits.

[0090] In typical split dose embodiments, all multiple injection doses are administered within 1 hour of each other. In some embodiments, all multiple injection doses are administered within 1.5, 2, 2.5, or 3 hours of each other.

[0091] In various embodiments of this method, the total dose of the polynucleotide construct, whether administered in a single unitary dose or divided into multiple injection doses, is administered to the subject only once.

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

[0093] In some embodiments, the cycle may be repeated. The cycle may be repeated 2, 3, 4, 5, 6 or more times.

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

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

[0096] In currently preferred embodiments, the nucleic acid construct is administered at a dose of 8 mg per affected limb, equally divided into multiple intramuscular injections and multiple visits, with each of the multiple injections at any single visit being at a separate injection site. In certain embodiments, the nucleic acid construct is administered at 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 are equally divided into multiple injection doses.

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

[0098] The plasmid DNA may be administered alone or in combination with other plasmid DNAs, either simultaneously or sequentially depending on the condition to be treated.

[0099] In some embodiments, the liquid pharmaceutical composition comprises a plasmid DNA encoding human HGF. The liquid pharmaceutical composition may be administered to treat a variety of diseases, including diseases previously demonstrated to be treatable by administration of plasmid DNA. The plasmid DNA may encode a therapeutic gene, such as human HGF. The diseases include, but are not limited to, ischemic or liver 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, neuropathies may be induced by diabetes, vitamin deficiencies, autoimmune diseases, genetic or hereditary disorders, amyloidosis, uremia, toxins or poisons, trauma or injury, tumors, or may be idiopathic. All references provided herein are hereby incorporated by reference in their entirety.

[0100] (blank) Effect of the Invention

[0101] The liquid dosage form containing the 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 therefore be usefully used as a pharmaceutical composition with excellent product stability and safety.

[0102] (blank) [Brief description of the drawings]

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

[0104] [Figure 2A] FIG. 1 is a diagram showing the results of a statistical analysis of the effects of each factor 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] FIG. 1 is a diagram showing the results of a statistical analysis of the effects of each factor 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] FIG. 2 is a diagram showing the composition derived as a result of analysis using statistical processing to derive the optimal liquid dosage form of the present invention. [Figure 3B] FIG. 2 is a diagram showing the composition derived as a result of analysis using statistical processing to derive the optimal liquid dosage form of the present invention.

[0106] [Figure 4] FIG. 1 shows the results of a statistical analysis conducted to derive an optimal liquid formulation for improving the long-term stability of the plasmid DNA of the present invention. [Diagram 5] FIG. 1 shows the results of a statistical analysis conducted to derive an optimal liquid formulation for improving the long-term stability of the plasmid DNA of the present invention.

[0107] [Figure 6] FIG. 10 is a diagram comparing the stability of plasmid DNA when the liquid dosage form of the present invention is prepared as a vial and a pre-filled syringe in order to confirm whether there is a difference in storage stability depending on the unit dosage form.

[0108] (blank) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0109] (blank) EXAMPLES

[0110] (blank)

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

[0112] (blank)

[0113] [Table 1] [Table 1]

[0114] Example 1. Development of plasmid DNA liquid dosage form A

[0115] The present inventors screened the 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 the five screenings were all the same. In this example, the 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, and the rubber stoppers were sterilized by a vendor (West-DAIKYO SEIKO) and immediately used in the test after opening.

[0116] (blank)

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

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

[0119] In the case of 1× buffer, the composition was the same as that of the final formulation.

[0120] This test was the manufacture of a small-scale buffer (100 mL), and taking into account the manufacturing error of an actual commercial production process, the 1X and 2X buffers were manufactured with a manufacturing error range of 2%.

[0121] The API used in this test was a VM202 DS (lot no. 88084) sample produced by Company A. It was transferred from a -70°C deep freezer to a 2-8°C refrigerator and thawed for 20-24 hours 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 formulations used in the first screening [Table 3]

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

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

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

[0130] The fourth screen was a duplicated experiment, so P40 ctrl was produced and analyzed in duplicate.

[0131] [Table 7] List of formulations used in five screening rounds (long-term stability studies) [Table 7]

[0132] Screening analysis method

[0133] Since high temperature conditions accelerate pDNA SC% decomposition, in this example, the liquid DP vials were stored in a 70°C oven, and samples were taken on days 0, 1, 3, and 5, and the SC% (using HPLC) was analyzed. The analytical samples were stored in a -70°C deep freezer immediately after sampling, and thawed in a 2-8°C cold room 1 hour before sample analysis, and then HPLC analysis was performed.

[0134] To save sample analysis time and costs, if a sample had a pDNA SC% below 10%, analysis was not performed on any specimens sampled after that sample.

[0135] (blank)

[0136] Long-term Stability Study

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

[0138] To keep sampling conditions constant, only a maximum of four samples were taken per vial (5 ml filled) and discarded after the final pH measurement.

[0139] All other conditions were the same as those for the existing first to fourth screening rounds.

[0140] (blank)

[0141] Screening analysis results

[0142] Example 1-1. First Screening (Buffering Agent, pH Screening)

[0143] The present inventors attempted to determine the type of buffer and pH range that would enable stable maintenance of VM202pDNA as a starting material.

[0144] First, to confirm the effects of various buffers, potassium phosphate, Tris, histidine, and sodium citrate were used as buffers.

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

[0146] (blank)

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

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

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

[0150] Specifically, the SC% was maintained at 30% or more for 3 days in PC8.0 and PD8.0, and the SC% was maintained at 60% or more on the first day in PC7.4 and PD7.4, but was approximately 10% on the third day, confirming decreased SC% stability compared to PC8.0 and PD8.0.

[0151] Based on the above results, it was confirmed that when the same buffer was used, the higher the pH, the better the stability of SC% was maintained.

[0152] It was also confirmed that the higher the buffer strength, the longer the pH was maintained during manufacturing. (See PC8.0, PD8.0, TC8.0, TD8.0 results.) Therefore, it was confirmed that, for the same buffer, the higher the pH and buffer strength, the higher the stability of SC%.

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

[0154] To summarize the above results, it was determined that the most suitable buffer for the VM202 liquid formulation was potassium phosphate buffer at pH 8.0.

[0155] (blank)

[0156] Example 1-2. Second Screening (Excipient Screening)

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

[0158] (blank)

[0159] The basic composition was potassium phosphate, pH 8.0, which was the optimal buffer identified in the first screening above.

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

[0161] (blank)

[0162] The results of the SC% (using HPLC) analysis of the second screening were as 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 among PMS0.1, PMS0.5, PMS1, and PMS2. In addition, the SC% on day 1 was maintained at 70% or more in both PN45 and P20 / MS, but the SC% on day 3 showed an increase of 30% or more in stability in PN45 compared to P20 / MS.

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

[0166] In addition, since the Day 3 SC% of PN45 was about 30% higher than that of P20 / MS, it was determined that the buffer strength had a greater effect on the SC% stability of pDNA than the content of mannitol and sucrose (see PN45, P20 / MS pDNA SC% results).

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

[0168] (blank)

[0169] Example 1-3. Third Screening (Salt Screening)

[0170] Based on a reference that states that when pDNA depurination occurs, salt neutralizes the negative charge of the phosphate group of pDNA to stabilize it, the present inventors attempted to find 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, the basic composition was determined to be 40 mM potassium phosphate, 0.45% NaCl, pH 8.0.

[0172] Based on academic literature that salts such as NaCl and MgCl2 affect pDNA stability, the inventors performed experiments with various concentrations of NaCl and MgCl2.

[0173] Finally, the salt concentration range was set taking into consideration the osmolality that can be injected into the intramuscular injection of VM202 (ideally 280-360mOsm / kg, preferably <600mOsm / kg).

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

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

[0176] VM202 pDNA confirmed that the SC% stability of pDNA increased as the NaCl concentration increased (see PN45, PN9 results).

[0177] It was also confirmed that the SC% stability of pDNA was significantly increased when the NaCl concentration was 0.9%.

[0178] In the case of MgCl2, when NaCl was 0.45%, the SC% was increased by about 10%, but it was confirmed that over time, MgCl2 reacted with water and was precipitated as Mg(OH)2.

[0179] It was confirmed that the maximum solubility of MgCl2 in 40 mM potassium phosphate and 0.9% NaCl was 0.01%, and any amount greater than this would result in precipitation.

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

[0181] Based on the above results, it was confirmed that the higher the NaCl concentration, the greater the increase in SC% stability. (See PN45 and PN9 results)

[0182] In addition, MgCl2 was precipitated as Mg(OH)2 at a certain concentration (PN45M) or higher in aqueous solution. This is because the conditions used in this round were high pH, ​​pH 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 Mg(OH)2 could not improve the SC% safety of pDNA at salt concentrations of PN45M or higher.

[0183] To summarize the above results, when the salt was added alone at 0.9% NaCl, the SC% stability of pDNA was highest (Day 5 SC%: 44%), and based on the results up to the third screening, the optimal composition for the liquid formulation of VM202 pDNA (Company A) was determined to be 40 mM potassium phosphate, 0.9% NaCl.

[0184] (blank)

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

[0186] The inventors attempted to confirm whether pDNA SC% was more stable when two types of buffers, potassium phosphate and Tris (double buffer), were used compared to when a single buffer, potassium phosphate, was used. Based on the fact that the higher the pH in the first to third screenings, the more stabilized the pDNA was, the inventors attempted to confirm whether pDNA would be more stabilized after upward adjustment of the pH using two types of buffers (double buffer).

[0187] (blank)

[0188] First, two types of buffers, a phosphate buffer and a Tris buffer, which were previously used, were mixed in different ratios to prepare pDNA in a liquid formulation.

[0189] Based on the results of the first screening, which showed that the higher the pH, the more stable the pDNA was, we attempted to test the pDNA stability by mixing phosphate and Tris to produce 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 of the fourth screening were as shown in Table 11 below.

[0193] [Table 11] 4th screening result (double buffer screening) [Table 11]

[0194] In an experiment in which the pH was fixed at 8.0 and the phosphate and Tris ratio was varied, it was confirmed that the higher the phosphate ratio, the more stable the pDNA. (See P / T 3:1 and P / T 1:3 results.) In an experiment in which the phosphate and Tris ratio was fixed and the pH was varied, it was confirmed that the higher the pH, the more stable the pDNA.

[0195] Finally, when double buffers were used, pDNA stability at a 1:1 phosphate to Tris ratio, pH 8.7, showed similar results to those at 40 mM phosphate, pH 8.0.

[0196] (blank)

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

[0198] It was found that at the same ratio of phosphate to Tris, the higher the pH, the more stable the pDNA.

[0199] Therefore, in the case of the double buffer formulation, it was confirmed that the higher the ratio of phosphate buffer and the higher the pH of the formulation, the higher the stability of pDNA.

[0200] However, the SC% stability of P40 and P / T8.7 was found to be similar. This means that the effect of the double buffer is not significantly different from the existing single buffer formulation in terms of pDNA SC% stability. In addition, when using the double buffer, the osmolality increases because each buffer strength is used by 40mM.

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

[0202] (blank)

[0203] Example 1-5.5th Screening (Long-term Stability of Final Candidates)

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

[0205] (blank)

[0206] To confirm the saturation point of the buffer and salt in the selected 40 mM phosphate, 0.9% NaCl, pH 8.0 condition, a long-term stability test was conducted using an additional condition of 60 mM phosphate, 0.9% NaCl / 100 mM phosphate, 1.1% NaCl.

[0207] First, samples were taken at different times at the DP storage temperature of 2~8℃, accelerated conditions of 25℃, and stress conditions of 37℃.

[0208] To keep sampling conditions constant, only a maximum of four samples were taken per vial (5 ml filled), and the remaining samples were discarded after the final pH measurement.

[0209] The long-term stability study plan is shown in Table 12 below.

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

[0211] The results of SC% (using HPLC) analysis of five screening rounds are shown in Table 13 below and in FIG.

[0212] [Table 13] 5th screening (long-term stability of final candidate group) SC% results [Table 13]

[0213] - N / A means not analyzed.

[0214] It was confirmed that the compositions P40, P60 and P100 all had similar SC% after storage at the respective temperature conditions.

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

[0216] As predicted, this composition was determined to be stable for more than 18 months when stored at 2-8°C. In fact, it was confirmed that when stored at 2-8°C for 6 months, the SC% did not differ by more than 1% from the TO point.

[0217] It was confirmed that the SC% was maintained at 80% for approximately two months under the stressful condition of 37℃.

[0218] (blank)

[0219] From the above results, it was confirmed that the analysis results of SC% were similar in all conditions of P40, P60, and P100. Therefore, it was determined that the VM202 pDNA SC% stability of phosphate buffer strength and NaCl was maintained at its maximum at 40mM, 0.9%, pH 8.0, and that NaCl or pH stability had no further effect on SC%.

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

[0221] (blank)

[0222] Conclusion of Example 1

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

[0224] This example confirmed that VM202 pDNA stability was affected not only by pH but also by the type of buffer. MgCl2, which was expected to maintain DNA more stably than NaCl based on academic data, precipitated while forming Mg(OH)2 and did not contribute to DNA stability. This is because, contrary to academic data, VM202 formulation had a relatively high pH of pH 8.0 and high (OH) - This is believed to be due to the formation of Mg(OH)2 ions.

[0225] In addition, 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 of 40 mM, NaCl 0.9%, pH 8.0.

[0226] If necessary, the final derived composition will be further tested for storage stability at 2-8°C through long-term stability testing (18 months), and the composition range applicable to future commercial production will be identified based on the final derived composition.

[0227] (blank)

[0228] The composition finally derived by the fifth screening in this Example 1 is 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 at 25°C storage conditions (89.5% SC), and this dosage form is deemed stable for 18 months or more at 2-8°C storage conditions. Based on the final derived composition, we plan to determine the composition range 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 liquid dosage form composition derived from Example 1 was applied to VM202 DS produced by Company C, Company W, and Helixmith, it was confirmed that the SC% stability was different from that of VM202 DS produced by Company A. Therefore, this experiment was performed to expand the limits of the liquid dosage form that were limited to VM202 DS produced by Company A. The liquid dosage form applied to Company A's VM202 DS established in Example 1 was named Liquid Dosage Form A.

[0235] (blank)

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

[0237] Assuming that the basic properties of VM202 DS are the same, screening in this example was performed based on the composition of liquid dosage form A selected in Example 1 above.

[0238] (blank)

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

[0240] The stability goal for this screening was to maintain the DP Spec (SC% 85%) standard for more than 3 months at a storage condition of 20-25°C. This goal was set based on research showing that the higher the storage temperature, the faster the decomposition of the SC% of pDNA accelerates, and was selected on the assumption that if a product is stable for 3 months at 20-25°C, it will be stable for more than 18 months at the DP storage temperature of 2-8°C.

[0241] To shorten the development period, the SC% was analyzed after accelerating the rate of pDNA decomposition by exposing pDNA to 70°C for 5 days (accelerated conditions), and for long-term stability testing, samples were taken every 2 weeks (monthly for 2-8°C) after storage at 2-8°C, 20-25°C, and 37°C, and the SC% was measured for 3 months (3-year plan for 2-8°C).

[0242] (blank)

[0243] A total of four screenings in this example were performed in the same manner as in Example 1, including 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 the same manner as the actual production process, and stored in a 70°C dryer before use. The rubber stoppers were sterilized by the vendor (West-DAIKYO SEIKO) and used in the test immediately after opening.

[0245] Manufacturing of dosage forms

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

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

[0248] In the case of 1X buffer, the composition is the same as the final formulation.

[0249] In this example, a small-scale buffer (100 mL) was produced. Taking into account the manufacturing error of the actual commercial production process, the 1X and 2X buffers were produced with a manufacturing error range of 2%.

[0250] The API used in this example was VM202 DS (lot no. 88084) produced by company A, VM202 DS (lot no. 2017#0042U) produced by company C, VM202 DS produced by Helixmith itself, and VM202 DS (lot nos. DEV19-035, 049, 061, 068) produced by company W. The API was transferred from a -70°C deep freezer to a 2-8°C refrigerator and thawed for 20-24 hours 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 Production and Sampling Procedures [Table 15]

[0255] The dosage forms used in the first to fourth screenings are listed in Tables 16 to 21 below.

[0256] [Table 16] 1st Pre-screening List 1 (Excipients) [Table 16]

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

[0258] (blank)

[0259] [Table 17] 1st Pre-screening List 2 (Arginine) [Table 17]

[0260] [Table 18] 1st Pre-screening List 3 (Range) [Table 18]

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

[0262] [Table 20] Third long-term stability test and comparative dosage form list A [Table 20]

[0263] [Table 21] 4th scale-up procedure adjustment [Table 21]

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

[0265] (blank)

[0266] ●Analysis

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

[0268] The analytical samples were stored in a -70°C deep freezer immediately after sampling, and then thawed in a 2-8°C refrigerator for 1 hour before analysis, followed by HPLC analysis.

[0269] To save sample analysis time and costs, samples with pDNA SC% less than 10% were not analyzed for specimens sampled after the 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, and samples were taken at two-week intervals (one month at 2-8°C) and the SC% was analyzed.

[0273] The 2~8°C samples were stored in the HX refrigerator, the 25°C samples were stored at room temperature in laboratory No. 530, and the 37°C samples were stored in a 37°C incubator.

[0274] To keep sampling conditions constant, only five samples of a maximum of 200 ul per vial (filled with 5 ml) were taken and then discarded.

[0275] (blank)

[0276] ●Scale-up process adjustment study

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

[0278] In order to confirm whether the presence or absence of a filter process affects the SC% stability of pDNA in each liquid formulation A and C, experiments were conducted after BDP production with and without the use of a filter.

[0279] (blank)

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

[0281] Considering that the efficacy of the composition of liquid dosage form A 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 tried to confirm the type and concentration range of excipients that can increase the stability of SC% for VM202 pDNA produced at various production sites other than Company A by adding various excipients.

[0282] (blank)

[0283] Study Design

[0284] To confirm the effect of various excipients, mannitol, sucrose, sorbitol, trehalose, leucine, and arginine were treated at appropriate concentrations and the significant range was confirmed.

[0285] The concentration range of each excipient was selected based on a survey of FDA-approved components of biosimilars (Reference) and literature (Rev Bras Otorrinolaringol, 2006;72(3):400-6).

[0286] (blank)

[0287] ●Results

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

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

[0290] Osmolality was measured only in prescreen 1 to 3 to account for overlapping excipient concentrations.

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

[0292] [Table 23] 1st Pre-screening 2 (Arginine) Results [Table 23]

[0293] Pre-screening 2 was performed with a NaCl concentration of 0.38%. The purpose of pre-screening 2 was to confirm whether the increase in SC% stability of A005 confirmed in pre-screening 1 was due to an increase in pH caused by arginine or an effect of arginine itself.

[0294] To confirm this, the subjects were divided into a group in which arginine (0.0025%) was added to the existing excipient (Excipient) and a group in which no arginine was added, and the pH was adjusted to 8.0 for both groups (to eliminate the effect of increased pH) to confirm the effect of arginine itself in increasing the SC%. As a result, the addition of arginine did not have any effect on increasing the stability of the SC%.

[0295] (blank)

[0296] It was confirmed that the increase in SC% stability of A005 confirmed in prescreening 1 was due to an increase in pH, and that arginine itself had no effect. Therefore, arginine was excluded from the subsequent formulation composition.

[0297] [Table 24] 1st Pre-screening 3 (Range) Results [Table 24]

[0298] In pre-screening 3, the test was performed at pH 7.9 to confirm stability under more severe conditions. When compared to the control liquid dosage form A composition (Control), M025 showed the highest increase in SC%, and the remaining excipients also showed a significant increase in SC% stability.

[0299] (blank)

[0300] ● Consideration

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

[0302] In addition, we selected liquid dosage form B, which was made by adding only 0.25% mannitol to the existing liquid dosage form A, and attempted to compare it with the composition of liquid dosage form C, which was being developed using DoE.

[0303] The remaining excipients were also set to their concentration ranges (maximum 1.5%) by taking their saturation points according to the same principle. In the case of sorbitol and leucine, the concentrations were set to 0.05-0.25 and 0.1-0.5%, respectively, taking into consideration that the lower the concentration, the higher the SC%.

[0304] Furthermore, it was confirmed that pH buffering was more stably maintained in disaccharides such as sucrose and trehalose than in monosaccharides such as mannitol and sorbitol.

[0305] In summary, the above results confirmed the possibility of developing a new formulation that can be applied to VM202 DS produced at other production plants such as Company C and Company W by adding an additional excipient to the composition of the existing formulation A, which was limited to Company A's VM202 DS. In addition, we attempted to maximize the efficiency of the DoE experiment by setting the concentration range in which each excipient affects the SC% stability.

[0306] (blank)

[0307] Example 2-2.2nd Study Design (Design of Experiment, DoE)

[0308] Using the excipient candidate group and its concentration range selected in the first prescreening, we will select the optimal excipient composition for stabilizing VM202 pDNA through DoE.

[0309] (blank)

[0310] Study Design

[0311] Based on the pDNA stability enhancing effects of various excipients identified from the first excipient screening, pDNA stability was compared across a range of concentrations to identify the effects of mutual combinations and the main excipient factors.

[0312] (blank)

[0313] The DOE was randomized in design and performed with two replicates.

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

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

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

[0317] [Table 25] Second study design results [Table 25-1]

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

[0319] In the case of SR (sucrose) and LC (leucine), it was determined that the lower the concentration within the selected range, the greater the pDNA stability.

[0320] In the case of MT (mannitol), we determined that due to an interaction with pH, ​​pDNA stability was most increased when the MT concentration was low at high pH values.

[0321] As a result, a model equation was derived that embodied the effect of each factor on %SC, and is shown in Figure 2B.

[0322] Based on the above analysis, the optimum composition was derived and is shown in FIG. 3A and FIG. 3B.

[0323] (blank)

[0324] ● Consideration

[0325] As shown in Figure 3A, there are seven factors that affect %SC: KP, N, pH, MT, SR, TH, and LC. Using these, the optimal model was derived, and it was confirmed to be solution 1. By applying the model using solution 1 and statistically analyzing the spinning equation, it was estimated that the range in which the average value, which is the %SC statistical value, would be distributed was approximately 19.209 to 22.845 at a 95% confidence level. In addition, it was predicted that in the future, individual values ​​of %SC would be distributed in the range of 17.451 to 26.603 at a 95% confidence level.

[0326] From the above results, the composition with the highest SC% (solution 1) was selected from the three compositions derived from the data.

[0327] Furthermore, the present inventors determined the concentration or value of each factor in the direction that can maximize the y value, i.e., % SC, within the range of each factor (FIG. 3B).

[0328] In the case of sorbitol, the DOE results showed that sorbitol had a slight effect on the SC% stability of pDNA, and the effect was fixed at 0.15%, which was the intermediate value, in the DOE sorbitol condition.

[0329] [Table 25-2]

[0330] Example 2-3. Manufacture of liquid formulation C (Formulation C: potassium phosphate 40 mM, NaCl 0.9%, pH 8.0, mannitol 0.05%, sorbitol 0.15%, sucrose 0.3%, trehalose 1.5%, leucine 0.1%) and comparison of formulations 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] The aim of the study was to confirm the long-term pDNA stability of the liquid dosage forms B and C compositions selected through the previous screening, and to confirm the pDNA stability of the combination of the VM202 DS production facility and liquid dosage forms A, B, and C.

[0333] (blank)

[0334] Study Design

[0335] The first prescreening confirmed that mannitol had the greatest pDNA stabilizing effect as a single excipient.

[0336] Liquid dosage form B composition, which was the existing liquid dosage form A composition with only 0.25% mannitol added, and liquid dosage form C composition, developed through DoE, were applied to 2 lots of VM202 DS produced by HX and W companies, and compared with liquid dosage form A, which was the existing control condition.

[0337] In a long-term stability test at 70°C for liquid dosage forms A, B, and C, samples were taken on days 0, 1, 3, and 5 to confirm the SC%.

[0338] (blank)

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

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

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

[0342] ●Results

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

[0344] [Table 27] Comparison of formulations A, B, and C at 70℃ [Table 27]

[0345] [Table 28] Third long-term stability study (formulations B and C) [Table 28]

[0346] - In the case of the samples from day 1, 3, and 5 and the samples from 2 to 8°C, only some of the samples were analyzed. - If the sample could not be analyzed, it was marked as N / A.

[0347] (blank)

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

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

[0350] The long-term stability results at 25°C showed that liquid dosage form C had an average SC% of 79.4% over six months, while liquid dosage form B had 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, which has a composition developed by DoE, has less deviation between DS lots compared to liquid dosage form B, and maintains VM202 pDNA SC% more stably at room temperature (20~25℃).

[0354] In addition, in the case of liquid dosage form C, the SC% of 85% or more after storage for 3 months at 25°C, which was the target of the test, was met (average of 86.7% after storage for 3 months).

[0355] Therefore, through the third screening, we successfully developed liquid dosage form C, which was previously limited to VM202 DS produced by Company A, but is also applicable to VM202 DS produced by Companies HX and W.

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

[0357] (blank)

[0358] (blank)

[0359] Example 2-4. 4th scale-up procedure adjustment (scale-up A, C)

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

[0361] (blank)

[0362] Study Design

[0363] A liquid formulation procedure previously performed at a 20 mL scale was scaled up and adjusted to a 300 mL scale.

[0364] In order to confirm the effect of the filter process, 300 mL of the produced BDP was divided into 150 mL portions, 150 mL of which was filtered and 150 mL of which was filled into vials without filtration, and a stress test was conducted.

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

[0366] In the fourth scale-up procedure adjustment, 70°C stress test, samples were taken on days 0, 1, 3, and 5 to confirm the SC%.

[0367] (blank)

[0368] ●Results

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

[0370] [Table 29] 4th scale-up procedure adjustment [Table 29]

[0371] As can be seen from Table 29, in the case of liquid dosage form C, the SC% was maintained constant regardless of whether or not the filter process was used. In contrast, in the case of liquid dosage form A, when the filter process was used, the day 3 SC% was 8.9%, a clear difference of more than 30% in pDNA SC% compared to when there was no filter process (40.6%).

[0372] (blank)

[0373] ● Consideration

[0374] In actual production, a filter process is indispensable due to problems of bioburden or sterility, so it is necessary to develop a liquid dosage form that can be applied to the filter process.

[0375] This test confirmed that the SC% of liquid dosage form C remained stable even after the filter process was applied. (In contrast, it was confirmed that the SC% stability of liquid dosage form A could not be maintained after the filter process was applied.)

[0376] Therefore, through this embodiment, liquid dosage form C, which is a liquid dosage form that can be applied to practical manufacturing procedures, was successfully developed.

[0377] (blank)

[0378] Conclusion of Example 2

[0379] Through a total of four screening rounds, we successfully developed a VM202 liquid dosage form that was applicable to VM202 DS produced at various production sites (Rounds 1-2), met the target SC% stability of the test (Third round), and was finally applicable to the actual production process (Fourth round).

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

[0381] In addition, this example confirmed that the stability of VM202 pDNA differed depending on the manufacturing environment and lot, and that the presence or absence of a filter process also affected pDNA stability. Therefore, when introducing liquid dosage forms into actual production in the future, it is considered that the process by which VM202 DS was produced, the filter material introduced into production, or the process time may affect the stability of the final liquid DP.

[0382] The final derived composition, liquid dosage form C composition, will be further confirmed for storage stability at 2-8°C through long-term stability testing (36 months) if necessary, and the composition range applicable to future commercial production will be identified based on the final derived composition.

[0383] (blank)

[0384] The composition derived from a total of four tests is as 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 at 20-25°C storage conditions (86.7% SC), and this dosage form is deemed to be stable for more than 18 months at 2-8°C storage conditions.

[0387] Based on the final derived composition, we plan to develop a composition applicable to commercial production in the future.

[0388] (blank)

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

[0390] It was confirmed that when the liquid dosage forms B and C compositions derived from Examples 1 and 2 were applied to VM202 DS produced by W Company, Helixmith, a liquid dosage form having a comparable level of stability to the lyophilized dosage form was developed.

[0391] In this example, a test was conducted to derive the most stable and optimal composition for VM202 using the RSM (response surface design) method for the composition of liquid dosage form C, and the optimal liquid dosage form derived by RSM was named liquid dosage form D.

[0392] (blank)

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

[0394] This study, which was conducted by improving the composition of liquid dosage form C, confirmed the main effects, interactions, and curvature effects between the factors of each composition, and the optimal composition was derived and applied through additional improvements in order to carry out this test.

[0395] In the previous example, liquid dosage form C was selected in which each excipient had an optimal concentration through DoE, and the composition was further improved taking into consideration the inter-factor reactions identified from liquid dosage form C.

[0396] The stability goal of this test is to maintain the DP Spec (SC% 85%) standard for more than 3 months at a storage condition of 20-25°C. This goal was set based on research showing that the higher the storage temperature, the faster the decomposition of SC% of pDNA accelerates, and was selected on the assumption that if it is stable for 3 months at 20-25°C, it will be stable for more than 18 months at the DP storage temperature of 2-8°C.

[0397] A long-term stability test was conducted on liquid dosage form D, which has an improved composition, using liquid dosage form C as a control. Samples were stored at 2-8°C every 4 weeks, 20-25°C, and 37°C, and then sampled every 2 weeks to measure the SC% for 3 months (2-8°C was a 3-year plan).

[0398] The test workflow was as shown in Table 31 below.

[0399] [Table 31] Test sequence [Table 31]

[0400] Test Overview

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

[0402] The vials used in this test were washed according to the vial washing protocol up to 2 days before BDP production, in the same manner as the actual production process, and stored in a 70°C dryer before use. The rubber stoppers were sterilized by the vendor (West-DAIKYO SEIKO) and used in the test immediately after opening.

[0403] (blank)

[0404] ●Manufacturing

[0405] The 1X and 2X buffers used in the tests were prepared up to 3 days before the start of the next BDP production for each test.

[0406] In the case of 2X buffer, the buffer strength, mannitol, sucrose, sorbitol, trehalose, leucine, and arginine concentrations were made to be 2X. However, the NaCl concentration was calculated based on the NaCl concentration (A) in the final composition by substituting it into the following formula: *(A×2)-0.9=NaCl concentration in 2X buffer

[0407] In the case of 1X buffer, the composition is the same as the final formulation.

[0408] This test was carried out on a small scale (100 mL) of buffer, and taking into account the manufacturing error of the actual commercial production process, the 1X and 2X buffers were carried out with a manufacturing error range of 2%.

[0409] The API samples used in the test were VM202 DS (lots: 8th and 9th) produced by Company A, Company C, Helixmith itself, and VM202 DS (Company W 1: 1912063, Company W 2: 1912073) produced by Company W. The samples were transferred from a -70°C deep freezer to a 2-8°C refrigerator and thawed for 16-18 hours 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 analytical procedures were as shown in Table 32 below.

[0412] [Table 32] BDP Production and Sampling Procedures [Table 32]

[0413] The list of RSM formulations used in the first screening was as shown in Table 33 below.

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

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

[0416] [Table 34] Second Long-Term Stability Dosage Form List [Table 34]

[0417] ●Analysis

[0418] Since high temperature accelerates pDNA SC% degradation, the liquid DP vials were stored in a 70°C oven, and samples were taken on days 0 and 5 and analyzed for SC% (using HPLC). The analytical samples were stored in a -70°C deep freezer immediately after sampling, and thawed in a 2-8°C refrigerator for 1 hour before analysis, and then analyzed by HPLC.

[0419] (blank)

[0420] Long-term stability test

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

[0422] The 2-8°C samples were stored statically in the HX refrigerator, the 25°C samples were stored statically at room temperature in Laboratory No. 530, and the 37°C samples were stored statically in a 37°C incubator.

[0423] To keep sampling conditions constant, only 10 samples were performed with a maximum of 100 ul per vial (5 ml filled) and then discarded.

[0424] (blank)

[0425] (blank)

[0426] Test results and discussion

[0427] Example 3-1: First test design (RSM; Response Surface Design)

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

[0429] (blank)

[0430] Study Design

[0431] The equation for the final composition of liquid dosage form C derived in Examples 1 and 2 was established, and the degree to which each factor affects stability was confirmed.

[0432] Using the obtained data, the final composition was improved taking into consideration the interactions between each factor that can be confirmed during the RSM design, and the combination of applicable factors in terms of pDNA stability, i.e., the optimal composition (best case), was obtained.

[0433] The optimum composition goals to be identified by DOE are as follows:

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

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

[0436] [Table 35] First study design (RSM) results [Table 35]

[0437] ●Results

[0438] The above result data analyzed by statistical processing are shown in FIG. 4 and FIG.

[0439] (blank)

[0440] ● Consideration

[0441] Figure 4 shows the effect of each of the factors (e.g., KP, NaCl, pH, etc.) used in pDNA preparation on %SC. Referring to Figure 4, it was found that the remaining factors other than SB have a certain degree of effect on %SC. Among these, the factors with the strongest effect could be considered to be potassium phosphate (KP), sodium chloride (N), and leucine (LC) when considering the degree of visual evaluation. Therefore, these three factors were used for optimizing the dosage form by RSM design, and the remaining excipients other than the three factors were made to have the same composition as liquid dosage form C, since the optimal composition was secured by the liquid dosage form C DOE in the previous study.

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

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

[0444] The composition of the final liquid dosage form D was as follows:

[0445] Liquid formulation D: Potassium phosphate 37 mM, NaCl 1.0%, pH 8.0, mannitol 0.05%, sorbitol 0.15%, sucrose 0.3%, trehalose 1.5%, leucine 0.05%

[0446] (blank)

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

[0448] Test Overview

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

[0450] (blank)

[0451] Study Design

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

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

[0454] The DS used were 2 lots of HX DS and 2 lots of W company DS, for a total of 4 lots.

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

[0456] (blank)

[0457] [Table 36] Second long-term stability test plan (formulations D and C) [Table 36]

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

[0459] Test results

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

[0461] [Table 37] Second long-term stability test results (formulations D and C) [Table 37]

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

[0463] [Table 38] Second long-term stability test results (average %SC) [Table 38]

[0464] As can be seen from Table 38, liquid dosage form C showed an average SC stability of 84.1% at 20-25°C after 24 weeks, whereas liquid dosage form D showed an average SC stability of 89.8% at 20-25°C after 24 weeks. This also confirmed that liquid dosage form C showed an average SC stability of 66.4% at 37°C after 12 weeks, whereas liquid dosage form D showed an average SC stability of 71.9% at 37°C after 12 weeks.

[0465] (blank)

[0466] ● Consideration

[0467] Analysis based on the above results confirmed that liquid dosage form D, which is a composition obtained by improving liquid dosage form C using RSM, has a 5.9% increased SC stability at room temperature (20-25°C) compared to liquid dosage form C, and a 5.5% increased SC stability at accelerated conditions of 37°C.

[0468] In addition, in the case of liquid dosage form C, the condition of SC% of 85% or more was met (89.4% SC) after 3 months of storage at 25℃, which was the target of the test, and liquid dosage form D was confirmed to have an even higher SC stability of 92.8%, ensuring an improved final composition (average % after 3 months of storage).

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

[0470] (blank)

[0471] Conclusion of Example 3

[0472] This Example 3 confirmed that liquid dosage form D, which is the final composition derived from the perspective of pDNA stability, is a composition that can maintain pDNA much more stably under accelerated conditions of 25°C and harsh conditions of 37°C, compared to liquid dosage form C confirmed in a previous study.

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

[0474] It was confirmed that the liquid dosage form C, which is the composition before the improvement, is applicable to VM202 DS produced at various production sites, meets the target SC% stability of the test, and is applicable to the actual production process. Therefore, it was confirmed that the liquid dosage form D composition, which was developed by improving this composition, also shows the same effect while showing higher stability in terms of stability.

[0475] This was confirmed to be an optimal combination that could enhance the safety of pDNA through synergy between the liquid formulation components, and it is predicted that it can be maintained more stably for up to two years or more under refrigerated conditions, which is the DP storage temperature.

[0476] (blank)

[0477] (blank)

[0478] Example 4: Long-term stability test of prefilled syringe dosage form

[0479] The present inventors conducted a test on the long-term stability of the liquid dosage forms C and D developed in Examples 1 to 3 when they were prepared into pre-filled syringe dosage forms.

[0480] Liquid dosage forms C and D containing plasmid DNA at 0.5 mg / ml were manufactured in vials and prefilled syringes, respectively, and the SC% was measured over a 3-month period at 2-8°C by sampling 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 FIG. 6, both the vial and the prefilled syringe maintained an SC% of approximately 95% or more even after storage at 2 to 8°C for more than 6 months (24 weeks), indicating that there is no difference in storage stability even when prepared in the form of a prefilled syringe.

[0483] (blank)

[0484] Example 5: Stability test in dosage forms C and D using various plasmids

[0485] Experiments were conducted to confirm whether the liquid dosage forms C and D developed in Examples 2 and 3 show the same stability in other plasmids other than VM202. 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 previous examples. The liquid dosage forms C and D of the three types of 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, it was confirmed that all three types of plasmids exhibited high stability in the C or D liquid dosage form. In particular, in the case of pCK-SDF-1a, the stability was significantly reduced even when exposed to the initial composition of 0.9% NaCl for a short period of time (SC% rapidly decreased to 80%), but it was found that it was maintained very stably in the C or D dosage form for 4 weeks without a significant decrease in SC%. This demonstrated that the C or D liquid dosage form, especially the D liquid dosage form, is a dosage form that can stably maintain even very unstable plasmid DNA.

[0489] (blank)

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

[0491] The three types of plasmids used in Example 5, pCK-SDF-1a (WO2016 / 048105A1), pTx-IGF-1X10 (WO2020 / 016655A2), and pCMV3-cMet-flag (Sino Biological Inc., China), were prepared in prefilled syringes using liquid formulation D and their stability after storage for 4 weeks was measured in terms of SC%.

[0492] The results are shown in Table 41.

[0493] [Table 41] [Table 41]

[0494] As shown in Table 41, similar to the results of Example 5, it was confirmed that all three types of plasmids were very stable even when the D formulation was stored for 4 weeks in a prefilled syringe formulation.

Claims

1. A liquid formulation of plasmid DNA comprising 30-50 mM potassium phosphate, 0.5-1.5% NaCl, and plasmid DNA, with a pH of 7.5-8.

5.

2. 2. The liquid formulation of claim 1, wherein the liquid formulation comprises 40 mM potassium phosphate.

3. 2. The liquid formulation of claim 1, wherein the liquid formulation contains 0.9% NaCl.

4. 2. The liquid dosage form of plasmid DNA of claim 1, wherein the pH of the liquid dosage form is 8.

0.

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

6. 10. The liquid dosage form of claim 1, wherein the liquid dosage form further comprises sucrose, trehalose, mannitol, sorbitol, leucine, or a combination thereof.

7. 2. The liquid dosage form of plasmid DNA of claim 1, wherein the 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.

8. 2. The liquid formulation of plasmid DNA according to claim 1, wherein the plasmid DNA is present at a concentration of 0.01 to 5 mg / mL.

9. 2. The liquid dosage form of plasmid DNA of claim 1, wherein the liquid dosage form further comprises 0.15-1.5% mannitol.

10. 10. The liquid dosage form of plasmid DNA of claim 9, wherein the plasmid DNA in the liquid dosage form maintains 90-100 SC% even after storage at 2-8°C for at least 6 months.

11. 10. The liquid dosage form of plasmid DNA of claim 9, wherein the plasmid DNA in the liquid dosage form maintains 60-100 SC% after storage at 15-30° C. for at least 3 months, 4 months, 5 months, or 6 months.

12. 2. The liquid dosage form of plasmid DNA of claim 1, further comprising 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.

13. 13. The liquid dosage form of plasmid DNA of claim 12, wherein the plasmid DNA in the liquid dosage form maintains 90-100 SC% after storage at 2-8° C. for at least 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, or 9 months.

14. 13. The liquid dosage form of plasmid DNA of claim 12, wherein the plasmid DNA in the liquid dosage form maintains 80-100 SC% after storage at 20-30° C. for at least 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, or 9 months.

15. 2. The liquid dosage form of plasmid DNA of claim 1, wherein the 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.

16. 16. The liquid formulation of claim 15, wherein the liquid formulation comprises 37-37.3 mM potassium phosphate.

17. 16. The liquid formulation of claim 15, wherein the liquid formulation comprises 0.975-1.0% NaCl.

18. 16. The liquid dosage form of plasmid DNA of claim 15, wherein the plasmid DNA in the liquid dosage form maintains 90-100 SC% after at least 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, or 9 months of storage at 2-8°C.

19. 16. The liquid dosage form of plasmid DNA of claim 15, wherein the plasmid DNA in the liquid dosage form maintains 80-100 SC% after storage at 20-30° C. for at least 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, or 9 months.

20. The liquid dosage form of plasmid DNA according to any one of claims 1 to 19, wherein the liquid dosage form is prepared as a vial, an ampoule, a bottle, or a pre-filled syringe.

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