CN114903922B - Pharmaceutical formulations comprising adenoviruses and methods of preserving same - Google Patents

Abstract

The application discloses a pharmaceutical preparation containing adenovirus and a preservation method thereof. The pharmaceutical preparation containing adenovirus adopts a citric acid buffer system, combines a nonionic surfactant and small-molecule saccharides with the C atom number less than or equal to 24 or sugar alcohols with the C atom number less than or equal to 24, and realizes the stability of the preparation. The adenovirus-containing pharmaceutical preparation can be prepared into a stable liquid preparation, the osmotic pressure is in a reasonable range, the injection requirement is met, the pH value is close to neutral, the adverse reaction probability of an organism can be effectively reduced, the raw materials are simple, and the safety is good.

Description

Pharmaceutical preparation containing adenovirus and method for preserving the same

Technical Field

The present application relates to the field of biomedicine technology, and in particular to a pharmaceutical preparation containing adenovirus and a method for preserving the same.

Background technique

Vaccines are auto-immune preparations for the prevention or treatment of infectious diseases that are made by artificially attenuating, inactivating or genetically modifying pathogenic microorganisms (such as bacteria, rickettsia, viruses, etc.) and their metabolites. They are the most common type of biological preparations. The common routes of administration of vaccines are oral and injection. Due to the molecular activity requirements of vaccines, current vaccine research is mainly focused on injections. Common vaccine injections are small water injections or freeze-dried powder injections. Freeze-dried preparations have become the basic dosage form of biological products due to their high stability, but their process is complex, the cycle is long and the cost is high. Although liquid preparations are more difficult to develop, they have simple processes, short cycles, avoid freeze-dried titer loss and are low in cost.

Adenovirus vectors are one of the most promising viral vectors and are widely used in various preclinical and clinical studies including vaccine development, gene therapy and oncolytic therapy. The biological activity of adenovirus depends on the integrity of the conformation of at least one core sequence of nucleotides surrounded by an icosahedral shell structure, which is composed of capsid proteins. Unlike traditional organic and inorganic drugs, adenovirus particles are very easy to degrade and have poor stability. Therefore, in order to ensure a reasonable shelf life, a good formulation of adenovirus preparations is crucial. Adenovirus research also focuses on adenovirus preparations intended for human administration. Such adenovirus preparations should not only be safe, sterile and of good manufacturing practice (GMP) grade, but these preparations should also show and promote the long-term stability of adenovirus, minimizing the loss of adenovirus titer during manufacturing, packaging and storage. The preparation should also further prevent adenovirus from adsorbing to the surface of the container for packaging and storing adenovirus and the mechanical surface used in the manufacturing process. Although a lot of research has been invested in the design of adenovirus preparations, there is still a demand for improved adenovirus preparations.

Summary of the invention

The present application provides a pharmaceutical preparation containing adenovirus, which can achieve long-term storage of adenovirus preparations, minimize the loss of adenovirus titer, and advantageously prevent the formation of visible particles and prevent the adsorption of adenovirus to the surfaces of packaging and storage containers and mechanical surfaces used in the manufacturing process.

The present application provides a pharmaceutical preparation, which comprises, based on the total volume of the pharmaceutical preparation:

(a) recombinant adenovirus;

(b) Citrate buffer 5 mM to 400 mM;

(c) nonionic surfactant (v/v) 0.01% to 1%; and

(d) protective agent (w/v) 4% to 15%;

Wherein, the protective agent is a combination of one or more of a carbohydrate compound with a carbon number of ≤24 or a sugar alcohol with a carbon number of ≤24;

The pH value of the pharmaceutical formulation is 5.0 to 7.0.

In some embodiments of the present application, the pharmaceutical preparation is a liquid preparation.

In some embodiments of the present application, the pharmaceutical preparation further comprises NaCl, and the concentration (w/v) of the NaCl is ≤35%, for example, the concentration (w/v) of the NaCl is 0.

In some embodiments of the present application, the concentration of the citrate buffer is 10 mM to 140 mM, such as 20 mM to 75 mM, such as 40 mM to 80 mM, 30 mM to 100 mM, or preferably 20 mM to 60 mM.

In some embodiments of the present application, the pH value of the pharmaceutical formulation is 5.5 to 6.8, for example, the pH value of the pharmaceutical formulation is in any range of 5.8 to 6.7, 6.0 to 6.8, or 6.2 to 6.7; for example, the pH value is in any range of 5.8 to 6.0, 6.0 to 6.2, 6.5 to 6.7, or 6.4 to 6.6.

In some embodiments of the present application, the nonionic surfactant is polysorbate, such as polysorbate 80 (Tween 80, PS80).

In some embodiments of the present application, in the pharmaceutical preparation, the concentration (v/v) of the nonionic surfactant is 0.05% to 0.5%, such as 0.08% to 0.2%, preferably 0.1%.

In some embodiments of the present application, the sugar compound is one or more of sucrose, trehalose or lactose.

In some embodiments of the present application, in the pharmaceutical formulation, the protective agent concentration (w/v) is 4.2% to 10%, for example, 4.4% to 7.5%, 4.4% to 7.2% or 4.8% to 7.5%.

In some embodiments of the present application, the pharmaceutical formulation further comprises glycine and/or mannitol.

In some embodiments of the present application, in the pharmaceutical preparation, the concentration (w/v) of glycine is 0.2% to 5%, for example, 0.5% to 1%; the concentration (w/v) of mannitol is 0.2% to 5%, for example, 0.5% to 1%.

In some embodiments of the present application, the pharmaceutical formulation comprises a recombinant adenovirus with a titer ranging from 7.0 lgIFU/mL to 12.0 lgIFU/mL; for example, a titer ranging from 7.0 lgIFU/mL to 11.0 lgIFU/mL, or a titer ranging from 8.0 lgIFU/mL to 11.0 lgIFU/mL, or a titer ranging from 8.0 lgIFU/mL to 10.0 lgIFU/mL.

In the recombinant adenovirus of the present invention, the adenovirus vector is derived from human adenovirus or non-human simian adenovirus. In one embodiment, the adenovirus vector of the present invention is derived from human adenovirus such as Ad1, Ad2, Ad4, Ad5, Ad6, Ad11, Ad 24, Ad34 or Ad35, in particular Ad5, Ad11 or Ad35.

In some embodiments of the present application, the recombinant adenovirus comprises an adenovirus vector derived from a non-human simian adenovirus, such as a chimpanzee adenovirus vector or a gorilla adenovirus vector.

In some embodiments of the present application, the adenoviral vector is a chimpanzee adenoviral vector, such as AdC68, ChAd3, ChAd63, ChAd83, ChAd155, Pan 5, Pan 6, Pan 7 or Pan 9. The adenoviral vector may also be derived from a non-human simian adenovirus isolated from a bonobo, such as PanAd1, PanAd2 or PanAd3.

In some embodiments of the present application, the recombinant adenovirus comprises a heterologous nucleic acid molecule.

In some embodiments of the present application, the recombinant adenovirus comprises at least one of the following heterologous nucleic acid molecules:

(A) a nucleic acid molecule encoding the PreS protein of SARS-CoV-2; or

(B) Nucleic acid molecule encoding the full-length S protein of the SARS-CoV-2 virus.

In a specific embodiment, the amino acid sequence of the PreS protein includes the amino acid sequence shown in SEQ ID NO.1, and the nucleic acid molecule encoding the PreS protein of SARS-CoV-2 includes the nucleotide sequence shown in SEQ ID NO.2;

In a specific embodiment, the amino acid sequence of the full-length S protein includes the amino acid sequence shown in SEQ ID NO.3, and the nucleic acid molecule encoding the full-length S protein of the SARS-CoV-2 virus includes the nucleotide sequence shown in SEQ ID NO.4.

In other embodiments of the present application, the heterologous nucleic acid molecule included in the recombinant adenovirus can be a gene encoding RSV antigen protein, a gene encoding VZV antigen protein, a gene encoding GFP protein, or a gene encoding IE63 protein.

In some embodiments of the present application, the pharmaceutical preparation is a nasal spray preparation, a nasal drop preparation, an aerosol inhalation preparation, an intramuscular injection preparation, a subcutaneous injection preparation, or an oral administration preparation.

In some embodiments of the present application, the pharmaceutical formulation consists of the following ingredients: recombinant adenovirus, 5 mM to 400 mM citrate buffer, 0.01% to 1% (v/v) of a nonionic surfactant, and 4% to 15% (w/v) of a protective agent.

In some embodiments of the present application, the pharmaceutical preparation is composed of the recombinant adenovirus, 5 mM to 400 mM citrate buffer, 0.01% to 1% (v/v) of the nonionic surfactant, 4% to 15% (w/v) of the protective agent, 0.2% to 5% (w/v) of glycine, and 0.2% to 5% (w/v) of mannitol. In a specific embodiment, the content of each component can be further preferred as described above.

The present application further provides a medical preparation, comprising, based on the total volume of the medical preparation, adenovirus, 20 mM to 60 mM citrate buffer, 4.4% to 7.2% (w/v) sucrose or trehalose, 1% (w/v) glycine, 1% (w/v) mannitol, and 0.1% (v/v) Tween 80, wherein the pH of the medical preparation is 5.8 to 6.7.

Accordingly, the present application also provides a method for preserving a pharmaceutical preparation containing adenovirus, comprising: storing the pharmaceutical preparation as described above at a temperature of -60°C to 37°C.

In one embodiment, the pharmaceutical formulation is stored at a temperature of 0° C. to 25° C. In another embodiment, the pharmaceutical formulation is stored at 2° C. to 8° C. In another embodiment, the pharmaceutical formulation is stored between -20° C. to -60° C.

Beneficial effects:

The adenovirus-containing pharmaceutical preparation provided by the present invention adopts a citrate buffer system, combined with low molecular weight sugars or sugar alcohols as protective agents, to achieve the stability of the preparation. The adenovirus-containing pharmaceutical preparation of the present invention can be prepared as a stable liquid preparation, the osmotic pressure is within a reasonable range, meets the injection requirements, and has good storage stability, can maintain a good virus infection rate under temperature conditions of 0 to 37°C, the virus particle size remains uniform and stable, there is no degradation or obvious aggregation, and the virus titer loss is effectively avoided. It can meet the storage and transportation requirements of the product, can greatly reduce costs, and achieve a breakthrough in the balance of stability and medicinal properties of adenovirus drug liquid preparations. In addition, the pH value of the adenovirus preparation provided by the present invention is close to neutral, the formula materials are safe and non-toxic, and the probability of adverse reactions in the subject's body is effectively reduced.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for describing the embodiments are briefly introduced below.

FIG1 is a particle size distribution (DLS) diagram of adenovirus preparation samples of groups B1 to B16 in Experimental Example 2 after being placed at 37° C. for 28 days; wherein the numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and 16 in the horizontal axis groups correspond to sample groups B1, B2, B3, B4, B5, B6, B7, B8, B9, B10, B11, B12, B13, B14, B15 and B16, respectively;

FIG2 is a particle size distribution (DLS) diagram of adenovirus preparation samples of groups C1 to C8 in Experimental Example 3 in a heat accelerated stability test at 37° C. and 42° C.;

FIG3 is the virus titer test results of adenovirus preparation samples of groups D3 and D4 in Experimental Example 4 after freezing and thawing 1 to 5 times;

FIG4 is a result of detecting the number of virus particles in adenovirus preparation samples of groups D3 and D4 after freezing and thawing 1 to 5 times in Experimental Example 4;

FIG5 is the test results of virus infection activity (VP/IFU specific activity) of adenovirus preparation samples of groups D3 and D4 in Experimental Example 4 after freezing and thawing 1 to 5 times;

FIG. 6 shows the particle size distribution of adenovirus preparation samples of groups D3 and D4 in Experimental Example 4 after being frozen and thawed 1 to 5 times.

Specific implementation plan

The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of this application.

The embodiments of the present application provide a pharmaceutical preparation including adenovirus. The following are described in detail. It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of the embodiments. Various embodiments of the present invention may be in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity, and should not be understood as a rigid limitation on the scope of the present invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values within the range. For example, it should be considered that the range description from 1 to 6 has specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the range, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated in this article, it is meant to include any cited numbers (fractions or integers) within the indicated range.

The adenovirus contained in the pharmaceutical preparation of the present invention is a recombinant adenovirus. In some embodiments, the pharmaceutical preparation comprises at least one recombinant adenovirus. The construction of the adenovirus vector in the recombinant adenovirus is well understood in the art based on the prior art and involves the use of standard molecular biology techniques. The adenovirus vector may be defective in at least one essential gene function in the E1 region (e.g., E1a region and/or E1b region) of the adenovirus genome, and the E1 region is essential for the replication of the virus. In some embodiments of the present invention, the construction of the adenovirus vector includes: constructing a replication-deficient adenovirus vector with a deletion in the E1 region, for example, using a low-melting point gel direct ligation method to construct a replication-deficient adenovirus molecular clone with a deletion in the E1 region, and packaging infectious virus particles in HEK293 cells. In some embodiments, the adenovirus vector of the present invention is derived from a non-human simian adenovirus, such as a chimpanzee adenovirus or a gorilla adenovirus. These adenovirus vectors generally have low seropositivity and/or low pre-existing neutralizing antibody titers in the human population. In other embodiments, the adenoviral vector of the present invention is derived from human adenovirus, such as at least one of Ad1, Ad2, Ad4, Ad5, Ad6, Ad11, Ad 24, Ad34, and Ad35, in particular Ad5, Ad11, or Ad35.

In other embodiments, the recombinant adenovirus of the present invention further includes a heterologous nucleic acid molecule. Suitable heterologous nucleic acid molecules are well known to those skilled in the art, and may include a transgenic open reading frame, such as an open reading frame encoding a polypeptide/protein. The present invention does not specifically limit the type or sequence of the heterologous nucleic acid molecule. For example, when preparing for the purpose of vaccination of an immune response, a nucleic acid molecule or a fragment thereof that has an immune response to certain viruses is selected, which is well known to technicians. In the present invention, it can be any heterologous nucleic acid molecule or fragment, for example, the heterologous nucleic acid molecule contains a gene encoding an RSV antigen protein, a gene encoding a VZV antigen protein, a gene encoding a GFP protein, or a gene encoding an IE63 protein, as long as it can achieve the immune and/or therapeutic effect of the preparation. In some embodiments, the heterologous nucleic acid molecule is a nucleic acid molecule or a fragment thereof derived from a SARS-CoV-2 virus; or any nucleic acid molecule or a fragment thereof that has an immune response to a SARS-CoV-2 virus. As an exemplary technical solution, the heterologous nucleic acid molecule is a nucleotide sequence shown in SEQ ID NO.2 or SEQ ID NO.4. However, these heterologous nucleic acid molecules are only examples and do not represent any limitation. Those skilled in the art should understand that the pharmaceutical formulation of the present invention may include a recombinant adenovirus containing any other heterologous nucleic acid molecules.

The pharmaceutical formulations comprising recombinant adenoviruses involved in the present invention further relate to related pharmaceutical products, such as gene drugs and/or vaccine preparations. In some embodiments, the pharmaceutical formulation is a formulation in liquid form, which shows excellent adenovirus stability. These formulations are suitable for storage at 2-8°C, and can also maintain good stability when stored at lower temperatures, for example, -20°C or lower, -40°C or lower, -65°C or lower. They can also be stably stored at temperatures above 8°C, for example, 25°C, 37°C or even higher. In the present invention, the term "stability" refers to the relative resistance to degradation of adenovirus particles in the formulation, retaining its efficacy over the time scale of its expected usefulness. In the present invention, the adenovirus stability refers to that the virus titer of a preparation or formulation containing adenovirus does not decrease or only decreases very slightly within an acceptable range in the art under specific conditions within a certain period of time, so as to achieve the virus titer maintaining the effective medicinal effect of the preparation and meet the storage and transportation requirements in the art; for example, under the conditions of an accelerated experiment at 37°C for 7 days or an accelerated experiment at 37°C for 14 days, the virus titer decreases by _lgTCID50 /mL≤0.5 or decreases by lgIFU/mL≤0.5; for another example, when stored at 2-8°C for 270 days, or stored at below -60°C for 1 year, 1.5 years, 2 years, 3 years or 5 years, the virus titer decrease level remains _lgTCID50 /mL≤0.5 or decreases by lgIFU/mL≤0.5.

The pharmaceutical preparation of the present invention comprises a citrate buffer, a nonionic surfactant (v/v) of 0.01% to 1%, and a protective agent, wherein the protective agent is a combination of one or more of a saccharide compound with a C (carbon) number of ≤24 or a sugar alcohol with a C number of ≤24.

The "citrate buffer" described in this application may also be referred to as "citrate buffer". The citrate buffer is prepared by mixing citric acid and/or sodium citrate and water; different contents of citric acid and/or sodium citrate in the citrate buffer will cause the pH value of the pharmaceutical preparation to change. It is well understood by those skilled in the art based on the prior art that the pH value of the pharmaceutical preparation can be adjusted by adjusting the amount of citric acid and/or sodium citrate in the citrate buffer to a specific value or a specific range. In some embodiments of the present application, the concentration of the citrate buffer can be understood as the concentration of citrate ions in the citrate buffer.

The "protective agent" described in this application may also be referred to as a "stabilizer", which can be used alone or in combination with other additives to prevent low-temperature damage to adenovirus vectors being frozen or stored at low temperatures, or to protect vaccines from high temperatures and extreme temperature changes (such as adverse conditions such as freeze-thaw). In the present invention, the protective agent is preferably a combination of one or more of a carbohydrate compound with a C (carbon) number of ≤24 or a sugar alcohol with a C number of ≤24. In some embodiments of the present invention, the carbohydrate compound is one or more of a monosaccharide, a disaccharide or a trisaccharide; in some embodiments, the carbohydrate compound has a C number of ≤12, such as 12 or 6, and the carbohydrate compound is preferably a monosaccharide or a disaccharide; in some embodiments, the carbohydrate compound is one or more of sucrose, trehalose or lactose. In some embodiments, other carbohydrate compounds or their derivatives may be added to the pharmaceutical preparation as protective agents, such as hydroxypropyl cyclodextrin, dextran, etc. Those skilled in the art will appreciate that when adding other carbohydrate compounds or their derivatives, the effect of the added substances on the safety of human pharmaceutical products should be considered. In the present invention, for the safety of human pharmaceutical products, one or more of sucrose, trehalose or lactose is preferably added as a protective agent. In some embodiments of the present invention, the total volume of the pharmaceutical preparation is calculated, and the concentration (w/v) of the protective agent is 4% to 15%, such as 4.2% to 10%, and another example is 4.4% to 7.5%. In some embodiments, the concentration (w/v) of the saccharide with a C number of ≤24 or the sugar alcohol with a C number of ≤24 is 4.4% to 9%, such as 4.8% to 7.5% or 4.4% to 7.2%. In other embodiments, the protective agent includes a saccharide with a C number of ≤24 or a sugar alcohol with a C number of ≤24 at a concentration (w/v) of 4.8% to 7.5%, and a saccharide with a C number of ≥30 at a concentration (w/v) of 2.5% to 5%.

In some embodiments, the molar concentration of the citrate buffer is 10 mM to 140 mM, for example, 20 mM to 75 mM, 20 mM to 60 mM, 30 mM to 100 mM, or 40 mM to 80 mM; exemplarily, the molar concentration of the citrate buffer is 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM or 80 mM.

In some embodiments, the pharmaceutical formulation is a liquid preparation having a pH value between 5.0 and 7.0, for example, a pH value in the range of 5.5 to 6.8, 5.8 to 6.7, 6.0 to 6.8, or 6.2 to 6.7; for example, a pH value in the range of 5.8 to 6.0, 5.8 to 6.2, 6.0 to 6.2, 6.5 to 6.7, or 6.4 to 6.6.

The pharmaceutical preparation of the present invention may further include glycine and mannitol, wherein the concentration (w/v) of glycine is 0.2% to 5%, for example, 0.5% to 1%, based on the total volume of the pharmaceutical preparation; the concentration (w/v) of mannitol is 0.2% to 5%, for example, 0.5% to 1%. In some embodiments, the concentrations of glycine and mannitol are 1%, respectively. The addition of glycine and mannitol has been confirmed to be beneficial to the stability of adenovirus.

In some embodiments, the pharmaceutical formulation does not contain NaCl or other forms of sodium salts, or the concentration of NaCl or other forms of sodium salts is maintained below 35% (w/v%) to achieve good recombinant adenovirus stability, such as 25% (w/v%) and below, or 10% (w/v%) and below. In some embodiments, the concentration of NaCl in the pharmaceutical formulation is 0.

In some embodiments, the addition of ethanol, disodium EDTA, and glycerol to the pharmaceutical formulation is acceptable, but the addition of these ingredients does not significantly improve the performance of the formulation.

In some embodiments, the pharmaceutical preparation is composed of the above-mentioned recombinant adenovirus, citrate buffer, non-ionic surfactant and one or more of a carbohydrate compound with a C (carbon) number ≤ 24 or a sugar alcohol with a C (carbon) number ≤ 24.

In other embodiments, the pharmaceutical preparation is composed of the above-mentioned recombinant adenovirus, citrate buffer, a non-ionic surfactant, one or more of a carbohydrate compound with a C (carbon) number ≤ 24 or a sugar alcohol with a C (carbon) number ≤ 24, glycine and mannitol.

The "preparation" and "preparation" of the present invention are used interchangeably and refer to a composition containing active ingredients with preventive or therapeutic effects and pharmaceutically acceptable excipients and other ingredients. In some embodiments, the active ingredient with preventive or therapeutic effects refers to a recombinant adenovirus that can induce an immune response in the subject (e.g., a mammal) or a recombinant adenovirus that can be used for gene therapy; in some embodiments, the preparation or preparation is prepared into various dosage forms through different types of pharmaceutically acceptable excipients and processes, such as liquid preparations, lyophilized preparations, tablets, ointments, etc.; in some embodiments, the preparation or preparation is administered by nasal spray, nasal drops, aerosol inhalation, intramuscular injection, subcutaneous injection, or oral administration. The pharmaceutical preparation of the present invention provides stability for recombinant adenoviruses at different virus concentrations, single or multivalent, and can be administered to various mammals. Suitable mammals falling within the scope of the present invention include, but are not limited to: primates, livestock (e.g., sheep, cattle, horses, monkeys, pigs, etc.), laboratory test animals (e.g., rabbits, mice, etc.), pets (e.g., cats, dogs, etc.) and captive wild animals (e.g., wolves, foxes, deer, etc.). In some embodiments, the mammal is an experimental mouse. In some embodiments, particularly those applicable to humans, the vaccine may be administered as a vaccine that can provide a prophylactic and/or therapeutic effect to individuals who have not been previously infected or do not have antibodies.

Unless otherwise specified, in the present invention, the concentration of each component is calculated based on the total volume of the prepared pharmaceutical preparation (preparation), representing the mass/volume percentage (w/v), volume/volume percentage (v/v), or volume molar concentration (mM, mmol/L) of each component in the final formulated adenovirus-containing liquid preparation.

The present invention is specifically described below in conjunction with examples, but it is not intended to limit the present invention to the following examples.

The preparations used in the examples of the present invention are shown in Table 1. Unless otherwise specified, the instruments, reagents or materials used in the present invention are commercially available or can be prepared using existing technologies.

Table 1 Preparation source and basic information

The recombinant adenoviruses used in the embodiments of the present invention include: rAdC68XY3-GFP adenovirus, rAdC68XY3-PreS adenovirus, and rAdC68XY3-S adenovirus. The embodiments of the present invention use the purified solution of the above adenoviruses, and the virus information corresponding to each experimental batch is shown in Table 2.

Table 2 Relevant information about recombinant adenovirus used in the present invention

The detection method used in the embodiment of the present invention is an operation method known in the art, and can be completed by ordinary technicians based on the existing technology without creative work. For example, the TCID50 method is used to detect adenovirus titer, which has a wide detection range and can detect not only samples with higher concentrations, but also samples with lower concentrations. The adenovirus titer-TCID50 method reference ( G., Archiv f experiment Pathol u Pharmakol, 162:480-483, 1931).

The ICC method can also be used to detect adenovirus titers, which has a higher sensitivity when compared within the same order of magnitude. The ICC method can be performed as follows: Referring to the instructions of the kit Adeno-X Rapid Titer Kit (Clontech, 632250), the adenovirus is gradient diluted and inoculated on 293A cells grown into a monolayer, and then the cells are cultured. After a certain period of culture, an antibody that can recognize the adenovirus Hexon protein and a secondary antibody that can recognize the antibody are used to form staining spots in the cells infected by the adenovirus through antibody binding and staining with a staining solution. The staining spots are observed and counted under a microscope.

The OD ₂₆₀ method can be used to calculate the number of adenovirus particles: add lysis buffer to the sample to be tested, and measure the absorbance at 260nm with a UV spectrophotometer. According to the formula: adenovirus particles/mL = OD value × dilution factor × 1.1 × 1012, calculate the number of adenovirus particles. The HPLC method can also be used to calculate the number of adenovirus particles.

Example 1 Preparation of recombinant adenovirus stock solution

1.1 Construction of recombinant plasmid

In this embodiment, pShuttle-CMV plasmid without exogenous gene insertion is selected as the vector of recombinant plasmid. pShuttle-CMV is a shuttle plasmid containing CMV enhancer, CMV promoter, T7 promoter, chimeric intron and bGH poly (A) tailing signal, NotI and KpnI double restriction sites, and kanamycin (Kana) resistance. Insert the target heterologous nucleic acid molecule into the pShuttle-CMV plasmid multiple cloning site to obtain a recombinant plasmid containing the target gene, wherein the heterologous nucleic acid molecules used in this embodiment are: nucleic acid molecules encoding GFP protein (amino acid sequence as shown in SEQ ID NO.5) (nucleotide sequence as shown in SEQ ID NO.6), nucleic acid molecules encoding SARS-CoV-2 PreS protein (amino acid sequence as shown in SEQ ID NO.1) (nucleotide sequence as shown in SEQ ID NO.2), nucleic acid molecules encoding the full-length S protein of SARS-CoV-2 virus (amino acid sequence as shown in SEQ ID NO.3) (nucleotide sequence as shown in SEQ ID NO.4).

1.2 Construction of recombinant adenovirus plasmid

In this example, self-constructed recombinant plasmids and commercially available adenovirus vector plasmids were selected to construct recombinant adenovirus plasmids.

1.3 Linearization of recombinant adenovirus plasmid

The recombinant adenovirus plasmid obtained in 1.2 is digested with restriction endonucleases to linearize the recombinant adenovirus plasmid.

1.4 Preparation of recombinant adenovirus

The gene fragments recovered after enzyme digestion in 1.3 were transfected. In this example, LipofectamineTM2000 kit was used for transfection, and HEK293 cells were selected as the expression system. According to the operating instructions of LipofectamineTM2000 kit, the gene fragments recovered after enzyme digestion in 1.3 were transfected into HEK293 cells with a confluence of 60% to 70%.

In the "Inoculation of Cells" step of the LipofectamineTM2000 kit operating instructions, the medium used to culture HEK293 cells is MEM medium, and the MEM medium is replaced with DMEM medium two hours before transfection. Five hours after transfection, the medium is replaced with DMEM medium containing 10% (volume percentage) fetal bovine serum.

The day after transfection, the cell lesions were observed under an inverted microscope until 60% of the HEK293 cells showed plaques, and the cells were collected. The collected cells were repeatedly frozen and thawed three times between room temperature (25°C) and -80°C, and then centrifuged at 1200×g for five minutes to collect the supernatant, which contained the recombinant adenovirus. The supernatant was aliquoted and stored in a -80°C ultra-low temperature refrigerator for later use. The gene in the recombinant adenovirus genome was identified as the target gene.

1.5 Amplification and purification of recombinant adenovirus

The recombinant adenovirus prepared in step 1.4 of the amplification was purified by anion chromatography. The adenovirus purification can be performed according to the adenovirus purification steps described in Sofiya Fedosyuk et al, Vaccine 37 (2019). After that, the recombinant adenovirus purified solution was replaced with a 4FF molecular sieve chromatography column for preparation buffer and further purification. The obtained recombinant adenoviruses were named: rAdC68XY3-GFP, rAdC68XY3-PreS, and rAdC68XY3-S.

Example 2 Preparation of recombinant adenovirus preparation

Take the purified adenovirus solution prepared in Example 1, add different excipients according to different groups, and finally form the corresponding liquid preparation formula. After fully mixing, filter and package, and then put the sample into a biochemical incubator at the corresponding temperature for accelerated stability test.

Experimental Example 1 Preparation and stability test of adenovirus liquid preparations in different buffer systems

The preparation containing rAdC68XY3-GFP adenovirus was prepared according to the formula shown in Table 3, and the prepared rAdC68XY3-GFP adenovirus preparation samples A1 to A14 were placed in a 37°C biochemical incubator (equipment number PERD1702020), and samples were taken on days 0, 7, 14 and 28 to investigate the changes in virus titer ( _1gTCID50 /mL) at each time point. The experimental results detected by TCID50 method are shown in Table 4.

Table 3 Composition of the preparation containing rAdC68XY3-GFP adenovirus in Experimental Example 1

Remark:

1. The concentration of the buffer solution is based on the molar concentration of citrate ions in the resulting preparation;

2. w/v means the weight of solute added to every 100 ml of solvent;

3. The pH of the pharmaceutical preparation solution is adjusted by the ratio of salt and corresponding acid in the buffer, for example, adjusting the ratio of citric acid and sodium citrate, adjusting the ratio of histidine and sodium histidine. Those skilled in the art know how to adjust the ratio of salt and corresponding acid in the buffer so that the pharmaceutical preparation reaches a specific pH.

The pharmaceutical preparations including A1 to A14 were subjected to 37°C accelerated stability test virus titer test respectively. The test results are shown in Table 4 below:

Table 4 Changes in virus titer in accelerated stability test at 37°C for preparations from A1 to A14 (1 g TCID _s0 /mL)

Group 0d 37℃7d 37℃14d 37℃28d A1 7.8 7.9 6.8 5.7 A2 7.8 7.9 6.9 5.6 A3 7.8 7.8 6.6 5.8 A4 7.8 7.9 6.9 5.8 A5 7.8 8 7.5 6.4 A6 7.8 7.8 7.9 6.8 A7 7.8 7.9 7.2 6 A8 7.8 8.0 7.3 6.2 A9 8.4 6.7 5.6 3.6 A10 8.3 6.5 4.4 <3.6 A11 8.5 6 3.8 <3.6 A12 8.2 5.6 3.9 <3.6 A13 8.5 4.5 <3.6 <3.6 A14 8.5 6.8 4.6 <3.6

As can be seen from Table 4, compared with the adenovirus liquid preparation containing a histidine buffer system (HISTIDINE), the adenovirus liquid preparation containing a phosphate buffer system (EBSS) and the adenovirus liquid preparation containing a Tris buffer salt system, the adenovirus liquid preparation containing a citrate buffer system has a better preservation effect on adenovirus, that is: the decrease in virus titer of A1 to A8 after 28 days at 37°C is significantly smaller than that of A10 to A14, indicating that the adenovirus liquid preparation containing a citrate buffer system is more conducive to improving the stability of adenovirus, which may be because citric acid has the effect of inhibiting free radical oxidation.

Experimental Example 2 Preparation and Stability Test of Adenovirus Liquid Preparations Containing Different Types of Excipients

Preparations containing rAdC68XY3-PreS adenovirus (batch number: PE20200914) were prepared according to the formula shown in Table 5, and the prepared rAdC68XY3-PreS adenovirus preparation samples B1 to B16 were placed in a 37°C biochemical incubator (equipment number PERD1702020), and samples were taken on days 0, 7, 14 and 28 to investigate the virus titer, changes in the number of virus particles, changes in particle size and osmotic pressure at each time point.

Table 5 Compositions of adenovirus preparation containing rAdC68XY3-PreS in Experimental Example 2

Notes: Refer to Table 3 notes.

(1) Changes in virus titer

Table 6 shows the results of the accelerated stability test of the preparation samples of groups B1 to B16 at 37°C using the TCID50 method. According to the data in Table 6, it can be seen that the virus titer (lgTCID ₅₀ /mL) of all samples from B1 to B16 did not change by more than 0.6 when placed at 37°C for 14 days, and the virus titer (lgTCID ₅₀ /mL) of the samples of groups B2, B4, B6, B8, B9, B11, B15 and B16 did not change by more than 0.7 when placed for 28 days, indicating that these groups of liquid preparations all have good stability.

Table 6 Changes in virus titer of preparation samples from groups B1 to B16 in 37°C accelerated stability test (1g TCID ₅₀ /mL)

Specifically, according to Table 6, the changes in virus titer of the preparation samples of group B10 on the 14th and 28th days were significantly greater than those of the preparations of other groups. It can be seen that when small molecular sugars such as sucrose and trehalose are not added and only dextran is added, its protective effect on adenovirus is significantly reduced. Comparing the virus titers of the preparation samples of groups B4, B5 and B6, it can be seen that after being placed at 37°C for 28 days, the virus titer (lgTCID ₅₀ /mL value) of group B6 (15% sucrose) changed the least, followed by group B4 (7.5% sucrose), and the virus titer of group B5 (4.8% sucrose) decreased the most. When the sucrose concentration dropped to 4.8% (w/v), the virus titer of group B5 on the 7th, 14th and 28th days decreased more significantly, which shows that high concentration of sucrose has a positive effect on the stability of the adenovirus preparation of this embodiment.

After 28 days of storage at 37°C, the virus titer (lgTCID ₅₀ /mL value) of group B2 (containing 1% glycine and 1% mannitol) decreased by 0.5, and the virus titer (lgTCID 50 /mL value) of group B3 (without glycine and mannitol) decreased by 1.1. Compared with group B1 (20mM citrate), the virus titer ( _{lgTCID 50 /} mL value) of group B15 (60mM citrate) decreased by 0.5 and 1, respectively, indicating that the addition of glycine, mannitol and high concentration of citrate are more conducive to the stability of chimpanzee adenovirus.

Compared with the B5 group formulation without glycerol, the virus titer (lgTCID ₅₀ /mL value) of the B12 group containing glycerol decreased slowly to a certain extent (0.9 and 1.6, respectively), indicating that glycerol has a certain enhancing effect on the stability of adenovirus preparations.

There was no significant difference in virus titer between group B4 and group B8. Surface sucrose and trehalose had comparable effects on enhancing formulation stability, but sucrose was more preferred in terms of cost.

Compared with the groups without the above excipients, the decrease in virus titer (lgTCID ₅₀ /mL) at each time point of histidine, ethanol and disodium EDTA was very small, indicating that the enhancement effect of histidine, ethanol and disodium EDTA on adenovirus stability in the adenovirus preparation system of the present invention was not obvious. After being placed at 37°C for 28 days, the decrease in virus titer (lgTCID ₅₀ /mL) of group B2 without sodium chloride was smaller than that of group B7 containing sodium chloride, further indicating that low content or no sodium chloride is more conducive to adenovirus stability in the adenovirus preparation system of the present invention.

(2) Number of virus particles

The number of virus particles in rAdC68XY3-PreS adenovirus preparation samples from groups B1 to B16 was detected by HPLC, and the test results are shown in Table 7. Compared with day 0, the number of virus particles in samples from groups B1 to B16 was less than 25% after being placed at 37°C for 7 days, and the number of particles was relatively stable; the number of virus particles in most groups remained relatively stable on days 14 and 28.

Table 7 Test results of virus particle count (10 ¹² VP/mL, HPLC method) of adenovirus preparation samples from groups B1 to B16

(3) Particle size detection

DLS was used to detect the particle size of chimpanzee adenovirus stock solution (containing different stabilizers) after being placed at 37°C for 28 days. The test results are shown in Table 8. As can be seen from Table 8 and Figure 1, the particle size of most samples is between 125 and 175 nm. The results show that after being placed at 37°C for 28 days, the adenovirus in the adenovirus preparation samples B1 to B16 has no obvious aggregation and is relatively stable.

Table 8 Virus particle size test results of adenovirus preparation samples B1 to B16 (DLS)

(4) Osmotic pressure detection

The blank stabilizer formulation without adenovirus was tested for osmotic pressure, and the results are shown in Table 9. From the results in Table 9, it can be seen that the osmotic pressure of the formulation containing 15% sucrose (B6 group) and 2.5% (B12 group) glycerol exceeded the predetermined standard. The osmotic pressure of groups B4 and B5 was appropriate, and the addition of excipients was simple, and the corresponding virus titer of the preparation sample added with rAdC68XY3-PreS adenovirus changed little.

Table 9 Osmotic pressure test results of blank stabilizers in groups B1 to B16

According to Experimental Example 2, it can be seen that sucrose, trehalose, dextran or hydroxypropyl cyclodextrin can improve the stability of adenovirus to a certain extent, but the safety of dextran and hydroxypropyl cyclodextrin as pharmaceutical excipients has yet to be confirmed; high concentrations of sucrose and high concentrations of citrate buffer have better effects on improving the stability of adenovirus. Glycine, mannitol and glycerol have positive effects on improving the stability of adenovirus in this experimental example, but the osmotic pressure of the formula containing glycerol is higher; when the content of NaCl and histidine is low or does not contain NaCl and histidine, the stability of the recombinant adenovirus is better, so the recombinant adenovirus preparation of the present invention may not contain NaCl.

Experimental Example 3 Preparation and stability test of adenovirus liquid preparations containing different amounts of excipients

Preparations containing rAdC68XY3-PreS adenovirus (PE20201026-1-A3) were prepared according to the formula shown in Table 10, and the prepared rAdC68XY3-PreS adenovirus preparation samples C1 to C7 groups were placed in a 37°C biochemical incubator or a 42°C biochemical incubator (equipment numbers PERD1702020, PERD1711056). Samples were taken on days 0, 7, 14 and 28 to investigate the virus titer, changes in the number of virus particles, changes in particle size and osmotic pressure at each time point.

Table 10 Composition of rAdC68XY3-PreS adenovirus preparation samples from groups C1 to C7

(1) Virus titer

The results of the accelerated thermal stability test at 42°C and 37°C for the rAdC68XY3-PreS adenovirus formulation samples from groups C1 to C7 detected by the ICC method are shown in Table 11.

Table 11 Changes in virus titer of samples from groups C1 to C7 at 37°C/42°C accelerated stability test (ICC method, lgIFU/mL)

According to Table 11, the adenovirus preparation samples of groups C1 to C7 are a preparation system formed by sucrose, Tween 80 and citrate buffer, and all have good stability. In addition, groups C1, C3 and C6 all contain glycine and mannitol, and the virus titer (lgIFU/mL) changes less than 0.5 after being placed at 42°C for 7 days. The protection effect is significantly better than that of groups C2, C4 and C7 that do not contain glycine and mannitol, indicating that glycine and mannitol have a further enhancing effect on the stability of rAdC68XY3-PreS adenovirus.

(2) Number of virus particles

The HPLC method was used to detect the number of viral particles in the rAdC68XY3-PreS adenovirus preparation samples from groups C1 to C7. The test results are shown in Table 12.

Table 12 Virus particle count of adenovirus formulations from Group C1 to C7 (HPLC method)

According to the data in Table 12, after samples from groups C1 to C7 were placed at 37°C for 28 days, the number of virus particles did not decrease significantly, indicating that the above formulations had good stability. After being placed at 42°C for 7 days, the number of viruses in group C4 decreased significantly compared with groups C1 and C3. The results of the virus particle number test were basically consistent with the virus titer test results.

(3) Particle size detection

DLS was used to detect the particle size of samples from groups C1 to C7 after being placed at 37°C and 42°C, and the test results are shown in Figure 2. As can be seen from Figure 2, there is no significant difference in the particle size distribution of each group under the two conditions, and the particle size of adenovirus particles in the samples of group A at day 0 (120 to 160nm) is close. The results show that after being placed at 42°C for 7 days or at 37°C for 28 days, there is no obvious aggregation behavior of adenovirus in samples from groups C1 to C7.

(4) Osmotic pressure detection

The osmotic pressure test was performed on the preparation samples C1 to C7 containing rAdC68XY3-PreS adenovirus. The results are shown in Table 13.

Table 13 Osmotic pressure test results of adenovirus preparation samples from groups C1 to C7

Group C1 C2 C3 C4 C5 C6 C7 Osmotic pressure of stock solution (mOsmol/kg) 659 440 529 321 547 748 552

Comparing groups C1 and C2, C3 and C4, and C6 and C7, it can be seen that the addition of 1% glycine and 1% mannitol increased the osmotic pressure by 120mOsmol/kg to 200mOsmol/kg. Comparing groups C1 and C5, it can be seen that when the citrate buffer solution is increased from 20mM to 60mM, the osmotic pressure increases by about 110mOsmol/kg. In the accelerated stability experiment, groups C1, C3, and C6 have better protection effects, but the osmotic pressure of group C6 is high, and that of group C1 is slightly higher than the general standard of osmotic pressure for injections. Therefore, groups C1 and C3 are more preferred injection formulations.

According to Experimental Example 3, it can be seen that the adenovirus preparations of groups C1 to C7 provided by the embodiments of the present invention adopt a citrate buffer system, which can achieve good preparation stability effects. In particular, in the accelerated thermal stability test at 37°C and 42°C, the rAdC68XY3-PreS adenovirus samples of group C1 and group C3 had a 14-day virus titer value of lgIFU/m decreased by less than 0.5, and a 28-day virus titer value of lgIFU/mL decreased by less than 1, and the number and size of virus particles changed little, showing good stability effects.

Experimental Example 4 Preparation and stability test of different batches of rAdC68XY3-PreS adenovirus preparations

To further confirm the formula of the adenovirus preparations determined in Experimental Examples 1 to 3, the rAdC68XY3-PreS chimpanzee adenovirus stock solution with an internal batch of PE20201026-1-B3 was used to prepare D1-D5 group preparations according to Table 14, and placed in a 37°C carbon dioxide incubator. Samples were taken on days 0, 7, 14 and 28 to examine the changes in virus titer, virus particle number, particle size, and osmotic pressure of the stock solution at each time point; at the same time, some samples were frozen and thawed 1 to 5 times below -60°C and at room temperature, and samples were taken according to the number of freeze-thaw cycles to examine the changes in virus titer, virus particle number, specific activity and particle size under different freeze-thaw cycles.

Table 14 Composition of adenovirus formulations of groups D1 to D5

(1) Acceleration stability

The results of the accelerated stability test of rAdC68XY3-PreS adenovirus preparation samples from groups D1 to D5 at 37°C using the TCID50 method are shown in Table 15.

Table 15 Virus titer of adenovirus preparation samples from groups D1 to D5 in the 37°C thermal stability test (1g TCID ₅₀ /mL)

Group 0d 37℃7d 37℃14d D1 7.83 7.87 7.63 D2 7.83 7.73 7.43 D3 7.93 7.90 7.83 D4 7.9 7.87 7.83 D5 7.97 7.77 7.47

As can be seen from Table 15, the virus titer of groups D1 to D5 did not change much after being placed at 37°C for 14 days, indicating that the formulations of this experimental group have good stability. Among them, the virus titer value (lgTCID ₅₀ /mL) of the samples in groups D3 and D4 changed by less than 0.1, and the virus titer (lgTCID ₅₀ /mL) of group D5 decreased by 0.5 after 14 days. Compared with D1 and D4, and D2 and D3, the only difference is the amount of buffer used, which further proves that the higher concentration of buffer has a better effect on the stability of the formulation.

(2) Number of virus particles

The OD ₂₆₀ method was used to detect the number of virus particles in the samples of groups D1 to D5. The test results are shown in Table 16. After 28 days at 37°C, the number of chimpanzee adenovirus particles in the five groups of formulations ranged from 4.0E+11VP/mL to 6.0E+11VP/mL. Relative to the test results on day 0, the percentage change in the number of particles ranged from 75% to 115%. It can be seen that there was no significant difference in the number of particles in the samples of groups D1-D5 after 28 days at 37°C.

Table 16 Number of virus particles in groups D1 to D5 (OD ₂₆₀ )

(3) Particle size detection

DLS was used to detect the particle size of samples from groups D1 to D5 at 37°C for 0 days, 7 days, 14 days and 28 days, and the test results are shown in Table 17. As can be seen from Table 17, except for group D2, the particle size of samples in other groups did not change significantly, indicating that the virus particles were basically not aggregated and had good stability.

Table 17 Particle size of adenovirus preparation samples from groups D1 to D5 (nm)

Group 0d 37℃7d 37℃14d D1 130.0 137.3 145.0 D2 119.8 135.3 1850.0 D3 123.9 124.8 124.5 D4 131.8 133.3 132.4 D5 119.3 117.1 118.3

(4) Osmotic pressure detection

The osmotic pressure test results of samples from groups D1 to D5 are shown in Table 18. The osmotic pressure of samples in each group did not exceed 600 mOsmol/kg, which met the general standards for injections in this field.

Table 18 Osmotic pressure of adenovirus preparation samples from groups D1 to D5

Group D4 D3 D5 D1 D2 Osmol/kg 587 522 233 510 417

(5) Freeze-thaw experiment

The effects of freeze-thaw on the stability of adenovirus were investigated in terms of virus titer, number of virus particles, specific activity (infectious activity) and particle size change in the preparations of groups D4 and D3, and the results are shown in Figures 3 to 6. As can be seen from Figures 3 to 6, after repeated freeze-thaw for 5 times, the virus titer value of the preparations of groups D4 and D3 did not change by more than _0.2lgTCID50 /mL, the number of virus particles changed by less than 10%, and the particle size was stable (about 135nm and 123nm, respectively). By calculation, the specific activity (VP/IFU) was in the range of 80 to 150, and most of it was in the range of 100 to 120. The results show that the adenovirus in the preparations of groups D4 and D3 showed good stability during the 5 freeze-thaw processes.

In summary, adenovirus preparations containing sucrose, Tween 80, mannitol, glycine and citrate buffer salt system can maintain good stability, and the stability is better when the concentration of citrate buffer salt is relatively high. Specifically, in the accelerated experiment, the chimpanzee adenovirus preparation samples of group D4 and group D3 showed good stability for 14 days. The number and particle size of virus particles in the two groups of samples changed little after 28 days, the specific activity was in the range of 80 to 160, and the osmotic pressure was also appropriate, showing good stability during 5 freeze-thaw processes. At the same time, it also verifies that when this formula is applied to different batches of adenovirus preparations, it has a good enhancing effect on the stability of the preparation.

Experimental Example 5 Preparation and Stability of Adenovirus Formulations at Different pH Values

The difference between Experimental Example 5 and the D3 group in Experimental Example 4 is only the pH value. The total concentration of citrate is 60 mM. A total of three groups with different pH values (D3-a, D3-b, D3-c) are set up: 5.9, 6.1 and 6.6. The formula of each group of preparations is shown in Table 19. The preparation samples of rAdC68XY3-PreS adenovirus of batch PE20201026-1-B3 were placed in a 37°C or 42°C biochemical culture box (equipment number: PERD1702020 & PERD1711056), and samples were taken on days 0, 7, 14 and 28, respectively, to examine the changes in virus titer, virus particle number and particle size at each time point.

Table 19 Adenovirus formulations at different pH

Notes: Refer to Table 3 notes.

The ICC method was used to detect the virus titer of D3-a, D3-b and D3-c samples obtained from the accelerated stability test at 42°C, and the OD ₂₆₀ method was used to detect the number of virus particles in the samples. The test results are shown in Tables 20 and 21, respectively. As shown in Tables 20 and 21, the virus titer (lgIFU/mL) of rAdC68XY3-PreS adenovirus changed by about 0.3 to 0.4 after being placed at 42°C for 7 days under three pH values (5.8 to 6.0, 6.0 to 6.2, and 6.5 to 6.7). The difference under different pH values was very small, and the number of virus particles did not change significantly among the three groups after being placed at 42°C for 7 days. This shows that based on the existing formula, the chimpanzee adenovirus has good stability in the pH range of 5.8 to 6.7.

Table 20 Summary of changes in virus titer of adenovirus preparations at different pH values in 42°C accelerated test (lgIFU/mL)

Group D3-a D3-b D3-c pH 5.9 6.1 6.6 0d 9.36 9.47 9.45 42℃7d 9.09 9.08 9.10

Table 21 Detection results of virus particle count of adenovirus preparations at different pH values (OD ₂₆₀ method)

Group pH 42℃7 days virus particle count (VP/mL) D3-a 5.9 3.10E+11 D3-b 6.1 3.10E+11 D3-c 6.6 3.04E+11

The present invention has found through preparation research that the concentration of sugar compounds or sugar alcohols, citrate buffer system, glycine and mannitol play a major role in the stability of recombinant adenovirus. The liquid preparation formula containing adenovirus provided by the present invention is stable in multiple freeze-thaw cycles, and the possibility of stable storage at 2°C to 8°C will also greatly reduce the storage and transportation costs of adenovirus vaccines, which is of great value.

Experimental Example 6 Preparation and Stability of Adenovirus Formulations Containing Different Heterologous Nucleic Acid Molecules

Preparations containing rAdC68XY3-GFP, rAdC68XY3-PreS, and rAdC68XY3-S were prepared according to the formula shown in Table 22.

Table 22 Composition of the preparation samples of three adenoviruses from Group E1 to E3

Notes: Refer to Table 3 notes.

(1) Virus titer

The results of the accelerated thermal stability test at 42°C and 37°C for samples of groups E1 to E3 containing different recombinant adenovirus preparations detected by ICC method are shown in Table 23.

Table 23 Summary of changes in virus titer in 37℃/42℃ accelerated stability test for samples from groups E1 to E3 (ICC method, lgIFU/mL)

It can be seen from Table 23 that whether in the 37°C accelerated stability experiment virus titer experiment or in the 42°C accelerated stability experiment virus titer experiment, the samples in groups E1 to E3 showed ideal stability. Among them, the virus titer of samples in groups E1 to E3 changed very little in the 42°C accelerated stability experiment virus titer experiment, and the decrease or increase in the virus titer on day 7 compared with the virus titer on day 0 was less than 0.15.

The above is a detailed introduction to a pharmaceutical preparation containing adenovirus provided by the present application. Specific examples are used herein to illustrate the principles and implementation plans of the present application. The description of the above embodiments is only used to help understand the method of the present application and its core idea. At the same time, for technicians in this field, according to the ideas of the present application, there will be changes in the specific implementation plans and application scopes. In summary, the content of this specification should not be understood as a limitation on the present application.

Sequence Listing

<110> Wuhan Bowo Biotechnology Co., Ltd.

<120> Pharmaceutical preparations containing adenovirus and methods for preserving the same

<130> WHP220017CN

<141> 2022-01-28

<150> 202110181056.1

<151> 2021-02-09

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1248

<212> PRT

<213> Artificial sequence

<400> 1

Met Gly Lys Arg Ser Ala Gly Ser Ile Met Trp Leu Ala Ser Leu Ala

1 5 10 15

Val Val Ile Ala Cys Ala Gly Ala Ser Gln Cys Val Asn Leu Thr Thr

20 25 30

Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val

35 40 45

Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu His Ser Thr Gln

50 55 60

Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp Phe His Ala Ile

65 70 75 80

His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn Pro Val Leu

85 90 95

Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile

100 105 110

Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser

115 120 125

Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys Val Cys Glu

130 135 140

Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr Tyr His Lys Asn

145 150 155 160

Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn

165 170 175

Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu

180 185 190

Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn

195 200 205

Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile Asn Leu

210 215 220

Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu Val Asp

225 230 235 240

Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu

245 250 255

His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala

260 265 270

Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu

275 280 285

Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ala

290 295 300

Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr Val

305 310 315 320

Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr Glu

325 330 335

Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu

340 345 350

Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys

355 360 365

Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala

370 375 380

Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn

385 390 395 400

Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly

405 410 415

Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp

420 425 430

Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp

435 440 445

Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu

450 455 460

Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile

465 470 475 480

Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu

485 490 495

Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr

500 505 510

Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu

515 520 525

Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn

530 535 540

Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly

545 550 555 560

Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln

565 570 575

Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp Pro Gln

580 585 590

Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly Val Ser

595 600 605

Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val Leu Tyr

610 615 620

Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala Asp Gln

625 630 635 640

Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser Asn Val Phe Gln

645 650 655

Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val Asn Asn Ser Tyr

660 665 670

Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr Gln Thr

675 680 685

Gln Thr Asn Ser Pro Gly Ser Ala Ser Ser Val Ala Ser Gln Ser Ile

690 695 700

Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr Ser

705 710 715 720

Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val Thr Thr

725 730 735

Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys Thr Met

740 745 750

Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln Tyr

755 760 765

Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr Gly Ile Ala Val

770 775 780

Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys Gln Ile

785 790 795 800

Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe Asn Phe Ser Gln

805 810 815

Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp

820 825 830

Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Ile Lys Gln

835 840 845

Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys Ala

850 855 860

Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Glu

865 870 875 880

Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr Ser

885 890 895

Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala Met

900 905 910

Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu

915 920 925

Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly

930 935 940

Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu

945 950 955 960

Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys

965 970 975

Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile

980 985 990

Leu Ser Arg Leu Asp Pro Pro Glu Ala Glu Val Gln Ile Asp Arg Leu

995 1000 1005

Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu

1010 1015 1020

Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys

1025 1030 1035 1040

Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly

1045 1050 1055

Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala Pro His Gly Val

1060 1065 1070

Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr

1075 1080 1085

Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu

1090 1095 1100

Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn

1105 1110 1115 1120

Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly

1125 1130 1135

Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro

1140 1145 1150

Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe

1155 1160 1165

Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile

1170 1175 1180

Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu

1185 1190 1195 1200

Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly

1205 1210 1215

Lys Tyr Glu Gln Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln

1220 1225 1230

Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu

1235 1240 1245

<210> 2

<211> 3769

<212> DNA

<213> Artificial sequence

<400> 2

ggccgccgcc atgggcaaga ggtccgccgg cagcatcatg tggctggcct ccctggccgt 60

cgtgattgcc tgcgccggcg cttctcagtg tgtgaatctg acaacacgga cccagctgcc 120

tcccgcctat accaactcct tcaccagggg ggtgtactac cctgataagg tgtttcggtc 180

atctgtgctg catagcaccc aggatctgtt cctgcccttc tttagcaacg tgacttggtt 240

ccacgccatc cacgtgagtg gcacaaatgg aaccaagagg ttcgacaatc ctgtgctgcc 300

tttcaacgac ggagtgtact tcgccagcac tgaaaagtcc aatatcatta ggggctggat 360

ctttggcaca accctggact ccaaaaccca gtccctgctg atcgtgaaca acgccaccaa 420

cgttgtgatt aaggtgtgtg agtttcagtt ttgcaatgac cccttccttg gcgtgtatta 480

tcataagaat aataagtctt ggatggagtc tgagttcaga gtgtactcct cagccaataa 540

ttgcaccttc gaatacgtga gccagccatt cctgatggat ctggagggta aacagggcaa 600

ttttaagaac ctgcgcgaat ttgtgtttaa gaatattgat ggatatttca agatctactc 660

taaacatacc cccatcaatc tcgtgagaga tctgccacag ggctttagcg ccctggaacc 720

actcgtggac ctgccaatcg gcatcaatat tacacggttc cagacccttc tggccctgca 780

tcggtcttac ctgacccctg gcgatagttc ctccggctgg actgccgggg ccgccgccta 840

ttacgtggga tacctgcagc ccaggacatt tctcctgaaa tataatgaga acggcaccat 900

caccgacgca gtggattgtg ctctggaccc actgtccgag accaaatgca cactgaagtc 960

tttcacagtg gagaaaggca tctatcagac ttccaacttt cgcgttcagc ccacagagag 1020

catcgttagg tttcctaata tcactaacct gtgcccattc ggagaagtgt ttaatgccac 1080

caggttcgcc agtgtctacg cttggaaccg caagaggatt tctaactgcg tcgccgacta 1140

ctcagtgctg tacaacagcg ctagtttctc cacattcaaa tgttacggag tgtctccaac 1200

caagctgaat gacctgtgtt tcactaacgt gtacgccgat agtttcgtta tcagaggcga 1260

cgaggtgcgc cagatcgctc ccggacagac tggcaaaatt gctgactaca actacaagct 1320

ccctgacgac ttcaccgggt gcgtgattgc atggaacagc aacaatctgg attccaaagt 1380

aggagggaat tataactacc tgtaccgcct ctttagaaag tccaacctga aaccctttga 1440

aagggatatt tccacagaga tctatcaggc cggctctacc ccttgtaacg gcgtggaggg 1500

ctttaattgt tactttcctc tgcagagcta tgggttccag ccaacaaatg gcgtgggcta 1560

tcagccatat agggtggtgg tgctgagttt cgaactcctg catgcccctg ctaccgtgtg 1620

cggccctaag aagtctacca atctggtgaa aaataagtgc gtgaacttta acttcaatgg 1680

cctgacagga accggcgtgc tgacagaaag caacaaaaag ttcctgcctt tccagcaatt 1740

cggcagagat atcgccgata ccactgacgc tgtgagagac ccccagaccc tggagattct 1800

cgacataaca ccctgctcct tcggcggagt gagcgtcatt acaccaggaa caaacacttc 1860

caatcaggtg gccgtgctgt atcaggatgt gaactgtaca gaggtgcctg tggcaatcca 1920

cgctgaccag ctgaccccaa cctggcgggt ttatagtaca ggtagtaatg tgtttcagac 1980

aagagccggt tgcctgattg gggccgaaca cgttaacaat tcttacgaat gtgacatccc 2040

tatcggagcc ggcatttgcg cctcctatca aacccagacc aacagcccag gaagtgctag 2100

cagcgtggct agtcagtcca tcatcgcata tactatgagt ctgggagccg agaatagcgt 2160

ggcctactcc aataacagca ttgccatccc aaccaatttt accatctctg tgaccactga 2220

gattctgcca gtgtcaatga ctaaaacctc agttgattgc acaatgtata tctgtggcga 2280

ctctaccgag tgctctaacc tgctcctgca gtatgggtct ttttgtaccc agctgaacag 2340

ggctctgacc ggaattgccg tggagcagga taaaaacact caagaggtgt tcgcacaggt 2400

gaagcagatc tataagactc cccctatcaa ggatttcgga ggcttcaact tcagccagat 2460

cctgcctgac ccatccaaac ccagcaagag atcctttatt gaagatctgc tgttcaacaa 2520

ggtgaccctc gccgacgccg gctttatcaa gcagtacggt gactgcctgg gggacattgc 2580

cgctagagat ctcatttgcg ctcagaagtt taacggcctg accgtgctgc caccactcct 2640

caccgatgag atgatcgccc agtatacttc tgccctgctg gctggcacca tcacctccgg 2700

atggaccttc ggcgccggcg cagcactgca gatcccattc gccatgcaga tggcttatcg 2760

cttcaatggg atcggcgtga cccagaatgt gctgtacgag aatcagaagc tgatcgccaa 2820

ccagtttaat agcgccatcg gaaagatcca ggacagcctg agcagcactg ccagcgctct 2880

gggcaagctc caggacgttg tgaaccagaa cgctcaggcc ctgaacactc tggtgaaaca 2940

gctgtcatcc aattttggag ccatcagttc agtcctgaat gatatcctgt ctagactgga 3000

cccacccgag gctgaggttc agatcgaccg gctgatcacc ggcagactgc agagcctcca 3060

gacttatgtg acccagcagc tgatcagggc cgccgagatt agagccagcg ccaacctcgc 3120

tgctaccaaa atgagcgagt gcgttctggg acagtctaag cgcgtggact tttgtgggaa 3180

aggataccac ctgatgagct ttcctcagag cgctcctcat ggcgtggtgt tcctgcacgt 3240

gacttatgtg cctgcccagg aaaagaactt cacaacggcc cctgccattt gtcacgacgg 3300

aaaggcccac ttcccacgcg aaggcgtgtt tgtgtctaat ggaactcact ggttcgtgac 3360

tcagagaaat ttctatgagc cacagattat cacaactgat aacacctttg tgagcgggaa 3420

ttgtgacgtg gttatcggca ttgtgaataa taccgtctat gaccccctgc agccagaact 3480

ggacagcttt aaggaagagc tggataagta cttcaagaac cacacatccc cagacgtgga 3540

tctgggggac atcagtggca tcaacgcctc tgtggtgaat atccagaagg agatcgatcg 3600

gctgaatgag gtggccaaga acctgaacga gtctctgatc gacctgcagg agctggggaa 3660

atacgagcag ggcggctaca tccctgaggc ccccagggac ggccaggcct acgtgaggaa 3720

ggacggcgag tgggtgctgc tgtccacctt cctgtagtga taaggtacc 3769

<210> 3

<211> 1285

<212> PRT

<213> Artificial sequence

<400> 3

Met Gly Lys Arg Ser Ala Gly Ser Ile Met Trp Leu Ala Ser Leu Ala

1 5 10 15

Val Val Ile Ala Cys Ala Gly Ala Ser Gln Cys Val Asn Leu Thr Thr

20 25 30

Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val

35 40 45

Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu His Ser Thr Gln

50 55 60

Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp Phe His Ala Ile

65 70 75 80

His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn Pro Val Leu

85 90 95

Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile

100 105 110

Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser

115 120 125

Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys Val Cys Glu

130 135 140

Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr Tyr His Lys Asn

145 150 155 160

Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn

165 170 175

Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu

180 185 190

Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn

195 200 205

Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile Asn Leu

210 215 220

Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu Val Asp

225 230 235 240

Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu

245 250 255

His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala

260 265 270

Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu

275 280 285

Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ala

290 295 300

Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr Val

305 310 315 320

Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr Glu

325 330 335

Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu

340 345 350

Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys

355 360 365

Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala

370 375 380

Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn

385 390 395 400

Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly

405 410 415

Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp

420 425 430

Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp

435 440 445

Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu

450 455 460

Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile

465 470 475 480

Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu

485 490 495

Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr

500 505 510

Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu

515 520 525

Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn

530 535 540

Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly

545 550 555 560

Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln

565 570 575

Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp Pro Gln

580 585 590

Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly Val Ser

595 600 605

Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val Leu Tyr

610 615 620

Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala Asp Gln

625 630 635 640

Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser Asn Val Phe Gln

645 650 655

Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val Asn Asn Ser Tyr

660 665 670

Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr Gln Thr

675 680 685

Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala Ser Gln Ser Ile

690 695 700

Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr Ser

705 710 715 720

Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val Thr Thr

725 730 735

Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys Thr Met

740 745 750

Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln Tyr

755 760 765

Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr Gly Ile Ala Val

770 775 780

Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys Gln Ile

785 790 795 800

Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe Asn Phe Ser Gln

805 810 815

Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp

820 825 830

Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Ile Lys Gln

835 840 845

Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys Ala

850 855 860

Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Glu

865 870 875 880

Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr Ser

885 890 895

Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala Met

900 905 910

Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu

915 920 925

Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly

930 935 940

Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu

945 950 955 960

Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys

965 970 975

Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile

980 985 990

Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp Arg Leu

995 1000 1005

Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu

1010 1015 1020

Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys

1025 1030 1035 1040

Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly

1045 1050 1055

Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala Pro His Gly Val

1060 1065 1070

Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr

1075 1080 1085

Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu

1090 1095 1100

Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn

1105 1110 1115 1120

Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly

1125 1130 1135

Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro

1140 1145 1150

Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe

1155 1160 1165

Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile

1170 1175 1180

Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu

1185 1190 1195 1200

Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly

1205 1210 1215

Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe

1220 1225 1230

Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu Cys Cys

1235 1240 1245

Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly Ser

1250 1255 1260

Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys Gly Val

1265 1270 1275 1280

Lys Leu His Tyr Thr

1285

<210> 4

<211> 3858

<212> DNA

<213> Artificial sequence

<400> 4

atgggcaaga ggtccgccgg cagcatcatg tggctggcct ccctggccgt cgtgattgcc 60

tgcgccggcg cttctcagtg tgtgaatctg acaacacgga cccagctgcc tcccgcctat 120

accaactcct tcaccagggg ggtgtactac cctgataagg tgtttcggtc atctgtgctg 180

catagcaccc aggatctgtt cctgcccttc tttagcaacg tgacttggtt ccacgccatc 240

cacgtgagtgcacaaatgg aaccaagagg ttcgacaatc ctgtgctgcc tttcaacgac 300

ggagtgtact tcgccagcac tgaaaagtcc aatatcatta ggggctggat ctttggcaca 360

accctggact ccaaaaccca gtccctgctg atcgtgaaca acgccaccaa cgttgtgatt 420

aaggtgtgtg agtttcagtt ttgcaatgac cccttccttg gcgtgtatta tcataagaat 480

aataagtctt ggatggagtc tgagttcaga gtgtactcct cagccaataa ttgcaccttc 540

gaatacgtga gccagccatt cctgatggat ctggagggta aacagggcaa ttttaagaac 600

ctgcgcgaat ttgtgtttaa gaatattgat ggatatttca agatctactc taaacatacc 660

cccatcaatc tcgtgagaga tctgccacag ggctttagcg ccctggaacc actcgtggac 720

ctgccaatcg gcatcaatat tacacggttc cagacccttc tggccctgca tcggtcttac 780

ctgacccctg gcgatagttc ctccggctgg actgccgggg ccgccgccta ttacgtggga 840

tacctgcagc ccaggacatt tctcctgaaa tataatgaga acggcaccat caccgacgca 900

gtggattgtg ctctggaccc actgtccgag accaaatgca cactgaagtc tttcacagtg 960

gagaaaggca tctatcagac ttccaacttt cgcgttcagc ccacagagag catcgttagg 1020

tttcctaata tcactaacct gtgcccattc ggagaagtgt ttaatgccac caggttcgcc 1080

agtgtctacg cttggaaccg caagaggatt tctaactgcg tcgccgacta ctcagtgctg 1140

tacaacagcg ctagtttctc cacattcaaa tgttacggag tgtctccaac caagctgaat 1200

gacctgtgtt tcactaacgt gtacgccgat agtttcgtta tcagaggcga cgaggtgcgc 1260

cagatcgctc ccggacagac tggcaaaatt gctgactaca actacaagct ccctgacgac 1320

ttcaccgggt gcgtgattgc atggaacagc aacaatctgg attccaaagt aggagggaat 1380

tataactacc tgtaccgcct ctttagaaag tccaacctga aaccctttga aagggatatt 1440

tccacagaga tctatcaggc cggctctacc ccttgtaacg gcgtggaggg ctttaattgt 1500

tactttcctc tgcagagcta tgggttccag ccaacaaatg gcgtgggcta tcagccatat 1560

agggtggtgg tgctgagttt cgaactcctg catgcccctg ctaccgtgtg cggccctaag 1620

aagtctacca atctggtgaa aaataagtgc gtgaacttta acttcaatgg cctgacagga 1680

accggcgtgc tgacagaaag caacaaaaag ttcctgcctt tccagcaatt cggcagagat 1740

atcgccgata ccactgacgc tgtgagagac ccccagaccc tggagattct cgacataaca 1800

ccctgctcct tcggcggagt gagcgtcatt acaccaggaa caaacacttc caatcaggtg 1860

gccgtgctgt atcaggatgt gaactgtaca gaggtgcctg tggcaatcca cgctgaccag 1920

ctgaccccaa cctggcgggt ttatagtaca ggtagtaatg tgtttcagac aagagccggt 1980

tgcctgattg gggccgaaca cgttaacaat tcttacgaat gtgacatccc tatcggagcc 2040

ggcatttgcg cctcctatca aacccagacc aacagcccac ggcgggctcg gagcgtggct 2100

agtcagtcca tcatcgcata tactatgagt ctgggagccg agaatagcgt ggcctactcc 2160

aataacagca ttgccatccc aaccaatttt accatctctg tgaccactga gattctgcca 2220

gtgtcaatga ctaaaacctc agttgattgc acaatgtata tctgtggcga ctctaccgag 2280

tgctctaacc tgctcctgca gtatgggtct ttttgtaccc agctgaacag ggctctgacc 2340

ggaattgccg tggagcagga taaaaacact caagaggtgt tcgcacaggt gaagcagatc 2400

tataagactc cccctatcaa ggatttcgga ggcttcaact tcagccagat cctgcctgac 2460

ccatccaaac ccagcaagag atcctttatt gaagatctgc tgttcaacaa ggtgaccctc 2520

gccgacgccg gctttatcaa gcagtacggt gactgcctgg gggacattgc cgctagagat 2580

ctcatttgcg ctcagaagtt taacggcctg accgtgctgc caccactcct caccgatgag 2640

atgatcgccc agtatacttc tgccctgctg gctggcacca tcacctccgg atggaccttc 2700

ggcgccggcg cagcactgca gatcccattc gccatgcaga tggcttatcg cttcaatggg 2760

atcggcgtga cccagaatgt gctgtacgag aatcagaagc tgatcgccaa ccagtttaat 2820

agcgccatcg gaaagatcca ggacagcctg agcagcactg ccagcgctct gggcaagctc 2880

caggacgttg tgaaccagaa cgctcaggcc ctgaacactc tggtgaaaca gctgtcatcc 2940

aattttggag ccatcagttc agtcctgaat gatatcctgt ctagactgga caaagtcgag 3000

gctgaggttc agatcgaccg gctgatcacc ggcagactgc agagcctcca gacttatgtg 3060

acccagcagc tgatcagggc cgccgagatt agagccagcg ccaacctcgc tgctaccaaa 3120

atgagcgagt gcgttctggg acagtctaag cgcgtggact tttgtgggaa aggataccac 3180

ctgatgagct ttcctcagag cgctcctcat ggcgtggtgt tcctgcacgt gacttatgtg 3240

cctgcccagg aaaagaactt cacaacggcc cctgccattt gtcacgacgg aaaggcccac 3300

ttcccacgcg aaggcgtgtt tgtgtctaat ggaactcact ggttcgtgac tcagagaaat 3360

ttctatgagc cacagattat cacaactgat aacacctttg tgagcgggaa ttgtgacgtg 3420

gttatcggca ttgtgaataa taccgtctat gaccccctgc agccagaact ggacagcttt 3480

aaggaagagc tggataagta cttcaagaac cacacatccc cagacgtgga tctgggggac 3540

atcagtggca tcaacgcctc tgtggtgaat atccagaagg agatcgatcg gctgaatgag 3600

gtggccaaga acctgaacga gtctctgatc gacctgcagg agctggggaa atacgagcag 3660

tatatcaagt ggccctggta catctggctg gggtttatcg ctggactgat tgctatcgtg 3720

atggtgacca tcatgctgtg ttgcatgact agctgctgta gctgcctgaa gggatgttgc 3780

agctgcggca gctgctgtaa gttcgatgag gacgactctg agccagtgct gaaaggcgtg 3840

aaactgcact acacctag 3858

<210> 5

<211> 239

<212> PRT

<213> Artificial sequence

<400> 5

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu

1 5 10 15

Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly

20 25 30

Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile

35 40 45

Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr

50 55 60

Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys

65 70 75 80

Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu

85 90 95

Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu

100 105 110

Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly

115 120 125

Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr

130 135 140

Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn

145 150 155 160

Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser

165 170 175

Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly

180 185 190

Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu

195 200 205

Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe

210 215 220

Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys

225 230 235

<210> 6

<211> 720

<212> DNA

<213> Artificial sequence

<400> 6

atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60

ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120

ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180

ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240

cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300

ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360

gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420

aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480

ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540

gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600

tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660

ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720

Claims (9)

1. A pharmaceutical preparation, characterized in that, based on the total volume of the pharmaceutical preparation, the pharmaceutical preparation consists of the following ingredients: (a) Recombinant adenovirus; (b) Citrate buffer 60 mM to 140 mM; (c) nonionic surfactant v/v 0.01% to 1%; (d) Protectant w/v 4.4% to 9%; (e) glycine w/v 0.5% to 1%; and (f) mannitol w/v 0.5% to 1%; Wherein, the protective agent is selected from one or more of sucrose, trehalose or lactose; the pharmaceutical preparation is a liquid preparation, and the pH value of the pharmaceutical preparation is 5.8 to 6.6. 2. The pharmaceutical preparation according to claim 1, characterized in that the recombinant adenovirus is rAdC68XY3-PreS adenovirus or rAdC68XY3-S adenovirus; wherein the rAdC68XY3-PreS adenovirus comprises a nucleic acid molecule encoding the PreS protein of SARS-CoV-2, and the nucleic acid molecule encoding the PreS protein of SARS-CoV-2 comprises the nucleotide sequence shown in SEQ ID NO.2; the rAdC68XY3-S adenovirus comprises a nucleic acid molecule encoding the full-length S protein of SARS-CoV-2, and the nucleic acid molecule encoding the full-length S protein of SARS-CoV-2 comprises the nucleotide sequence shown in SEQ ID NO.4. 3. The pharmaceutical preparation according to claim 1, characterized in that the nonionic surfactant is polysorbate. 4. The pharmaceutical preparation according to claim 3, characterized in that the nonionic surfactant is polysorbate 80. 5. The pharmaceutical preparation according to any one of claims 1 to 4, characterized in that the titer of the recombinant adenovirus ranges from 7.0 lgIFU/mL to 12.0 lgIFU/mL. 6. The pharmaceutical preparation according to any one of claims 1 to 4, characterized in that the pharmaceutical preparation is a nasal spray preparation, a nasal drop preparation, an aerosol inhalation preparation, an intramuscular injection preparation, a subcutaneous injection preparation, or an oral preparation. 7. A method for preserving a pharmaceutical preparation containing an adenovirus, comprising: storing the pharmaceutical preparation as claimed in any one of claims 1 to 6 at a temperature of -60°C to 37°C. 8. The method for preserving a pharmaceutical preparation containing an adenovirus according to claim 7, wherein the pharmaceutical preparation is stored at a temperature of 0°C to 25°C. 9. The method for preserving a pharmaceutical preparation containing adenovirus according to claim 8, characterized in that the pharmaceutical preparation is stored at a temperature of 2°C to 8°C.