YY/T 1571-2017 English PDF (YYT1571-2017)
YY/T 1571-2017 English PDF (YYT1571-2017)
YY/T 1571-2017: Tissue engineering medical device products—Sodium hyaluronate
PHARMACEUTICAL INDUSTRY STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
Replacing YY/T 0606.9-2007
Tissue engineering medical device products - Sodium
ISSUED ON: MAY 02, 2017
IMPLEMENTED ON: APRIL 01, 2018
Issued by: China Food and Drug Administration
Table of Contents
Foreword ... 3
1 Scope ... 6
2 Normative references ... 6
3 Terms and definitions ... 7
4 Classification ... 7
5 Requirements ... 8
6 Test methods ... 10
7 Marking ... 14
8 Packaging, transportation and storage ... 15
Appendix A (Normative) Determination of sodium hyaluronate content ... 16 Appendix B (Normative) Determination of protein content ... 19
Appendix C (Normative) Determination of residual ethanol (headspace gas chromatography) ... 21
Appendix D (Informative) Determination of residual quaternary ammonium salt (cetylpyridinium chloride) ... 23
Appendix E (Normative) Determination of weight-average molecular weight and molecular weight distribution coefficient ... 26
References ... 28
Tissue engineering medical device products - Sodium
This Standard specifies the requirements and test methods for sodium hyaluronate used in surgical implants and tissue engineering medical device products.
This Standard applies to the preparation of sodium hyaluronate for tissue engineering medical device products and their scaffold materials.
2 Normative references
The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies to this document. For undated references, the latest edition (including any amendment) applies to this document.
GB/T 191, Packaging - Pictorial marking for handling of goods
GB/T 14518, Determination of the pH of adhesives
GB/T 16886.1, Biological evaluation of medical devices - Part 1: Evaluation and testing within a risk management process (GB/T 16886.1-2011, ISO 10993-1:2009, IDT)
GB 18278 (all parts), Sterilization of health care products - Moist heat GB 18279 (all parts), Sterilization of health care products - Ethylene oxide GB 18280 (all parts), Sterilization of health care products - Radiation YY/T 0313, Medical polymer products - Requirement for package and information supplied by manufacturer
YY/T 0606.25, Tissue engineered medical product - Part 25: Quantification of remnant DNA in biological materials utilizing animal tissues and their derivatives: Fluorescence method
YY/T 0771.1, Medical devices utilizing animal tissues and their derivatives - Part 1: Application of risk management (YY/T 0771.1-2009, ISO 22442-1:2007, IDT) 5 Requirements
White or off-white powder or granular or fibrous solid, without any visible foreign matter.
Typical Fourier transform infrared spectrum (FT-IR) frequencies (cm-1) of sodium hyaluronate are 3 275 ~ 3 390(b), 1 615(s), 1 405(m), 1 377(m), 1 150, 1 077, 1 045(s), 946(m), 893(w). The wavenumber error of the measured band in the fingerprint area shall be less than the specified value ±5 cm-1 (0.5%).
Note: s, strong; m, medium; w, weak; b, broad.
5.3 Sodium hyaluronate content
Based on the dry product, the content of sodium hyaluronate shall be 95.0% ~ 105.0% (mass fraction).
The pH of the 0.5% concentration solution shall be 5.0 ~ 8.5.
5.5 Intrinsic viscosity
It shall be 90% ~ 120% of its marked value.
5.6 Protein content
It shall not exceed 0.1% (mass fraction).
5.7 Nucleic acid content
5.7.1 Bacterial fermentation method
For solution of the concentration 0.33%, the absorbance value (OD value) at the wavelength of 260 nm shall not exceed 0.5.
5.7.2 Tissue extraction method
In accordance with YY/T 0606.25, the limit value of residual DNA is given according to the possible intended use.
5.8 Heavy metal content
It shall not exceed 10 μg/g (mass fraction).
The number of bacterial colonies per 1 g of the test product shall not exceed 102 CFU; the number of mold and yeast colonies shall not exceed 20 CFU. Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli shall not be detected.
Note: If sodium hyaluronate is provided in a non-sterile manner, this item shall be tested; if the final product is required to be sterile, it should be sterilized to meet the sterile requirements.
5.19 Bacterial endotoxins
The amount of endotoxin contained per 1 mg of sodium hyaluronate shall be less than 0.05 EU.
5.20 Raw material safety
5.20.1 The sodium hyaluronate prepared by the bacterial fermentation method shall be tested for hemolytic streptolysin, and the result shall be free from hemolytic ring. 5.20.2 The sodium hyaluronate prepared by the tissue extraction method shall be subjected to safety evaluation and quality control in accordance with the requirements of the series industry standards for medical devices utilizing animal tissues and their derivatives (YY/T 0771.1, YY/T 0771.2, YY/T 0771.3).
5.21 Biological evaluation
The sodium hyaluronate shall be subjected to biological evaluation according to the requirements of GB/T 16886.1; sodium hyaluronate shall not release substances that have adverse effects on the human body.
6 Test methods
Perform direct observation with the naked eye, which shall comply with the provisions of 5.1.
6.2 Fourier transform infrared spectrum (FT-IR)
Prepare the sample by the potassium bromide tablet method, and determine according to the method specified in the general rule 0402 of the Pharmacopoeia of the People’s Republic of China (2015 Edition, Volume IV), which shall meet the requirements of 5.2. 6.3 Determination of sodium hyaluronate content
Measure according to the method specified in Appendix A, which shall comply with the provisions of 5.3.
6.4 pH determination
According to the method stipulated in the general rule 0831 of Pharmacopoeia of the People’s Republic of China (2015 Edition, Volume IV), take 0.5 g of this product, use phosphorus pentoxide as a desiccant, and dry it at 105 °C for 6 h for determination, which shall meet the requirements of 5.10.
6.11 Determination of quaternary ammonium residues
Measure by referring to the method specified in Appendix D, which shall comply with the provisions of 5.11.
6.12 Determination of sulfated mucopolysaccharides
Test solution: Take about 50.0 mg of this product (based on the dry product); add 1.0 mL of perchloric acid to a stoppered test tube (length is 5 cm; inner diameter is 1.6 cm); dissolve it; use it as the test solution.
Control solution: Weigh 0.149 g of anhydrous sodium sulfate; add water to dilute to 100 mL, to dissolve it. Then, take 10.0 mL of the above solution; add water to dilute to 100 mL, to obtain it; take 1.0 mL of the solution; put it in a stoppered test tube; heat and evaporate to dryness at 90 °C ~ 95 °C; then, add 1.0 mL of perchloric acid as a control solution.
For both the test solution and the control solution, use glass wool to plug the test tube; heat at 180 °C until clear (about 12 h); then, cool to room temperature. Add 3.0 mL of 33.3 g/L barium chloride solution respectively; shake after sealing; place for 30 min. After shaking, follow the method specified in the general rule 0401 of Pharmacopoeia of the People’s Republic of China (2015 Edition, Volume IV); use water as a blank; measure the absorbance of the test solution and the control solution at a wavelength of 660 nm. If the absorbance of the test solution is less than the absorbance of the control solution, it meet the requirements of 5.12.
6.13 Determination of iron content
Take 0.25 g of this product (based on the dry product); add 1.0 mL of nitric acid; heat it in a water bath to dissolve; prepare 5 portions in parallel; after cooling, add water to one part to dilute to 10 mL as the test solution, and add 0.5 mL, 1.0 mL, 1.5 mL and 2.0 mL of iron standard solution (each 1.0 mL is equivalent to 10 μg/mL iron) to the other 4 portions, respectively; then, add water to dilute to 10 mL, as the control solution; according to the method stipulated in method II of the general rule 0406 of Pharmacopoeia of the People’s Republic of China (2015 Edition, Volume IV), measure it at a wavelength of 248.3 nm, which shall meet the requirements of 5.13. 6.14 Determination of chlorine content
Take 67 mg of this product; put it in a 100 mL measuring bottle; add water to dissolve and dilute to the mark. Take 15 mL and place it in a 25 mL Nessler colorimetric tube as the test solution; take another 10 mL of standard sodium chloride solution (1.0 mL is equivalent to 5 μg of Cl) and 5 mL of water as the control solution. Add 1.0 mL of dilute nitric acid (take 20 g of nitric acid and add water to dilute to 100 mL) and 1.0 mL of silver nitrate test solution to the test solution and the control solution, respectively; mix well; place in a dark place for 5 minutes; observe from the side on a black background. It shall comply with the provisions of 5.14.
6.15 Determination of molecular weight and molecular weight distribution Measure according to the method specified in Appendix E, which shall comply with the provisions of 5.15.
6.16 Determination of clarity and color of solution
Take 0.10 g of this product (based on the dry product); add 30 mL of 0.9% sodium chloride solution; shake to mix and dissolve, which shall meet the requirements of 5.16. 6.17 Sterility test
Measure according to the method specified in the general rule 1101 of Pharmacopoeia of the People’s Republic of China (2015 Edition, Volume IV), which shall meet the requirements of 5.17.
Note: If sodium hyaluronate is provided in a sterile manner, perform the test of this item, otherwise, perform the test of 6.18.
6.18 Microbial limits
Take 5.0 g of this product; add 100 mL of sterile phosphate buffered saline (pH 7.2) containing 45 000 units of hyaluronidase; shake and dissolve in a water bath at 42 °C for 1 h, to obtain a 1:20 test solution. Measure according to the method specified in the general rules 1105 and 1106 of Pharmacopoeia of the People’s Republic of China (2015 Edition, Volume IV), which shall meet the requirements of 5.18.
6.19 Bacterial endotoxin test
Use the water for detecting bacterial endotoxin to prepare 2 mg/mL sodium hyaluronate; measure according to the method specified in the general rule 1143 of Pharmacopoeia of the People’s Republic of China (2015 Edition, Volume IV), which shall meet the requirements of 5.19.
6.20 Hemolytic streptolysin test
Use normal saline to prepare 2 mg/mL sodium hyaluronate; take 1 mL and inoculate it directly on the blood agar plate medium; incubate at 37 °C for 24 h, which shall meet the requirements of 5.20.1.
Note: This clause applies to sodium hyaluronate by bacterial fermentation method. Appendix A
Determination of sodium hyaluronate content
The glucuronic acid produced by the hydrolysis of sodium hyaluronate reacts with the carbazole reagent to produce purple red, and the depth of the generated color is directly proportional to the content of glucuronic acid. Therefore, the content of sodium hyaluronate is represented by the measured glucuronic acid content.
A.2.1 Analytical balance.
A.2.2 UV spectrophotometer or equivalent equipment.
A.2.3 Vortex mixer or equivalent equipment.
A.3 Solution preparation
A.3.1 Carbazole test solution
Weigh 0.125 g of carbazole; add 100 mL of absolute ethanol to dissolve. Store in a dark place.
A.3.2 Glucuronic acid (GA) standard solution
Accurately weigh about 0.1 g of the glucuronic acid reference substance, that has been vacuum-dried to constant weight at 105 °C with phosphorus pentoxide, as a desiccant; put it in a 100 mL measuring bottle; add water to dissolve and dilute it to the mark; shake well; use it as a stock solution. Precisely measure 5.0 mL of the stock solution; put it in a 100 mL measuring bottle; add water to make a solution containing 50 μg per 1.0 mL; shake well; store at 4 °C.
A.3.3 0.025 mol/L sodium tetraborate sulfuric acid solution
Weigh 9.54 g of sodium tetraborate (Na2B4O7·10H2O); add it to 1 L of concentrated sulfuric acid; cover it. Shake occasionally until the sodium tetraborate is completely dissolved. Store at room temperature.
Note: The reagents used in the test are all analytical reagents, and the sulfuric acid should be guaranteed reagent.
A.4 Sample preparation
Determination of protein content
Foline-phenol test solution can react with the protein in the hyaluronic acid solution, and its color depth is proportional to the protein concentration.
Analytical balance, UV spectrophotometer or equivalent, vortex mixer or equivalent. B.3 Solution preparation
B.3.1 Alkaline cupric tartrate test solution
Weigh 0.5 g of copper sulfate and 1.0 g of sodium tartrate in a 50 mL volumetric flask; add water to dissolve and dilute to the mark. This is liquid A; weigh 2.0 g of anhydrous sodium carbonate in a 50 mL volumetric flask; add 0.4 g of sodium hydroxide; add water to dissolve and dilute to the mark, this is liquid B. Mix liquid A and liquid B at a ratio of 1:50 and shake well. Prepare the above test solutions when needed. B.3.2 Folin test solution (prepare as follow or purchase)
Weigh 100 g of sodium tungstate and 25 g of sodium molybdate; add 700 mL of water, 50 mL of 85% phosphoric acid and 100 mL of hydrochloric acid; put them in a flask; heat and reflux slowly for 10 h; let cool; add 150 g of lithium sulfate, 50 mL of water and a few drops of bromine solution; boil about 15 min, until the bromine is completely removed; let it cool to room temperature; add water to make it 1 000 mL. Filter; use the filtrate as a stock solution; put it in a brown bottle; store it in a refrigerator at 2 °C ~ 8 °C. Before use, take the stock solution; dilute it with water at 1:1; mix well. B.3.3 Preparation of standard solution
Accurately weigh an appropriate amount of bovine serum albumin reference substance; add water to make a solution containing about 50 μg per 1.0 mL; shake well. B.4 Sample preparation
Accurately weigh about 25 mg (m) of sodium hyaluronate; put it in a 15 mL test tube with a stopper; add 2.5 mL of water to swell the sample until it is completely dissolved; seal it with a stopper. Determine within 24 hours.
Determination of residual ethanol (headspace gas chromatography)
Dissolve the sample in 0.1 mol/L sodium hydroxide solution. At a certain temperature, draw the sample through the headspace and pass through the chromatographic column, so that the ethanol to be measured is separated from other components; detect by a hydrogen flame ionization detector; compare the obtained ethanol chromatographic peak with the chromatographic peak obtained by the external standard; calculate. C.2 Equipment
C.2.1 Gas chromatograph (equipped with FID detector).
C.2.2 Headspace sampler.
C.2.3 Capillary chromatographic column: DB-624 (0.45 mm × 30 m, film thickness 2.55 μm) or other chromatographic columns with the same separation effect. C.2.4 20 mL headspace vial.
C.2.5 Absolute ethanol: chromatographic pure.
C.3 Solution preparation
C.3.1 Blank solution
Pipette 5.0 mL of 0.1 mol/L sodium hydroxide solution into a 20 mL headspace vial, and seal it.
C.3.2 Standard solution
Accurately weigh about 0.1 g of ethanol into a 100 mL volumetric flask that has been added with 10 mL of water; add water to dilute to the mark; shake well; accurately measure 5 mL; put it in a 50 mL volumetric flask; add 0.1 mol/L sodium hydroxide solution to dilute to the mark; shake well; accurately measure 5 mL into a 20 mL headspace vial; seal it; use it as a standard solution.
C.4 Sample preparation
Accurately weigh about 0.1 g of sodium hyaluronate; place it in a 20 mL headspace vial; add 5.0 mL of 0.1 mol/L sodium hydroxide solution; seal it; place it overnight to dissolve.
C.5 Operating conditions
C.5.1 Column temperature: Maintain the initial temperature at 40 °C, for 5 minutes; raise to 150 °C at 20 °C/min, and maintain for 3 minutes. Raise to 200 °C at 30 °C/min and maintain for 3 min.
C.5.2 Temperature of the sample injector: 200 °C.
C.5.3 Temperature of the detector: 250 °C.
C.5.4 Carrier gas: nitrogen, 5 mL/min.
C.5.5 Headspace vial equilibrium temperature: 80 °C, equilibrium time: 30 min. C.6 Determination method
Take the standard solution for headspace injection; inject 5 times continuously; record the chromatogram. The separation between the ethanol peak and other peaks shall be greater than 2.0, and the relative standard deviation (RSD) of the peak area shall be less than 10%. Take the blank solution and the sample solution for headspace injection; record the chromatogram.
C.7 Result calculation
Calculate residual ethanol according to Formula (C.1).
w – residual ethanol content, in micrograms per gram (μg/g);
Ai – peak area of ethanol in the spectrum of the sample solution;
A0 – peak area of ethanol in the spectrum of the blank solution;
Asi – peak area of ethanol in the spectrum of the standard solution;
m – sample mass of the sodium hyaluronate, in grams (g);
msi – mass of ethanol in the standard stock solution, in grams (g).
Determination of residual quaternary ammonium salt (cetylpyridinium chloride) D.1 Principle
Taking advantage of the characteristics of CPC being soluble in organic solvents, first use 25% sodium chloride solution to infiltrate the dry powder of sodium hyaluronate, then use acetonitrile to extract; then, use high performance liquid chromatography to separate cetylpyridinium chloride (CPC) from other components; detect by an ultraviolet detector; then, quantitatively determine.
D.2 Equipment and reagents
D.2.1 High performance liquid chromatography Agilent technologies 1260 infinity. D.2.2 Waters chromatographic column: Symmetry Shield RP18.5 μm, (2.1×150) mm. D.2.3 METTLER TOLEDO analytical balance.
D.2.4 Vortex mixer.
D.2.5 Acetonitrile: chromatographic pure (purity >99.9%).
D.2.6 Formic acid: chromatographic pure (purity >99.9%).
D.2.7 Ultrapure water.
D.2.8 Standard substance: cetylpyridinium chloride (purity >99.5%).
D.3 Chromatographic testing conditions
D.3.1 Mobile phase A: acetonitrile, mobile phase B: 0.25% formic acid aqueous solution, A: B = 38: 62.
D.3.2 Instrument settings: detection wavelength: 259 nm; injection volume: 10 μL; flow rate: 0.4 mL/min; column temperature: 25.0 °C; program time: 15 min.
D.4 Solution preparation
D.4.1 Preparation of acetonitrile: water (1:1) solution: add 100 mL of water to 100 mL of acetonitrile; mix well.
D.4.2 Preparation of 25% sodium chloride solution: add 100 mL of water to 25.0 g of sodium chloride; dissolve and mix well.