KR102527095B1 - Method for measuring the crosslink rate in the compositon including crosslinked hyaluronic acid - Google Patents

Abstract

In the present invention, among dermal filler products containing hyaluronic acid (HA) as a main component, the crosslinking rate between hyaluronic acid and crosslinking agent in a crosslinked hyaluronic acid composition was measured by high-performance liquid chromatography-mass spectrometry (LC-MS) and high-performance liquid It relates to a method that can be easily measured using a chromatographic (HPLC) analysis method, and can enable scientific quality control for filler products whose main ingredient is hyaluronic acid (HA).

Description

Method for measuring the crosslink rate of the crosslinked hyaluronic acid composition {Method for measuring the crosslink rate in the compositon including crosslinked hyaluronic acid}

In the present invention, among dermal filler products containing hyaluronic acid (HA) as a main component, the crosslinking rate between hyaluronic acid and crosslinking agent in a crosslinked hyaluronic acid composition was measured by high-performance liquid chromatography-mass spectrometry (LC-MS) and high-performance liquid It relates to a method that can be easily measured using a chromatographic (HPLC) analysis method.

Dermal Filler is a minimally invasive non-surgical procedure that complements shallow to deep wrinkles and adds volume to nasolabial folds, crow's feet, cheeks, forehead, cheeks, and chin. is the core product of In particular, cross-linked hyaluronic acid fillers developed through cross-linking using hyaluronic acid present in the human body are gaining popularity enough to occupy 90% of the filler market.

Hyaluronic acid filler is a material that is driving the explosive growth of the facial beauty market due to its clinical safety, low price, simple procedure, and immediate effect, along with the advantage of reducing the risk of surgery to consumers compared to plastic surgery. There is no resistance to filler treatment, so the market for hyaluronic acid filler is rapidly expanding, not only to the middle-aged but also to the newcomers to society, and many companies are launching products. However, due to intensifying price competition among companies to enter the market and differences in technology, there is a significant difference in quality among hyaluronic acid filler products, and as side effects are frequent, consumers prefer safe fillers rather than prices. there is.

It is understood that there are about 700 companies worldwide that manufacture these fillers and about 20 in Korea. Among them, almost all hyaluronic acid fillers take the form of cross-link, and each manufacturer has hyaluronic acid cross-link technology, which is a core technology of filler. The cross-linking technology of the filler directly affects the safety and performance of the product (hereinafter referred to as “viscoelasticity”), and controls the viscoelasticity by the cross-linking ratio and particle size, so it is secure enough that only core engineers share it. It is also managed very carefully.

Cross-linking uses various cross-linking agents for each manufacturer and undergoes a unique cross-linking process. Most of the currently marketed hyaluronic acid filler products use 1,4-butanediol diglycidyl ether (BDDE) as a crosslinking agent, and the LD ₅₀ of BDDE is 1,130mg/kg (dermal, Rabbit), which is known to be about 80 times less toxic than the LD ₅₀ (14mg/kg, dermal, Rabbit) of divinylsulfone, another crosslinking agent.

However, it is known that when even this is contained in a large amount, there is a high possibility of causing side effects caused by the body's immune response. In particular, as more fillers are administered to a wider area than approved indications and doses in the market, there is always a concern about toxicity caused by BDDE.

Looking at the patents related to cross-linking of hyaluronic acid registered in Korea, a method for manufacturing a hyaluronic acid gel with low content of cross-linking agent and excellent viscoelasticity (Korean Registered Patent No. 10-1239037), anti-aging using low-molecular and high-molecular hyaluronic acid and polysaccharide extracts of natural product, Rhododendron bark. Composition (Korean Registered Patent No. 10-1449687), hyaluronic acid cross-linked with a high-density network structure and method for preparing the same (Korean Patent Publication No. 10-2015-0029578), biocompatible cross-linked hyaluronic acid hydrogel carrying a drug, and a composition containing the same , and its manufacturing method (Korean Registered Patent No. 10-1818705), excellent viscoelasticity, low injection extrusion force, and high stability against heat and decomposition enzymes, hyaluronic acid-crosslinking showing excellent cartilage protection effect through lubricating and buffering (cushioning) action Method for preparing microparticles (Korean Registered Patent No. 10-1728012), method for preparing microparticles composed of ultra-high density crosslinked hyaluronic acid with low crosslinking rate (Korean Patent Publication No. 10-2018-0064715), hyaluronic acid crosslinked with one or more multifunctional crosslinking agents Efficient production method for the product (Korean Patent Publication No. 10-2016-0025026), method for producing a precipitated, shaped cross-linked hyaluronic acid product having a degree of modification of 1 to 40 units of cross-linking agent per 1000 units of disaccharide (Korean Patent Publication 10-2016-0025026) 2048395), a method for accurately and efficiently measuring the cross-linking rate between HA and a cross-linking agent, but a method for preparing cross-linked hyaluronic acid by cross-linking HA and a cross-linking agent safely and efficiently has been registered. etc. are not registered.

On the other hand, the cases reported so far for the method of measuring the cross-link rate between hyaluronic acid and the cross-linking agent are as follows. To measure the crosslinking rate between hyaluronic acid (HA) and BDDE, ^BDDE -modified HA hydrogel (three crosslinked A method for measuring the cross-linking rate for parameters, that is, total modification, t-MOD; cross-link modification, c-MOD; pendent modification, p-MOD, etc., has been reported (Yang et al., Carbohydrate Polymers 131, 233-239, 2015).

In addition, a method for analyzing oligosaccharides, which are partial HA decomposition products obtained through enzymatic hydrolysis, using gel filtration chromatography (GPC), NMR, and MS analyzers has been reported by utilizing the molecular size-dependent characteristics (Wemde et al. Carbohydrate Polymers, http://dx.doi.org/10.1016/j.carbpol.2015.09.112, 2015). Kablik et al. proposed a method for measuring the concentration of HA by using the characteristic that the viscoelasticity of the HA hydrogel varies depending on the degree of crosslinking (Kablik et al., Dermatologic Surgery, 35, 302-312 DOI: 10.1111/j.1524-4725.2008.01046.x, 2009). Fidalgo et al. reported that by-products were detected when 0.1% NaOH aqueous solution was added to HA hydrogel cross-linked with autoclaved BDDE using LC-MS. It was argued that confirmation is absolutely necessary when evaluating the quality of the rate (Fidalgo et al., Medical Devices: Evidence and Research 11 , 367-376, 2018).

However, the method for measuring the cross-link rate of filler (HA) known to date has problems such as complicated sample pretreatment and low reproducibility, so it is practically suitable for application in industry as it is. There is a problem that has not been done.

Korean Patent Registration No. 10-1239037, Manufacturing method of cross-linked hyaluronic acid gel, registered on February 25, 2013. Korean Patent Registration No. 10-1449687, anti-aging composition containing low-molecular and high-molecular hyaluronic acid and polysaccharide extract isolated from radishes root, registered on October 2, 2014. Korean Patent Publication No. 10-2015-0029578, cross-linked hyaluronic acid with a high-density network structure and its preparation method, published on March 18, 2015. Korean Registered Patent No. 10-1818705, Hyaluronic acid cross-linked hydrogel filled with drugs and its use, registered on January 9, 2018. Korean Patent Registration No. 10-1728012, method for producing hyaluronic acid-crosslinked microparticles, registered on April 12, 2017. Korean Patent Publication No. 10-2018-0064715, microparticles composed of ultra-high-density hyaluronic acid crosslinked products with low crosslinking rate and manufacturing method thereof, published on June 15, 2018. Korean Patent Publication No. 10-2016-0025026, method for producing a crosslinked hyaluronic acid product, published on March 07, 2016. Korean Patent Publication No. 10-2048395, Manufacturing method of molded cross-linked hyaluronic acid product, registered on November 19, 2019.

Fidalgo, J. et al., Detection of a new reaction by-product in BDDE cross-linked autoclaved hyaluronic acid hydrogels by LC-MS analysis, Medical Devices: Evidence and Research 11, 367-376, 2018 Kablik, J. et al., Comparative physical properties of hyaluronic acid Dermal Fillers, Dermatologic Surgery, 35, 302-312 DOI: 10.1111/j.1524-4725.2008.01046.x, 2009 Wende, F. J et al., Determination of substitution positions in hyaluronic acid hydrogelsusing NMR and MS based methods, Carbohydrate Polymers, http://dx.doi.org/10.1016/j.carbpol.2015.09.112, 2015 Yang et al., Determination of modification degree in BDDE-modified hyaluronic acid hydrogel by SEC/MS, Carbohydrate Polymers 131, 233-239, 2015

An object of the present invention is to provide a method for effectively measuring the cross-link rate between hyaluronic acid (HA) and a cross-linking agent in a cross-linking filler product.

In the present invention, in order to measure the crosslinking rate of the HA-crosslinking agent in crosslinking filler products, hydrolysis is performed using hyaluronidase (HAase), a hydrolytic enzyme, and the hydrolyzed oligosaccharide-crosslinking agent hydrolyzate is obtained by liquid chromatography -It is about a method for rapid and efficient measurement using mass spectrometry (LC-MS) and high-performance liquid chromatography (HPLC).

the present invention

(Step 1) Diluting the sample with a pH 5-7 buffer solution so that the final concentration of hyaluronic acid (HA) in the sample is 0.1-0.8% by weight;

(Step 2) adding hyaluronic acid hydrolase to the sample in step 1 to be 40 to 400 U/mL;

(Step 3) preparing a hydrolyzate by hydrolyzing the sample to which the hyaluronic acid hydrolase of the second step was added at 37 to 50 ° C. for 48 to 120 hours;

(Step 4) Identifying component peaks of non-crosslinked (linear HA) and cross-linked HA by using high-performance liquid chromatography-mass spectrometry (LC-MS) for the sample hydrolyzate from step 3 above. ; and

(Step 5) measuring the cross-linking rate of the sample by applying the analysis result of step 4 to Equation 1 below;

A method for measuring the crosslinking rate between hyaluronic acid-crosslinking agent, characterized in that it comprises, [Equation 1] is,

Total strain (crosslink rate; t-MOD, %) =

(Where, A _HA is the total peak area of sample hydrolysates that are not cross-linked (non-cross-linked), A _HA-X-HA is the total peak area of sample hydrolysates that are cross-linked, A _HA-X is the total peak area of the sample hydrolyzate cross-linked as a pendant).

In addition, the crosslinking rate of the present invention provides a method for measuring the crosslinking rate between hyaluronic acid and crosslinking agent, characterized in that the crosslinking degree of modification and the pendant degree of modification are expressed by Equations 2 and 3 below.

[Equation 2]

Crosslinking Modification (c-MOD, %) =

(Where, A _HA is the total peak area of sample hydrolysates that are not cross-linked (non-cross-linked), A _HA-X-HA is the total peak area of sample hydrolysates that are cross-linked, A _HA-X is the total peak area of the sample hydrolyzate cross-linked with a pendant)

[Formula 3]

Pendant strain (p-MOD, %) = t-MOD (%) - c-MOD (%)

As shown in Figure 1, the hyaluronic acid (HA) is β-D-glucuronic acid (GlcA) and β-D-acetylglucosamine (β-D-N-acetylglucosamine, GlcNAc) It is a disaccharide polymer with a repeating basic structure of (1 → 4) bonds and (1 → 3) glycosylation bonds.

On the other hand, hyaluronidase (HAase) is a hyaluronate lyase (EC 4.2.2.1) isolated and extracted from the microorganism Streptomyces hyalurolyticus. It is a commercial enzyme that strongly hydrolyzes

Such hyaluronic acid (HA) itself is difficult to use industrially due to its water insoluble properties. Therefore, a crosslinking agent is added to increase the water solubility of HA. When the crosslinking agent is added under alkaline conditions, three types of HA-crosslinking agent crosslinking forms appear. As shown in Figure 2, the form of HA-crosslinking agent (BDDE is used in one embodiment of the present invention) is a pendant modification (p-MOD) bonded to only one side of the crosslinker (BDDE), bonded to both ends of the crosslinker The total modification (t-MOD) is the sum of the cross-link modification (c-MOD) and these pendant and cross-link modifications, and the cross-linking rate is represents the result value.

When measuring the HA-crosslinking agent, the HA concentration of the filler product is adjusted to a weight ratio of 0.1% to 0.8%, diluted in a buffer pH 5 to 7 (preferably pH 5), and 50 to 300 units (preferably 100 units) After adding 10 to 320 μL (preferably 160 μL) of hyaluronidase (HAase), a diluted hydrolytic enzyme, to 40 to 400 U/mL, the temperature is 37 to 50° C. (preferably 50 μL). C) for 48 to 120 hours on a water bath to determine the crosslinking rate of the HA-crosslinking agent. The concentration of the hyaluronidase is preferably 100 to 320 U/mL, more preferably 150 to 320 U/mL.

The buffer solution may be one selected from the group consisting of sodium phosphate, potassium phosphate, ammonium acetate and ammonium formate.

The crosslinking agent capable of measuring the crosslinking rate between the crosslinked hyaluronic acid (HA) and the crosslinking agent is butanediol diglycidyl ether (1,4-butandiol diglycidyl ether), ethylene glycol diglycidyl ether, hexane Diol diglycidyl ether (1,6-hexanediol diglycidyl ether), propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol di Glycidyl ether (polytetramethylene glycol diglycidyl ether), neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether ), glycerol polyglycidyl ether, tri-methylpropane polyglycidyl ether, bis-epoxypropoxyethylene (1,2-(bis(2,3-epoxypropoxy)ethylene) ), pentaerythritol polyglycidyl ether, sorbitol polyglycidyl ether, divinyl sulfone, epichlorohydrin, epibromohydrin And crosslinking agents capable of crosslinking with the filler product, such as a group consisting of combinations thereof, are all crosslinking agents that can be analyzed by high-performance liquid chromatography and liquid chromatography-mass spectrometry.

The hyaluronic acid may be one selected from the group consisting of an insert for treating arthritis, an insert for treating wrinkles, a cosmetic filler, and a drug carrier.

The present invention is a crosslinking method that can efficiently measure the crosslinking rate between the added crosslinking agent and hyaluronic acid in a filler product using hyaluronic acid (HA) as a basic raw material by using high performance liquid chromatography and liquid chromatography mass spectrometry. Filler (HA) 　 By enabling scientific quality control of products, it is possible to greatly contribute to enhancing the international competitiveness of related industries.

1 is a disaccharide repeating structure of hyaluronic acid (HA), which is the main component of a cross-linked filler (HA) product.
Figure 2 is a schematic diagram of the HA-BDDE bond form derived when hyaluronic acid and BDDE, a crosslinking agent, are reacted under alkaline conditions.
3 is an HPLC chromatogram analyzed for HA hydrolyzed under the concentration of hydrolase in Example 1 (159.0 U/mL).
Figure 4 is an HPLC chromatogram pattern of HA hydrolyzed under the hydrolase concentration (9.1 U//mL μL) of Example 2.
5 is an HPLC chromatogram pattern of HA hydrolyzed under the hydrolase concentration of Example 3 (79.0 U/mL).
6 is an HPLC chromatogram pattern of HA hydrolyzed under the hydrolase concentration (319.0 U/mL) of Example 4.
7 is an HPLC chromatogram pattern according to the hydrolysis time (24 h) after adding the amount of hydrolase (159.0 U/mL) of Example 5.
8 is an HPLC chromatogram pattern according to the hydrolysis time (72 h) after adding the amount of hydrolase (159.0 U/mL) of Example 6.
9 is an HPLC chromatogram pattern according to the hydrolysis time (120 h) after adding the amount of hydrolase (159.0 U/mL) of Example 7.
10 is an HPLC chromatogram pattern for the hydrolyzed HA ID and cross-linked filler product of Example 8.
11 is a total ion chromatogram (TIC) of the hydrolyzed cross-linked filler (HA) sample of Example 8 analyzed by LC-MS.
12 is a HPLC chromatogram comparison result of Comparative Example 1 to Example 1.
13 is a HPLC chromatogram comparison result of Comparative Example 2 to Example 8.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the disclosure herein is provided so that it will be thorough and complete, and will fully convey the spirit of the invention to those skilled in the art.

<Example 1. Preparation of HA standard sample (HA ID) and test solution for HPLC analysis>

First, 0.4 g of uncrosslinked HA (linear HA) powder was dissolved in 100 mL of 100 mM sodium acetate buffer (pH 5.0) to prepare a standard stock solution having a weight ratio of 0.4%. 100 μL of the prepared HA stock solution was taken, hyaluronidase (HAase) was added to a concentration of 99.5 U/mL, followed by hydrolysis in a water bath at 50° C. for 3 hours. Then, hyaluronidase (HAase) was further added to a concentration of 99.8 U/mL, followed by further hydrolysis in a 50° C. water bath for 90 minutes. The hydrolyzed standard solution was heat-treated on a 95 °C water bath for 10 minutes to inactivate the enzyme (HAase) to prepare HA ID (standard sample).

On the other hand, in order to measure the crosslinking rate of HA-BDDE among crosslinked filler products, 1 g of product sample (about 2.4% by weight as HA) was taken and 5 mL of 100 mM sodium acetate buffer (pH 5.0) was added to HA It was diluted to a weight ratio of 0.4%. After taking 50 μL of the diluted sample, hyaluronidase (HAase) was added to a concentration of 159.0 U/mL, followed by hydrolysis in a 50° C. water bath for 96 hours. The sample solution after hydrolysis was stored in a freezer at -20 ° C until use.

50 μL of the pretreated sample was injected using an auto-sampler and analyzed by HPLC at a detection wavelength of UV 232 nm.

Specific HPLC analysis conditions are shown in Table 1 below.

Analytical instrument Shimadzu LC-20A system column Anion exchange resin (4.6 mm (ID) x 250 mm, CarboPac PA100, Thermo Fisher Scientific) mobile phase (A) water, (B) 400 mM sodium phosphate buffer (pH 5.8) detection wavelength UV 232 nm Gradient Elution Conditions time (minutes) B% 0 0 70 100 71 0 80 0 flow rate 0.8 mL/min injection volume 50 µL column temperature room temperature

As a result of HPLC analysis after pretreatment of the sample under the same conditions as in Table 1, the hydrolysis pattern of HA in HA ID, linear HA, and crosslinked filler products was shown in FIG. 3.

As shown in FIG. 3, the HA standard sample (HA ID) contains 4 sugar polymers (tetramer), 6 sugar polymers (hexamer), 8 sugar polymers (octamer), 10 sugar polymers (decamer) and 12 sugar polymers (dodecamer). In linear HA and cross-linked filler products, components that are very large in the vicinity of retention times (Rt) corresponding to tetramers and hexamers compared to the component peak patterns of HA ID showed a peak.

In addition, when analyzing the chromatogram of the hyaluronic acid hydrolyzate among the crosslinked filler products corresponding to the retention time of the 4-12 sugar polymer of HA ID, the component peaks of the hydrolyzate modified by pendant modification and cross-linking around the 6 sugar polymer was able to observe

< Example 2. HPLC HA for analysis in the sample solution Comparison of hydrolysis patterns according to the capacity of hydrolase for 1>

The following experiments were conducted to establish optimal hydrolysis conditions for the cross-linked hyaluronic acid composition. The amount of hyaluronidase (HAase), a hydrolytic enzyme, was added to the test solution (linear HA, cross-linked filler product sample) to 9.1 U/mL, and then HA was hydrolyzed. At this time, the remaining sample pretreatment conditions were treated in the same manner as in Example 1.

The pretreated samples were analyzed under the same HPLC analysis conditions as in Example 1 and are shown in FIG. 4 . As shown in Figure 4, the HA hydrolysis pattern in HA ID, linear HA, and crosslinked filler products showed a similar decomposition pattern to Example 1, but the HA-BDDE crosslinking rate was reduced due to the small amount of enzyme added. It was calculated relatively lower than (see Table 5). This is considered to be due to insufficient amount of HAase added and insufficient hydrolysis of the cross-linked filler product.

< Example 3. HPLC HA for analysis in the sample solution Comparison of hydrolysis patterns according to the capacity of hydrolase for 2>

The amount of hyaluronidase (HAase), a hydrolytic enzyme, was added to the test solution (linear HA, filler product sample (S500)) to be 79.0 U/mL, and then the hydrolysis patterns of HA were compared. At this time, the remaining conditions were pretreated in the same manner as in Example 1.

The pretreated sample was analyzed under the same HPLC analysis conditions as in Example 1 and is shown in FIG. 5 . As shown in FIG. 5, the hydrolysis pattern of HA in the cross-linked filler product showed a similar decomposition pattern to Example 1, but the HA-BDDE cross-linking rate was relatively higher than that of Example 1 as in Example 2 due to the small amount of enzyme added. appeared low. This is considered to be because hydrolysis of HA by HAase was not sufficiently performed as in Example 2.

< Example 4. HPLC HA for analysis in the sample solution Comparison of hydrolysis patterns according to the capacity of hydrolase for 3>

The test solution (linear HA, cross-linked filler product sample) was adjusted to 319.0 U/mL by adding hyaluronidase (HAase), a hydrolytic enzyme, and then the HA hydrolysis pattern was compared and examined. At this time, the remaining conditions were pretreated in the same manner as in Example 1.

The pretreated samples were analyzed under the same HPLC analysis conditions as in Example 1 and are shown in FIG. 6 . As shown in FIG. 6, the hydrolysis pattern of HA in HA ID, linear HA, and cross-linked filler products showed a similar decomposition pattern to Example 1. The HA-BDDE cross-linking rate measurement results were almost similar to those of Example 1. It was found that even if more hydrolytic enzymes were added than in Example 1, hydrolysis of HA was no longer possible. Therefore, it was shown that the optimal HAase addition amount for filler (HA) was 155.2 U/mL.

< Example 5. HPLC HA for analysis in the sample solution Comparison of HA hydrolysis patterns according to hydrolysis time for 1>

The amount of hyaluronidase (HAase), a hydrolytic enzyme, was added to the test solution (linear HA, cross-linked filler product) to be 159.0 U/mL, and then the HA was hydrolyzed for 24 hours. The decomposition patterns were compared and reviewed. At this time, the remaining conditions were pretreated in the same manner as in Example 1.

The pretreated sample was analyzed under the same HPLC analysis conditions as in Example 1 and is shown in FIG. 7 . As shown in FIG. 7, the HA hydrolysis pattern in the HA ID, linear HA, and crosslinked filler products was similar to Example 1, but the HA-BDDE crosslinking rate in the filler product was relatively higher than that in Example 1. appeared low. This shows that 24 hours is not sufficient to hydrolyze the HA.

< Example 6. HPLC HA for analysis in the sample solution Comparison of HA hydrolysis patterns according to hydrolysis time for 2>

The amount of hyaluronidase (HAase), a hydrolytic enzyme, was added to the test solution (linear HA, cross-linked filler product) to a level of 159.0 U/mL, and then hydrolysis was performed for 72 hours to decompose the cross-linked HA. I tried to compare and review the patterns. At this time, the remaining conditions were pretreated in the same manner as in Example 1.

Samples pretreated as described above were analyzed under the same HPLC analysis conditions as in Example 1. As shown in FIG. 8, the HA hydrolysis pattern in HA ID, linear HA, and crosslinked filler products showed a similar decomposition pattern to Example 1, but the HA-BDDE crosslinking rate in the filler product was the same as in Example 5. Relatively lower than Example 1. This shows that 72 hours is not sufficient to hydrolyze cross-linked HA.

<Example 7. HA hydrolysis pattern comparison 3 according to hydrolysis time for HA sample solution for HPLC analysis>

The amount of hyaluronidase (HAase), a hydrolytic enzyme, was added to the test solution (linear HA, cross-linked filler product) to 159 U/mL, and the hydrolysis time of HA was hydrolyzed for 120 hours, and the degradation pattern was I wanted to do a comparative review. At this time, the remaining conditions were pretreated in the same manner as in Example 1.

Samples pretreated as described above were analyzed under the same HPLC analysis conditions as in Example 1. As shown in FIG. 9, the HA hydrolysis pattern in the HA ID, linear HA, and crosslinked filler products showed a similar decomposition pattern to Example 1, but the HA-BDDE crosslinking rate in the filler product was the result of Example 1. this was almost similar. This shows that HA hydrolysis does not occur any longer even when the hydrolysis time is longer than that of Example 1.

< Example 8. HA oligosaccharide and HA- by LC-MS BDDE sympathy for the complex >

The volume of hyaluronidase (HAase), a hydrolytic enzyme, was added to the test solution (HA ID, linear HA, cross-linked filler products) to 159.0 U/mL, and then the sample was pretreated in the same manner as in Example 1, as follows It was analyzed according to the LC-MS analysis conditions. Specific HPLC analysis conditions are shown in Table 2 below.

Analytical instrument Shimadzu LCMS-8040 system with multi-wavelength detector column Hypercarb (4 mm (ID) x 250 mm, Thermo Fisher Scientific) mobile phase (A) 400 mM ammonium acetate buffer solution (pH 5.8), (B) acetonitrile detector 1. Multi-wavelength detector (PDAD) - UV 232 nm
2. Mass detector (MSD) - 100 ∼ 2000 amu Gradient Elution Conditions time (minutes) B% 0 0 70 80 71 95 80 95 80.01 0 95 0 flow rate 1.0 mL/min column temperature 60 ℃ injection volume 50 µL interface ESI positive / negative interface voltage 3.0 / -3.0 kV drying gas flow 15 L/min Nebulizing gas flow 3 L/min DL temperature 250℃ heat block temperature 400℃

After pre-treatment of the sample under the same conditions as in Example 1, the HPLC analysis result is shown in FIG. 10, and the LC-MS TIC (total ion chromatogram) pattern analysis result is shown in FIG. As shown in FIGS. 10 and 11, in the case of HA ID, component peaks of hydrolyzed oligosaccharides were detected at around 15 to 25 minutes of retention time. That is, the detected oligosaccharide was identified as a tetramer, a hexamer, an octamer, a decamer, a dodecamer, and the like. In addition, in the linear HA and crosslinked filler products, component peaks corresponding to tetrasaccharide and hexasaccharide polymers were detected at retention times of about 15 and 18 minutes, and HA- Component peaks corresponding to the BDDE complex were detected.

Table 3 summarizes the structural analysis of the HA oligosaccharide and HA-BDDE complex identified by LC-MS as described above.

turn retention time
(minute) molecular formula
(Molecular Weight) MS ¹ Identification result cation
(positive m/z) anion
(negative m/z) One 15.50 C ₂₈ H ₄₂ N ₂ O ₂₂
(758) [M+H] ⁺ 759
[M+Na] ⁺ 781
[M+K] ⁺ 797 [MH] ^- 757 tetrasaccharide polymer
(tetramer) 2 18.05 C ₄₂ H ₆₃ N ₃ O ₃₃
(1137) [MH ₂ O+H] ⁺ 1120
[M+H] ⁺ 1138
[M+NH ₄ ] ⁺ 1155
[M+Na] ⁺ 1160
[M+K] ⁺ 1176 [MH] ^- 1136 Hexaccharide Polymer
(hexamer) 3 19.21 C ₅₆ H ₈₄ N ₄ O ₄₄
(1516) [1/2M+Na+H] ⁺ 782
[M+H] ⁺ 1517
[M+NH ₄ ] ⁺ 1534
[M+Na+H] ⁺ 1540
[M+K] ⁺ 1555 8 sugar polymer
(octamer) 2a 19.23 C ₅₂ H ₈₃ N ₃ O ₃₈
(1357) [M+H] ⁺ 1358
[M+NH ₄ ] ⁺ 1375
[M+Na] ⁺ 1380 Hexaccharide Multipolymer (Pendant 1)
(hexamer-pendent complex 1) 4 20.32 C ₇₀ H ₁₀₅ N ₅ O ₅₅
(1895) [1/2M+Na] ⁺ 971
[M+NH ₄ ] ⁺ 1913 10 sugar polymer
(decamer) 5 20.95 C ₈₄ H ₁₂₆ N ₆ O ₆₆
(2275) [1/2M+H] ⁺ 1138
[1/2M+NH ₄ +H] ⁺ 1156 12 sugar polymer
(dodecamer) 2b 24.20 C ₆₆ H ₁₀₄ N ₄ O ₄₉
(1736) [M+NH ₄ ] ⁺ 1754 Hexaccharide multipolymer (cross-linking)
(hexamer-crosslink complex) 2c 24.62 C ₅₂ H ₈₃ N ₃ O ₃₈
(1357) [M+NH ₄ ] ⁺ 1375 Hexaccharide Multipolymer (Pendant 2)
(hexamer-pendent complex 2) 2d 25.23 C ₅₂ H ₈₃ N ₃ O ₃₈
(1357) [M+NH ₄ ] ⁺ 1375 Hexaccharide Multipolymer (Pendant 3)
(hexamer-pendent complex 3) 2e 25.45 C ₅₂ H ₈₃ N ₃ NaO ₃₈ ⁺
(1380) [M+Na] ⁺ 1380 Hexaccharide multipolymer (pendant)
(hexamer-pendent complex Na salt)

< comparative example 1. For LC-MS analysis HPLC Comparison of analysis conditions 1>

0.4M sodium phosphate buffer solution (pH 5.8), 0.4M ammonium acetate buffer solution (pH 5.8) was applied to the anion ion exchange resin column, and the analysis results are shown in FIG. 12 . The rest of the conditions at this time were applied in the same manner as in Example 1.

As shown in FIG. 12, unlike the results of Example 1, it was confirmed that the HA and HA-BDDE complexes were not well separated from the anionic ion exchange resin in 0.4M ammonium acetate buffer (pH 5.8), which is an organic salt.

< comparative example 2. For LC-MS analysis HPLC Comparison of analysis conditions 2>

In order to measure the molecular weight of the partially hydrolyzed HA and HA-BDDE complexes analyzed as in Example 1 by LC-MS, the LC-MS analysis conditions of Wemde et al. were reviewed. The HPLC analysis conditions at this time are shown in Table 4.

Analytical instrument Shimadzu LC-20A system (with multi-wavelength detector) column Hypercarb (150 x 4.6 mm (ID), 5 μm, Hypercarb, Thermo Fisher Scientific) mobile phase (A) Water containing 0.1% trifluoroacetic acid (TFA)
(B) Acetonitrile with 0.1% triproacetic acid (TFA) detection wavelength UV 235 nm Gradient Elution Conditions time (minutes) B% 0 20 70 40 71 20 80 20 flow rate 1.0 mL/min injection volume 50 µL column temperature 60 ℃

The results of comparing the above conditions with the results of Example 8 are shown in FIG. 13 . As shown in Figure 13, the results of Comparative Example 2 are relatively poor in detection sensitivity and component peak resolution and selectivity compared to the conditions of Example 8, making it very unsuitable for analyzing HA oligosaccharides and HA-BDDE complexes. .

So far, HA-BDDE cross-link rate among filler (HA) products has been comparatively analyzed according to the analysis conditions of each Example and Comparative Example.

The calculation formula required for analysis is as follows.

[Equation 1] Total deformation (t-MOD, %) =

[Equation 2] Cross-linking modification (c-MOD, %) =

[Equation 3] Pendant strain (p-MOD, %) = t-MOD (%) - c-MOD (%)

Here, A _HA is the total peak area of uncrosslinked HA oligosaccharide, A _HA-X-HA is the total peak area of HA-BDDE HA oligosaccharide crosslinked by cross-link, and A _HA-X is pendant ), X is the total peak area of HA oligosaccharides cross-linked with BDDE, a cross-linking agent.

Table 5 shows the HA-BDDE crosslinking rate measurement results in the filler products according to the sample pretreatment and analysis conditions of Examples and Comparative Examples according to the above calculation formula.

Crosslinking rate (%) note p-MOD
(%RSD) c-MOD
(%RSD) t-MOD
(%RSD) Example 1 9.74±0.43
(0.04) 1.21±0.20
(0.17) 10.95±0.49
(0.04) Example 2 9.01±0.45
(0.05) 0.76±0.16
(0.21) 9.77±0.62
(0.06) Example 3 9.24±0.56
(0.06) 1.01±0.23
(0.23) 10.25±0.60
(0.06) Example 4 9.75±0.55
(0.06) 1.22±0.21
(0.17) 10.97±0.45
(0.04) Example 5 8.87±0.43
(0.05) 0.80±0.18
(0.23) 9.67±0.65
(0.07) Example 6 9.05±0.43
(0.05) 0.97±0.23
(0.24) 10.02±0.56
(0.06) Example 7 9.76±0.67
(0.07) 1.20±0.21
(0.18) 10.96±0.76
(0.07) Comparative Example 1 - - - Crosslinking rate cannot be measured due to non-separation of the complex of target material HA and HA-BDDE Comparative Example 2 - - - Crosslinking rate cannot be measured due to non-separation of the complex of target material HA and HA-BDDE

Claims (6)

(Step 1) Diluting the sample with a pH 5-7 buffer solution so that the final concentration of hyaluronic acid (HA) in the sample is 0.1-0.8% by weight;
(Step 2) adding hyaluronic acid hydrolase to the sample of step 1 to be 40 to 400 U/mL;
(Step 3) preparing a hydrolyzate by hydrolyzing the sample to which the hyaluronic acid hydrolase of the second step was added at 37 to 50 ° C. for 48 to 120 hours;
(Step 4) Identifying non-crosslinked and crosslinked component peaks of the sample hydrolyzate from Step 3 using high-performance liquid chromatography-mass spectrometry (LC-MS); and
(Step 5) measuring the cross-linking rate of the sample by applying the analysis result of step 4 to Equation 1 below;
Method for measuring crosslinking rate between hyaluronic acid-crosslinking agent comprising a.
[Formula 1]
Total strain (crosslink rate; t-MOD, %) =

(Where, A _HA is the total peak area of sample hydrolysates that are not cross-linked (non-cross-linked), A _HA-X-HA is the total peak area of sample hydrolysates that are cross-linked, A _HA-X is the total peak area of the sample hydrolyzate cross-linked with a pendant) According to claim 1,
The method for measuring the crosslinking rate between hyaluronic acid and crosslinking agent, characterized in that the crosslinking rate is expressed as a degree of crosslinking deformation and a degree of pendant deformation by Equations 2 and 3 below.
[Formula 2]
Crosslinking Modification (c-MOD, %) =

(Where, A _HA is the total peak area of sample hydrolysates that are not cross-linked (non-cross-linked), A _HA-X-HA is the total peak area of sample hydrolysates that are cross-linked, A _HA-X is the total peak area of the sample hydrolyzate cross-linked with a pendant)
[Formula 3]
Pendant strain (p-MOD, %) = t-MOD (%) - c-MOD (%) According to claim 1,
The hyaluronic acid (HA) is a method for measuring crosslinking rate between hyaluronic acid-crosslinking agent, characterized in that the high molecular weight of the disaccharide having 20 ~ 20,000 kDa. According to claim 1,
Wherein the hyaluronic acid is one selected from the group consisting of an insert for treating arthritis, an insert for treating wrinkles, a cosmetic filler, and a drug delivery system. According to claim 1,
The crosslinking agent is butanediol diglycidyl ether (1,4-butandiol diglycidyl ether; BDDE), ethylene glycol diglycidyl ether, and hexanediol diglycidyl ether (1,6-hexanediol diglycidyl ether). ), propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, neopentyl glycol Diglycidyl ether (neopentyl glycol diglycidyl ether), polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, Tri-methylpropane polyglycidyl ether, 1,2-bis(2,3-epoxypropoxy)ethylene, pentaerythritol polyglycidyl ether, sorbitol Hyaluronic acid, characterized in that it is 1 selected from the group consisting of sorbitol polyglycidyl ether, divinyl sulfone, epichlorohydrin, epibromohydrin and combinations thereof Method for measuring crosslinking rate between ronic acid and crosslinking agent. According to claim 1,
The buffer solution in the first step is hyaluronic acid-crosslinking agent, characterized in that at least one selected from the group consisting of sodium phosphate, potassium phosphate, ammonium acetate and ammonium formate Method for measuring crosslinking rate.

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