YY 0954-2015Nonactive surgical implants - Type I collagen implants - Specific requirements (English PDF)

7.2 Preclinical evaluation

7.2.1 General preclinical for preclinical evaluation

The preclinical evaluation of collagen implants shall comply with the requirements in 7.2 of YY/T 0640-2008.

In accordance with the requirements of the experiment, all test samples shall be the finally sterilized equipment or components.

The number of samples shall be chosen in accordance with the requirements of this standard.

Note: The preclinical evaluation and the proven test methods in this standard reflect the current level of technological development.

7.2.2 Appearance

It shall be white, milky white or yellowish viscous liquid, without visible foreign matter.

7.2.3 Loading capacity

It shall not be less than 90% of the indicated capacity.

7.2.4 Dynamic viscosity

It shall be within the indicated range.

7.3 Chemical characteristics

7.3.1 Type I collagen identification

After the SDS-polyacrylamide gel electrophoresis analysis, COMPARE the electrophoretic bands of the samples and the type I collagen reference product,

AND the electrophoretic bands shall be consistent.

7.3.2 Collagen content

It shall be 80% ~ 120% of the indicated amount.

7.3.3 Protein impurity analysis

8.4 Identification of type I collagen

USE the 3% acetic acid or 0.01 mol/L hydrochloric acid to dilute the sample into a solution with a collagen concentration of 1 mg/mL ~ 2 mg/mL. FOLLOW the methods as specified in Appendix IV C of the Pharmacopoeia of the People's

Republic of China (volume III) (2010 version); USE the SDS-polyacrylamide gel electrophoresis, with the separation gel concentration in 7% AND the sample amount of 20 µL. Through analyzing the sample and type I collagen reference substance, the results shall comply with the provisions of 7.3.1.

8.5 Collagen content

FOLLOW the method of Appendix A, AND the results shall comply with the provisions of 7.3.2.

8.6 Protein impurity analysis

FOLLOW the method of Appendix B, AND the results shall comply with the provisions of 7.3.3.

8.7 pH

FOLLOW the methods as specified in Appendix VI H of Pharmacopoeia of the

People's Republic of China (volume II) (2010 version), AND the results shall comply with the provisions of 7.3.4.

8.8 Residues on ignition

FOLLOW the methods as specified in Appendix VI N of the Pharmacopoeia of the People's Republic of China (volume II) (2010 version), AND the results shall comply with the provisions of 7.3.5.

8.9 Total heavy metals and trace elements

8.9.1 Total amount of heavy metals (calculated as Pb)

The residues left under the residues on ignition shall follow the 2 nd method of

Appendix VIII H of the Pharmacopoeia of the People's Republic of China (volume II) (2010 version), AND the results shall comply with the provisions of

7.3.6.1.

8.9.2 Trace elements

FOLLOW the methods as specified in Appendix C; AND the results shall comply with the provisions of 7.3.6.2.

13.1 General principles

The requirements in Chapter 11 of YY/T 0640-2008 AND the following requirements apply.

13.2 Implants materials

The manufacturer shall provide a description of the implant material.

13.3 Nominal capacity

It shall indicate the nominal capacity of the implants.

13.4 Information on life expectancy

The manufacturer shall provide information relevant to the intended sustained performance of the device in accordance with the design. Such information includes indications that are important factors influencing the actual life of the implant.

Note: In practice, it is not possible to accurately predict the actual life of the implant. This prediction is only a design requirement.

13.5 Operating instructions

If applicable, the instruction manual shall include the following information:

a) The name and address of the manufacturer;

b) The description of the implant, including the type and the main chemical composition;

c) If applicable, the contents of the package as sterile and the indications of the sterilization method used;

d) The instructions for the intended disposable use of the implant;

e) Life expectancy, expressed in accordance with 13.4;

A.1 Method I Determination by amino acid analyzer

A.1.1 Principles

Hydroxyproline is a specific amino acid contained in collagen, AND the content is relatively stable. MAKE sample be hydrolyzed to a single amino acid at

110 °C under the reaction of c (HCl) = 6 mol/L hydrochloric acid, and USE amino acid analyzer to determine the hydroxyproline content in the sample, in order to calculate the collagen content in the sample .

A.1.2 Instruments and reagents

A.1.2.1 Instruments

Balance;

Amino acid analyzer;

Rotary evaporator or concentrator;

Oven or hydrolysis furnace.

A.1.2.2 Reagents

L-hydroxyproline reference substance: the national standard product.

Hydrochloric acid solution, c (HCl) = 6 mol/L: MIX the guaranteed reagent hydrochloric acid and water at equal volume.

Sodium citrate buffer for elution: PREPARE it in accordance with the instrument instruction manual.

Ninhydrin solution: PREPARE it in accordance with the instrument instruction manual.

A.1.3 Preparation of L-hydroxyproline reference substance solution

A.1.3.1 L-hydroxyproline reference substance stock solution

TAKE appropriate amount of L-hydroxyproline reference substance into a 100 mL volumetric flask;

B.1 Principle

Using the triple helix structure of collagen which does not found in other proteins, in combination of the specific collagenase activity, through determination of the bovine serum albumin (BSA) staining limit by Coomassie Brilliant Blue,

DETERMINE the total amount of protein impurity in the collagen sample.

B.2 Instruments and reagents

B.2.1 Instruments

B.2.1.1 Balance.

B.2.1.2 Electrophoresis apparatus.

B.2.1.3 Vertical plate electrophoresis tank and plastic mold.

B.2.1.4 Image analysis system.

B.2.2 Reagents

B.2.2.1 Collagenase.

B.2.2.2 Bovine serum albumin (BSA).

B.2.2.3 Collagen reference substance.

B.2.2.4 Sodium dodecyl sulfate (SDS).

B.2.2.5 Trimethylolaminomethane (Tris).

B.3 Preparation of solution

B.3.1 Preparation of collagenase digestion solution

USE 20 mmol/L NaH2 PO 4 solution (pH 7.4, containing 0.1 mmol/L CaCl 2 ) to dissolve the collagenase AND prepare the collagenase digestion solution containing appropriate activity units (for example, 1.25 Unit/mL).

B.3.2 Preparation of sample buffer solution

respectively MEASURE 2.5 mL of

0.5 mol/L Tris-HCl, 2.0 mL of glycerol, 4.0 mL of 10% SDS, and 0.5 mL of 0.1% bromophenol blue; USE the ultra-pure water to make the volume reach to 10 mL.

B.3.3 Preparation of electrophoresis buffer solution

respectively WEIGH 15.1 g of Tris, 72.0 g of glycine, and 5.0 g of SDS; DISSOLVE in the ultra-pure water;

MAKE the volume reach to 1000 mL; before use, DILUTE it for 5 times.

B.3.4 Preparation of Coomassie brilliant blue staining solution

WEIGH 1 g of

Coomassie Brilliant Blue R250; ADD 200 mL of methanol, 50 mL of glacial acetic acid, and 250 mL of water; MIX it uniformly.

B.3.5 Preparation of Coomassie Brilliant Blue decolorization solution: TAKE

400 mL of methanol, 100 mL of glacial acetic acid, and 500 mL of water; MIX it uniformly.

B.3.6 Separation gel concentration

7%.

B.3.7 Spacer gel concentration

4%.

B.4 Determination

B.4.1 BSA staining limit analysis by Coomassie Brilliant Blue

B.4.1.1 Preparation of BSA solution

USE the ultra-pure water to dilute the BSA a gradient concentration.

B.4.1.2 SDS-PAGE analysis

TAKE 20 µL of BSA solution at each concentration.

After loading sample, CONNECT the electrodes of the electrophoresis tank into the electrophoresis apparatus; ADJUST the voltage to 110 V; START electrophoresis. When the blue line of bromophenol blue is observed to fall below the metal wire of the electrophoresis tank, STOP electrophoresis. After electrophoresis, REMOVE the film and ADD appropriate amount of Coomassie

Brilliant Blue staining solution; SHAKE to stain it for 2 h and LET it standing for overnight. USE the decolorization liquid and SHAKE it until the gel bottom color is almost colorless, during which REPLACE the deionized water for 2 times;

after finishing electrophoresis, USE the image analysis system to analyze the decolorized film, in order to determine the BSA staining limit by Coomassie

Brilliant Blue.

B.4.2 Analysis of protein impurity in samples

B.4.2.1 Collagen reference substance

USE 3% acetic acid or 0.01 mol/L hydrochloric acid to dissolve the collagen reference substance, in order to make the collagen concentration reach to 1 mg/mL.

B.4.2.2 Sample a

USE the 3% acetic acid or 0.01 mol/L hydrochloric acid to […]

C.1 Principles

The sample is digested with nitric acid to prepare the test solution; USE the atomic absorption spectrophotometer to determine the content of cadmium, copper, iron, nickel, lead and molybdenum. USE the fluorescence spectrophotometry to determine the content of arsenic and mercury.

C.2 Reagents

Nitric acid: excellent grade pure.

C.3 Preparation of standard solutions

As for the chromium, cadmium, copper, iron, nickel, lead, molybdenum, arsenic, mercury standard solution, FOLLOW the aforementioned routine methods to prepare 1 mg/mL stock solution; before use, USE the nitric acid of volume fraction 1% to prepare it to the required concentration.

C.4 Preparation of test solution

TAKE sample and accurately WEIGH it; PLACE it into beaker; ADD 10 mL of nitric acid; HEAT it on the electric plate until the solution clarifies; CONTROL the temperature; OPEN the port to drain the acid to almost empty; ADD appropriate amount of nitric acid of volume fraction 1%; HEAT it to almost boiling; LET it cool down; TRANSFER the solution into the volumetric flask;

USE the nitric acid of volume fraction 1% to wash the beaker for several times;

Combine the wash solution into the volumetric flask; USE the 1% nitric acid to dilute it to the mark.

C.5 Determination

C.5.1 Determination of cadmium, iron, lead, chromium, nickel, copper and molybdenum

TAKE the test solution as prepared in accordance with C.4; FOLLOW the methods as specified in Appendix IV D of the Pharmacopoeia of the People's

Republic of China (volume 2) (2010 version).

E.1 Principles

At 110 °C and the effects of alkali, tryptophan can be hydrolyzed from the protein. The indole group in tryptophan can react with acetaldehyde to produce a purple-red material, which can be used for tryptophan examination.

E.2 Reagents

E.2.1 Alkaline solution-lithium hydroxide solution, c (LiOH) = 4 mol/L: WEIGH

167.8 g of lithium hydroxide; USE water to dissolve and dilute it to 1000 mL.

E.2.2 Sodium citrate buffer solution (pH2.2), c (Na

+

) = 0.2 mol/L: WEIGH 19.6 g of trisodium citrate; USE water to dissolve it; ADD 16.5 mL of guaranteed reagent hydrochloric acid; ADD water to make the volume reach to 1000 mL;

SHAKE it uniformly.

E.2.3 Hydrochloric acid solution, c (HCl) = 6 mol/L

MIX the guaranteed reagent hydrochloric acid and water at equal volume.

E.3 Alkaline hydrolysis

TAKE 1 mL of sample and PLACE it in a Teflon hydrolysis tube; ADD 1.5 mL of antalkali (E.2.1); CHARGE nitrogen (at least 5 min); SEAL the tube. PLACE the hydrolysis tube in a (11 ± 1) °C constant temperature oven for hydrolysis 20 h;

TAKE the hydrolysis tube out; COOL it to room temperature; OPEN the tube;

USE the sodium citrate buffer solution (E.2.2) to quantitatively transfer the hydrolyzate to a 10 mL or 25 mL volumetric flask; ADD about 1.00 mL of hydrochloric acid solution (E.2.3) for neutralization; USE the aforementioned sodium citrate buffer solution to make the volume reach to the mark. After using

0.45 µm membrane to filter it; TAKE the supernatant as the alkaline hydrolysis solution.

E.4 Inspection

TAKE a few drops of alkaline hydrolysis solution as prepared by E.3; ADD about

1mL of glacial acetic acid containing a small amount of glyoxylic acid; along the tube wall, slowly ADD about 1 mL of sulfuric acid; MAKE them overlap BUT not mix; PLACE it in water bath for warming up; after standing, OBSERVE the liquid […]