YY/T 1561-2017Tissue engineering medical device products - Residual alpha-Gal antigen determination in scaffold materials utilizing animal tissues and their derivatives (English PDF)

Foreword

This document was issued on 28 March 2017 by the China Food and Drug Administration, and has applied since 1 April 2018.

It is a YY/T standard: a recommended industry standard of China's pharmaceutical and medical device sector.

1 Scope

This standard provides a quantitative method for the determination of residual alpha-Gal antigen in biological materials derived from animal tissues and their derivatives used to prepare scaffold materials for tissue-engineered medical products.

This standard applies to the determination of alpha-Gal antigen in the various biological materials derived from animal tissues and their derivatives used to prepare scaffold materials for tissue-engineered medical products.

2 Normative references

The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies.

GB/T 16886.20, Biological evaluation of medical devices - Part 20: Principles and methods for immunotoxicology testing of medical devices (GB/T 16886.20-2015, ISO/TS 10993-20:2006, IDT)

YY/T 0606.25, Tissue engineered medical products - Part 25: Quantification of residual DNA in biological materials derived from animal tissues and their derivatives - Fluorescence method

3 Terms and definitions

For the purposes of this document, the terms and definitions given in GB/T 16886.20 and YY/T 0606.25 apply.

4 Reagents and instruments

4.1 Reagents include: a) phosphate buffered saline (PBS, pH 7.4); and the further reagents listed in the standard.

5 Test procedures

5.1.1 Test sample: weigh 2 mg to 10 mg of the test sample accurately into a 2.0 mL or 5 mL sterile centrifuge tube. Cut solid pieces or sheets into small fragments with sterile ophthalmic scissors; liquids and powders need no treatment. Add lysis solution (a commercial product may be used), with 1 % (volume fraction) of 1 mmol/L PMSF added before use, to a sample concentration of 2 mg/mL to 10 mg/mL; 2 mL of lysis solution per sample is recommended. Homogenize at low temperature for 2 min to 10 min until a uniform homogenate is formed, leave at room temperature (25 °C ± 5 °C) for 30 min to 3 h until fully lysed, centrifuge at 3,000 r/min to 5,000 r/min and keep the supernatant.

Note 1: Three parallel samples are tested for each sample and the result is reported as mean ± SD.

Note 2: At the client's request, three samples of different batches or of the same batch may be taken to evaluate the stability of the process between or within batches.

5.1.2 Standard curve samples: 2 mg of Gal-antigen-negative biological reference material (lyophilized powder) is lysed at 2 mg/mL in lysis solution containing Gal alpha 1-3 Gal-BSA, and at least five serial dilutions of Gal-BSA are prepared for the standard curve, down to a concentration whose OD equals that of the negative reference, which fixes the minimum detectable concentration of the test.

5.1.3 Gal-antigen-positive and negative reference samples: 2 mg of each reference material is lysed at 2 mg/mL in the same way and the supernatant kept.

5.1.4 Control wells: one M86/Gal-negative reference reaction sample gives the 100 % reaction value, and one lysis solution reaction sample gives the background.

5.2 M86 antibody incubation: 200 µL of each sample is mixed with an equal volume of M86 antibody diluted 1:100 to 1:200 (the antibody must be in excess, as confirmed by preliminary tests), shaken gently for 2 h at room temperature and incubated overnight at 4 °C; the supernatant is collected after centrifugation at 14,000 g and 4 °C for 30 min.

Note: M86 is a non-purified antibody whose activity may vary between batches; confirm the activity of each new batch and, if necessary, re-verify the dilution ratio.

5.3.1 Antigen-coated plates: Gal alpha 1-3 Gal-BSA is diluted in deionized water and then in carbonate buffer pH 9.5 to 2 µg/mL, added at 100 µL per well, shaken for 2 h at room temperature and coated overnight at 4 °C. The plates are washed at least three times with 0.05 % Tween-20/PBS, blocked with 200 µL per well of 1 % serum albumin at 37 °C for 2 h, washed and dried. Coated plates may be sealed and kept at 4 °C for use within one week.

6 Calculation of antigen content

The M86 binding inhibition rate of the test sample is entered in the exponential equation of the standard curve to obtain the corresponding Gal-BSA mass concentration X (µg/mL), from which the number of Gal epitopes per unit mass of the test sample, Nt, is calculated using the reaction volume V, the number of Gal epitopes per unit of Gal-BSA, NS = 1.82 x 10^14 per µg, and the mass of the test sample Mt.

Note: Gal-BSA mass is approximately equal to its BSA content (molecular mass 66.33 kD, 9.08 x 10^18 molecules per g), and each BSA molecule carries about 20 Gal residues, giving 1.82 x 10^14 Gal residues per µg of Gal-BSA.

Where the antigen in the test sample is at trace level and difficult to quantify, the result is reported as below the minimum detectable concentration of the test. Samples taken before decellularization or antigen removal can be tested at the same time as a reference for the effectiveness of the process.