KR20210081284A - Plasma expanders comprising gelatin of succinic acid - Google Patents

Abstract

The present invention relates to a method for producing succinate gelatin that can be used as a plasma bulking agent and a plasma bulking agent prepared using the same, wherein the succinate gelatin prepared by the preparation method of the present invention shows heavy metals, endotoxins, and ignition residues that are below standard to be applicable as the plasma bulking agent, and the succinate gelatin according to the preparation method of the present invention has a certain molecular weight compared to conventional gelatin, and the plasma bulking agent including the same has superior stability compared to existing plasma bulking agents and is expected to be widely used in the pharmaceutical market.

Description

Plasma expanders comprising gelatin of succinic acid

The present invention relates to a method for producing succinate gelatin that can be used as a plasma bulking agent, and to a plasma bulking agent manufactured using the same.

Plasma bulking agent is also called plasma replacement solution and can be conveniently used to restore and maintain circulating blood volume when plasma is lost due to hemorrhage, and it is divided into crystal solution and colloid solution. The crystal solution is an electrolyte solution with a low molecular weight and basically contains sodium chloride. The crystallization solution currently used in clinical practice uses physiological saline (hypertonic, isotonic, hypotonic) to form an electrolyte similar to that of plasma, Lactated Ringer's solution, and magnesium ions instead of calcium ions. Normosol, Plasma-Lyte, Isolyte, and Plasma solution A are similarly produced and adjusted to pH 7.4. On the other hand, the colloid solution contains a solute with a relatively large molecular weight that forms colloid oncotic pressure, and thus, unlike the crystalloid solution, it cannot move freely through the blood vessel wall, so it remains in the blood vessel and retains water, thereby increasing the plasma volume.

Gelatin used as a plasma extender is a plasma extender produced by decomposing animal-derived collagen protein. Gelatin is one of the first synthetic colloidal solutions prepared for use in patients, and has been used in intensive care units and operating rooms. Gelatin formulations currently used include cross-linked gelatin such as gelofundiol and urea-cross-linked gelatin such as Haemacel.

However, in general, the amino group of gelatin has a problem of causing a disintegration delay by causing a crosslinking reaction with a specific agent of the drug. Accordingly, since the amino group is substituted with a carboxyl group, the need for the production of succinic acid gelatin to solve the problem of delay in disintegration according to the change over time is increasing (Korean Patent Laid-Open Publication No. 10-2012-0046160).

In order to meet the above needs, the inventors of the present invention established a manufacturing method for separating and purifying succinic acid gelatin from atelocollagen, and confirmed the efficacy of succinic acid gelatin prepared by the above manufacturing method as a plasma extender. The present invention was completed.

Accordingly, an object of the present invention is to recover the collagen solution from which the telopeptide is removed by pre-treating pig skin and inactivating pepsin; (b) centrifuging the recovered collagen solution to obtain an intermediate layer solution from which fat and impurities in the upper and lower layers are removed, adding sodium chloride (NaCl) solution to the intermediate layer solution, and centrifuging to extract atelocollagen; (c) adding and stirring the atelocollagen to the ethanol solution and centrifuging to obtain a precipitate, then adding sodium hydroxide (NaOH) to the precipitate and stirring to base the collagen solution; (d) acid-treating the collagen solution by adding hydrochloric acid to the base-treated collagen solution and stirring, and supplying purified water to the acid-treated solution to obtain a collagen solution whose pH is adjusted to 4.0-5.5; (e) extracting gelatin by applying heat at 55 to 70° C. to the collagen solution whose pH is adjusted to 4.0 to 5.5, and adjusting the pH to 6.7 to 8.2 to obtain an extract containing gelatin succinate; (f) separating, purifying, and concentrating the extract containing the gelatin succinate using a tangential flow filtration (TFF) method to separate, purify and concentrate only gelatin having a molecular weight or higher; And (g) to provide a method for producing succinic acid gelatin comprising the step of lyophilizing the concentrated gelatin after filtration after rapid freezing in a deep freezer (deep freezer).

Another object of the present invention is to provide succinic acid gelatin prepared by the above method.

Another object of the present invention is to provide a plasma bulking agent comprising the succinate gelatin.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention comprises the steps of: (a) pretreating pig skin and inactivating pepsin to recover a collagen solution from which telopeptide is removed; (b) centrifuging the recovered collagen solution to obtain an intermediate layer solution from which fat and impurities in the upper and lower layers are removed, adding sodium chloride (NaCl) solution to the intermediate layer solution, and centrifuging to extract atelocollagen; (c) adding and stirring the atelocollagen to the ethanol solution and centrifuging to obtain a precipitate, then adding sodium hydroxide (NaOH) to the precipitate and stirring to base the collagen solution; (d) acid-treating the collagen solution by adding hydrochloric acid to the base-treated collagen solution and stirring, and supplying purified water to the acid-treated solution to obtain a collagen solution whose pH is adjusted to 4.0-5.5; (e) extracting gelatin by applying heat at 55 to 70° C. to the collagen solution whose pH is adjusted to 4.0 to 5.5, and adjusting the pH to 6.7 to 8.2 to obtain an extract containing gelatin succinate; (f) separating, purifying, and concentrating the extract containing the gelatin succinate using a tangential flow filtration (TFF) method to separate, purify and concentrate only gelatin having a molecular weight or higher; and (g) filtering the concentrated gelatin and then freeze-drying the concentrated gelatin in a deep freezer.

In one embodiment of the present invention, sodium chloride (NaCl) in step (b) may be added in an amount of 5 to 15M.

In another embodiment of the present invention, the base treatment in step (c) may be stirring the collagen solution for 24 hours to 48 hours at a pH of 11.5 to 13.5.

In another embodiment of the present invention, the acid treatment in step (d) may be stirring the collagen solution for 15 to 30 hours at a pH of 2 to 4.

In another embodiment of the present invention, adjusting the pH to 6.7 to 8.2 in step (e) may be by administration of succinic anhydride.

In addition, the present invention provides a succinate gelatin prepared by the above production method.

In addition, the present invention provides a plasma bulking agent comprising succinate gelatin.

The succinate gelatin produced by the production method of the present invention shows heavy metals, endotoxins, and ignition residues below the standard, so it can be applied as a plasma extender, and the succinate gelatin according to the production method of the present invention has a constant molecular weight compared to the existing gelatin. In addition, the plasma extender containing the same is expected to be widely used in the pharmaceutical market because of its superior stability compared to the existing plasma extender.

1 shows a schematic schematic diagram of the manufacturing process of succinic acid gelatin.
Figure 2 shows the succinic acid gelatin manufacturing process.
Figure 3 shows the analysis of succinate gelatin prepared by the production method of the present invention through SDS-PAGE analysis, and Figure 3a is a protein marker, gelatin of other companies (comparative group) and succinate gelatin prepared by the production method of the present invention The results of SDS-PAGE analysis according to the reaction time are shown, and FIG. 3b is a protein marker, atelocollagen, gelatin after TFF filtration/purification, gelatin before TFF filtration/purification (other manufacturing methods), and imported gelatin (control group). ) shows the results of SDS-PAGE analysis.
4 shows the raw material test results of succinic acid gelatin prepared by the present invention, FIG. 4a is the property result, FIGS. 4b to 4d are the confirmatory property result, FIGS. 4e to 4m are the purity test result, FIG. 4n is the pH result, FIG. 4o is the ignition residual result, FIG. 4p is the drying loss result, FIGS. 4q and 4r are the endotoxin content results, and FIGS. 4s to 4v are the microbial limit test results.
Figure 5 shows the raw material test result for the component of the plasma extender containing gelatin succinate, Figure 5a is the raw material test result of sodium lactate, Figure 5b is the raw material test result of magnesium chloride, Figure 5c is the raw material of potassium chloride The test result, Figure 5d shows the raw material test result of sodium chloride.
Figure 6a shows that the stability of the plasma extender is divided into Initial, 1 month, 2 months, 3 months, and 6 months according to the period and analyzed before/after sterilization under accelerated conditions, refrigeration conditions and experimental conditions, and FIG. The stability of the plasma bulking agent containing the prepared succinate gelatin was confirmed, and FIG. 6b is the stability of the plasma bulking agent control.

Hereinafter, the present invention will be described in detail.

The inventors of the present invention established a manufacturing method for isolating and purifying succinic acid gelatin from atelocollagen, and confirmed the efficacy of the succinic acid gelatin prepared by the above manufacturing method as a plasma extender, and thus completed the present invention.

Accordingly, the present invention comprises the steps of: (a) treating pre-treated pig skin with pepsin to remove telopeptide and inactivating pepsin to recover a collagen solution from which telopeptide is removed; (b) centrifuging the recovered collagen solution to obtain an intermediate layer solution from which fat and impurities in the upper and lower layers are removed, adding sodium chloride (NaCl) solution to the intermediate layer solution, and centrifuging to extract atelocollagen; (c) adding and stirring the atelocollagen to the ethanol solution and centrifuging to obtain a precipitate, then adding sodium hydroxide (NaOH) to the precipitate and stirring to base the collagen solution; (d) acid-treating the collagen solution by adding hydrochloric acid to the base-treated collagen solution and stirring, and supplying purified water to the acid-treated solution to obtain a collagen solution whose pH is adjusted to 4.0-5.5; (e) extracting gelatin by applying heat at 55 to 70° C. to the collagen solution whose pH is adjusted to 4.0 to 5.5, and adjusting the pH to 6.7 to 8.2 to obtain an extract containing gelatin succinate; (f) separating, purifying, and concentrating the extract containing the gelatin succinate using a tangential flow filtration (TFF) method to separate, purify and concentrate only gelatin having a molecular weight or higher; and (g) filtering the concentrated gelatin and then freeze-drying the concentrated gelatin in a deep freezer.

Hereinafter, the succinic acid gelatin manufacturing method of the present invention will be described in detail by subdividing each step.

[(a) step: recovery step of collagen solution from which telopeptide has been removed]

After washing, swelling, and sterilizing pig skin, it is pre-treated by stirring with an acid solution, and then the solution homogenized by putting a homogenizer is treated with pepsin to remove telopeptide, and then inactivated pepsin to recover the collagen solution from which telopeptide has been removed is a process to

[(b) step: atelocollagen extraction step]

This is a process of centrifuging the collagen solution from which the telopeptide has been removed, adding sodium chloride to the intermediate layer solution obtained by removing the fat and impurities from the upper and lower layers, stirring, and centrifuging to extract atelocollagen.

The amount of sodium chloride in this step can be added in an amount used in a conventional collagen production method, but it is preferably added in an amount of 5M to 15M.

[(c) step: base treatment step of collagen solution]

It is a base treatment process in which the extracted atelocollagen is added to an ethanol solution, stirred at room temperature, centrifuged to obtain a precipitate, and sodium hydroxide is added thereto and stirred under basic pH conditions.

In this step, the base treatment is preferably performed by adding sodium hydroxide and stirring the collagen solution for 24 hours to 48 hours at a pH of 11.5 to 13.5.

[(d) step: acid treatment and pH adjustment step of collagen solution]

It is a process of adding hydrochloric acid to the base-treated collagen solution, stirring it under acidic pH conditions to perform acid treatment, and adding purified water to the acid-treated solution to adjust the pH to 4.0 to 5.5.

[Step (e): Step of obtaining succinic acid gelatin extract]

This is a process of extracting gelatin by applying heat to the solution adjusted to pH 4.0 to 4.5, adjusting the pH to 6.7 to 8.2, and reacting at room temperature to obtain an extract containing gelatin succinate.

Adjusting the pH to 6.7-8.2 in this step may be due to administration of succinic anhydride.

[(f) step: purification and concentration step]

It is a process of separating, purifying, and concentrating the obtained succinic acid gelatin extract by using a tangential flow filtration method to separate, purify and concentrate only gelatin having a molecular weight or higher.

[(g) step: freeze-drying step]

It is a process of filtering the purified and concentrated gelatin and freeze-drying after rapid freezing in a lyophilizer (deep freezer).

As another aspect of the present invention, the present invention provides succinic acid gelatin prepared by the above preparation method.

The present inventors confirmed through Examples that the succinate gelatin prepared according to the preparation method of the present invention has a certain molecular weight and exhibits excellent stability when used as a plasma extender.

Specifically, in one embodiment of the present invention, before and after the tangential flow filtration (TFF) process and imported gelatin were compared, it was confirmed that the foreign imported gelatin, which is a control gelatin, showed a band of degradation rather than a certain molecular weight, and gelatin before the TFF process. In comparison, in the case of gelatin after the tangential flow filtration (TFF) process, filtration, purification, and concentration were performed, and it was confirmed that a constant band of 20-30 kDa appeared (see Example 2).

In addition, in another embodiment of the present invention, as a result of confirming the stability of the plasma extender containing gelatin succinate prepared by the method of the present invention, it was confirmed that the stability of the plasma extender prepared by the present invention was superior to that of the control drug. (see Example 4).

From the above Example results, the succinate gelatin prepared by the manufacturing method of the present invention shows high purity and stability, and the succinate gelatin according to the manufacturing method of the present invention can be used as a plasma extender in the pharmaceutical market.

Accordingly, as another aspect of the present invention, the present invention provides a plasma bulking agent comprising the succinate gelatin.

When the succinate gelatin of the present invention is a plasma bulking agent, it contains the succinate gelatin prepared by the preparation method of the present invention as an active ingredient, and contains sodium lactate, magnesium chloride, potassium chloride, sodium chloride, sodium acetate hydrate, potassium chloride hydrate, and 1N sodium hydroxide solution. and 1M hydrochloric acid solution, and the like, and may further include therapeutic drugs such as water for injection, glucose, commercially available nutritional solutions, bactericidal antibacterial agents, and antiviral agents.

The plasma increasing agent according to the present invention may include pharmaceutical components included in general intravenous injections in addition to the above components. For example, it may further include one or more components selected from the group consisting of amino acids, sugars, lipids, vitamins, electrolytes, pH adjusters, stabilizers, osmotic pressure adjusters, and solubilizers. Depending on the type of components such as amino acids, sugars or lipids, the plasma bulking agent of the present invention can be used as an intravenous preparation of various compositions.

The plasma increasing agent of the present invention may be administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat or diagnose a disease at a reasonable benefit/risk ratio applicable to medical treatment or diagnosis, and the effective dose level is determined by the patient's disease type, severity, and drug activity, sensitivity to drugs, administration time, administration route and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field. The plasma increasing agent according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered single or multiple. In consideration of all of the above factors, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

[Example]

Example 1. Method for preparing succinic acid gelatin

The present invention prepares succinate gelatin by the process shown in FIGS. 1 and 2 . The manufacturing method for each step is shown below.

1-1. (a) step: recovery step of collagen solution from which telopeptide has been removed

After washing, swelling and sterilizing pig skin conforming to the Korea Food Safety Control Certification (HACCP), it was pre-treated and stirred with 0.1 M acetic acid solution. Thereafter, the homogenized solution was treated with pepsin using a homogenizer to remove telopeptide, and then pepsin was inactivated to recover a collagen solution from which telopeptide was removed.

1-2. (b) step: atelocollagen extraction step

According to Example 1-1, the recovered solution was centrifuged at 4° C., 10 minutes, and 7,800 rpm, and then the fat and impurities in the upper and lower layers were removed to obtain a solution in the middle layer. A 10 M NaCl solution was added to the centrifuged intermediate layer solution, stirred for 1 hour, and centrifuged at 4° C., 10 minutes, and 7,800 rpm to extract atelocollagen.

1-3. (c) step: base treatment step of collagen solution

Atelocollagen extracted in Example 1-2 was added to a 99% ethanol solution, stirred at 4° C. for 12 hours, centrifuged to obtain a precipitate, and then 5M sodium hydroxide (NaOH) was added to obtain a pH of 12.5. Under the conditions of stirring for 24 hours, it was treated with a base.

1-4. (d) step: acid treatment and pH adjustment step of the collagen solution

According to Example 1-3, 1M hydrochloric acid (HCl) was added to the base-treated solution through sodium hydroxide (NaOH) stirring for 24 hours, followed by acid treatment by stirring under pH 2.5 conditions for 24 hours. D.W. was supplied to the solution after the 24-hour HCl acid treatment was completed to adjust the pH to 4.0 ~ 5.5.

1-5. (e) step: obtaining a succinic acid gelatin extract

The solution obtained in Examples 1-4 was heated at 55-70° C. to extract gelatin for 10 hours, and the pH was adjusted to 6.7-8.2 by adding 4-5% succinic anhydride, and reacted at room temperature to form succinic acid gelatin. was prepared and extracted.

1-6. (f) step: purification and concentration step

The solution from which the succinic acid gelatin was extracted according to Example 1-5 was separated/purified and concentrated only with gelatin having a certain molecular weight or more through a tangential flow filtration (TFF) method.

1-7. (g) step: freeze-drying step

The gelatin separated, purified and concentrated according to Example 1-6 was filtered and then rapidly frozen in a lyophilizer (deep freezer) and then freeze-dried to complete succinic acid gelatin.

Example 2. SDS-PAGE analysis of the prepared succinic acid gelatin

In order to analyze the SDS-PAGE of the succinic acid gelatin prepared in Example 1, the protein marker (1), the comparator's gelatin comparison group (2), and the succinic acid gelatin of the present invention were reacted for 12 hours (3), the present invention The case of reacting with succinate gelatin for 16 hours (4) was analyzed. As a result, as shown in FIG. 3a , in the case of the comparative group, a band of degradation rather than a certain molecular weight appeared, and in the case of the succinic acid gelatin of the present invention, a clearer band was formed when reacted for 16 hours compared to when reacted for 12 hours confirmed that

In addition, the difference in succinate gelatin according to whether tangential flow filtration (TFF) was performed was analyzed through SDS-PAGE. There are a total of 5 lanes: ① Protein marker lane, ② atelocollagen lane, ③ Gelatin lane after TFF filtration/purification, ④ Gelatin (other manufacturing method) lane before TFF filtration/purification, and ⑤ Imported gelatin (control) lane The bands were identified by dividing by .

As a result, as shown in FIG. 3b , when comparing the tangential flow filtration (TFF) process before and after the succinic acid gelatin production and the imported gelatin from abroad, filtration, purification and concentration were performed through tangential flow filtration (TFF), resulting in a band of 20-30 kDa. was confirmed to appear. On the other hand, it was confirmed that in the control gelatin, imported gelatin (⑤ lane), a band in the form of degradation rather than a certain molecular weight appeared.

Example 3. Confirmation of specifications for the manufacture of plasma extenders

3-1. Confirmation of specifications of succinate gelatin

In order to confirm the specifications and standards of the succinic acid gelatin produced through the present invention, internal QC and external test institutes were requested to confirm the properties, confirmation tests, pH, purity, heavy metal content, ignition residue, free succinic acid content, loss on drying, endotoxin The content, the amount of bacteria, the amount of fungi and the content of specific microorganisms were checked, and the results are shown in FIG. 4 below.

Test Items Specifications and standards Secondary raw material test result appearance pale yellow light yellow confirmation test yellow precipitate yellow and brown precipitate pH 5.5 to 6.5 5.9 water SDS-PAGE, HPLC fitness heavy metal ≥50 ppm 50ppm or less remnant of ignition ≥3.5% 0.0(0.02)% free succinic acid ≥0.5% - dry weight loss ≥15% 10.8% endotoxin ≥0.25 EU/ml 0.25 EU/mg or less Germ ≥1Х10 ³ cfu 0 CFU/g fungus ≥1Х10 ² cfu 0 CFU/g specific microorganisms non-detection non-detection

From the above results, it was confirmed that the properties, confirmation test, heavy metal content, ignition residue, free succinic acid content, loss on drying, and endotoxin content represent suitable pharmaceutical raw material standards within the pharmaceutical raw material standards range.

3-2. Confirmation of specifications for plasma extender components

In order to confirm the specifications and standards of components that may be additionally included in the plasma extender, the properties, confirmation tests and purity tests of sodium lactate, magnesium chloride, potassium chloride and sodium chloride were analyzed, and the results are shown in FIGS. 5A to 5D .

As shown in FIGS. 5A to 5D , it was confirmed that sodium lactate, magnesium chloride, potassium chloride and sodium chloride were all suitable for drug stability.

Example 4. Plasma bulking agent preparation and stability confirmation

4-1. Confirmation of stability of plasma bulking agent containing succinate gelatin prepared by the present invention

In order to confirm the stability of the plasma extender containing gelatin succinate prepared by the preparation method of the present invention, four types of plasma extender containing gelatin succinate prepared by the method of Example 1 were prepared. Each plasma extender was prepared by slightly varying the contents of gelatin succinate, sodium chloride, sodium acetate hydrate, potassium chloride, potassium chloride hydrate, magnesium chloride, 1N sodium hydroxide solution, 1M hydrochloric acid solution, and water for injection.

The plasma extenders of the four compositions were divided into Initial, 1 month, 2 months, 3 months, and 6 months according to the period, and the pH and properties before and after sterilization were analyzed under accelerated conditions, refrigeration conditions, and experimental conditions. The analysis results are shown in [Table 2] below, and the results of preparing prototypes and conducting stability tests based on plasma extender composition 1, plasma extender composition 2 and plasma extender composition 3 in [Table 2] are shown in FIG. 6a It was.

plasma bulking agent prototype Plasma bulking agent composition 1 Plasma bulk composition 2 Plasma bulking agent composition 3 Plasma bulking agent composition 4 Item Container/Material 50 ml Glass 50 ml Glass 50 ml Glass 50 ml Glass inspection day Condition Item before sterilization after sterilization before sterilization after sterilization before sterilization after sterilization before sterilization after sterilization Initial Acceleration pH 7.26 6.89 7.44 7.88 7.24 7.2 7.16 7.02 appearance Off-white, Transparent, Sol Off-white, Transparent, Sol Colorless, Transparent, Sol Colorless, Transparent, Sol Off-white, Transparent, Sol Off-white, Transparent, Sol Off-white, Transparent, Sol Off-white, Transparent, Sol 1 month Acceleration pH 6.59 6.72 7.3 7.94 6.9 6.86 6.81 6.76 appearance Off-white, Transparent, Sol Off-white, Transparent, Sol Colorless, Transparent, Sol Colorless, Transparent, Sol Off-white, Transparent, Sol Off-white, Transparent, Sol Off-white, Transparent, Sol Off-white, Transparent, Sol 2 months Acceleration pH 6.6 6.72 7.22 8.15 6.92 6.92 6.92 6.8 appearance Off-white, Transparent, Sol Off-white, Transparent, Sol Colorless, Transparent, Sol Colorless, Transparent, Sol Off-white, Transparent, Sol Off-white, Transparent, Sol Off-white, Transparent, Sol Off-white, Transparent, Sol 3 months Acceleration pH 6.61 6.69 7.05 7.15 6.88 6.94 6.98 6.81 appearance Off-white, Transparent, Sol Off-white, Transparent, Sol Colorless, Transparent, Sol Colorless, Transparent, Sol Off-white, Transparent, Sol Off-white, Transparent, Sol Off-white, Transparent, Sol Off-white, Transparent, Sol cold storage pH 6.99 6.79 7.04 6.93 6.86 6.9 7.04 6.94 appearance off-white, transparent, gel off-white, transparent, gel Colorless, Transparent, Sol Colorless, Transparent, Sol off-white, transparent, gel off-white, transparent, gel off-white, transparent, gel off-white, transparent, gel room temperature pH 6.43 6.78 7.28 7.59 6.9 6.88 7 6.85 appearance Off-white, Transparent, Sol Off-white, Transparent, Sol Colorless, Transparent, Sol Colorless, Transparent, Sol off-white, transparent, gel off-white, transparent, gel Off-white, Transparent, Sol Off-white, Transparent, Sol 6 months Acceleration pH 6.41 6.53 7.08 8.12 6.8 6.94 7.1 6.82 appearance Off-white, Transparent, Sol Off-white, Transparent, Sol Colorless, Transparent, Sol Colorless, Transparent, Sol Off-white, Transparent, Sol Off-white, Transparent, Sol Off-white, Transparent, Sol Off-white, Transparent, Sol cold storage pH 6.78 6.64 7.07 7.33 6.75 7.15 6.86 6.86 appearance Off-white, Transparent, Sol Off-white, Transparent, Sol Colorless, Transparent, Sol Colorless, Transparent, Sol off-white, transparent, gel off-white, transparent, gel off-white, transparent, gel off-white, transparent, gel room temperature pH 6.25 6.5 7.33 8.04 6.71 6.87 7 6.78 appearance Off-white, Transparent, Sol Off-white, Transparent, Sol Colorless, Transparent, Sol Colorless, Transparent, Sol Off-white, Transparent, Sol Off-white, Transparent, Sol Off-white, Transparent, Sol Off-white, Transparent, Sol

As the approval standard for plasma extenders is pH 6.9 to 7.9, it was confirmed from the results of [Table 2] that Plasma Extender Formulation 2 was the most stable condition.

4-2. Confirmation of stability of plasma bulking agent control group

In order to confirm that the stability of the plasma extender containing gelatin succinate prepared by the preparation method of the present invention is excellent, the stability of the plasma extender (control drug) containing gelatin succinate prepared differently from the preparation method of the present invention was compared. .

In the case of the control drug, three different plasma bulking agents including succinate gelatin prepared without performing steps (f) (purification and concentration step) and (g) (lyophilization step) in the manufacturing method of Example 1 were used. was used.

The three plasma extenders were divided into Initial, 6 months, and 12 months according to the period, and the pH and properties before and after sterilization were analyzed under accelerated conditions, refrigeration conditions, and experimental conditions. The analysis results are shown in Table 3 below, and the stability test results for each of the three plasma extenders are shown in FIG. 6B .

plasma bulking agent control drug Control drug 1 Control drug 2 Control drug 3 Item Container/Material 100 ml Glass 100 ml Glass 100 ml Glass inspection day Condition Item before sterilization after sterilization before sterilization after sterilization before sterilization after sterilization Initial Acceleration pH 5.78 5.82 5.52 5.68 5.94 6.13 appearance off-white, transparent, gel Off-white, Transparent, Sol off-white, transparent, gel Off-white, Transparent, Sol Off-white, Transparent, Sol Off-white, Transparent, Sol cold storage pH 5.78 5.82 5.52 5.68 5.94 6.13 appearance off-white, transparent, gel Off-white, Transparent, Sol off-white, transparent, gel Off-white, Transparent, Sol Off-white, Transparent, Sol Off-white, Transparent, Sol room temperature pH 5.78 5.82 5.52 5.68 5.94 6.13 appearance off-white, transparent, gel Off-white, Transparent, Sol off-white, transparent, gel Off-white, Transparent, Sol Off-white, Transparent, Sol Off-white, Transparent, Sol 6 months Acceleration pH 5.7 5.75 5.73 5.7 6.14 6.21 appearance colorless, precipitated, gel Light pink, transparent, Sol Off-white, precipitated, Sol Pale yellow, transparent, Sol Off-white, Transparent, Sol Light pink, transparent, Sol cold storage pH 5.7 5.75 5.73 5.7 6.14 6.21 appearance colorless, gel Light pink, transparent, Sol Pale color, Gel Pale yellow, precipitated, Sol colorless, gel Colorless, Transparent, Sol room temperature pH 5.7 5.75 5.73 5.7 6.14 6.21 appearance colorless, gel Light light color, transparent, Sol Pale color, Gel Off-white, Sol colorless, gel colorless, Sol 12 months cold storage pH 6 5.81 5.72 5.75 6.36 6.18 appearance colorless, gel colorless, gel colorless, gel colorless, gel colorless, gel colorless, Sol room temperature pH 5.86 5.79 5.64 5.74 5.99 6.18 appearance colorless, gel colorless, Sol Off-white, Sol colorless, Sol colorless, Sol colorless, Sol

The approval standard of the plasma extender is pH 6.9 to 7.9, and from the results of Table 3 above, it was confirmed that the control drug, the plasma extender, exhibited a pH value that did not meet the approval standard.

From the results of the above examples, it was confirmed that the plasma extender containing gelatin succinate prepared by the method of the present invention had excellent stability.

The above description of the present invention is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Claims (7)

(a) recovering a collagen solution from which telopeptide has been removed by pre-treating pig skin and inactivating pepsin;
(b) centrifuging the recovered collagen solution to obtain an intermediate layer solution from which the upper and lower layers of fat and impurities are removed, adding sodium chloride (NaCl) solution to the intermediate layer solution and centrifuging to extract atelocollagen;
(c) adding and stirring the atelocollagen to the ethanol solution and centrifuging to obtain a precipitate, then adding sodium hydroxide (NaOH) to the precipitate and stirring to base the collagen solution;
(d) acid-treating the collagen solution by adding hydrochloric acid to the base-treated collagen solution and stirring, and supplying purified water to the acid-treated solution to obtain a collagen solution whose pH is adjusted to 4.0-5.5;
(e) extracting gelatin by applying heat at 55 to 70° C. to the collagen solution whose pH is adjusted to 4.0 to 5.5, and adjusting the pH to 6.7 to 8.2 to obtain an extract containing gelatin succinate;
(f) separating, purifying, and concentrating the extract containing the succinate gelatin using a tangential flow filtration (TFF) method to separate, purify and concentrate only gelatin having a molecular weight or higher; and
(g) succinic acid gelatin manufacturing method comprising the step of lyophilizing the concentrated gelatin after filtration after rapid freezing in a deep freezer (deep freezer). According to claim 1,
Sodium chloride (NaCl) in step (b) is characterized in that the addition of 5 ~ 15M, succinic acid gelatin manufacturing method. According to claim 1,
The base treatment in step (c) is a method for producing succinic acid gelatin, characterized in that the collagen solution is stirred for 24 to 48 hours at a pH of 11.5 to 13.5. According to claim 1,
The acid treatment in step (d) is a method for producing succinic acid gelatin, characterized in that the collagen solution is stirred for 15 to 30 hours at a pH of 2 to 4. According to claim 1,
Adjusting the pH to 6.7 to 8.2 in step (e) is a method for producing succinic acid gelatin, characterized in that by administration of succinic anhydride. A succinic acid gelatin prepared by the method of any one of claims 1 to 5. A plasma bulking agent comprising the succinate gelatin of claim 6.

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