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YY/T 1561-2017 (YYT 1561-2017)

YY/T 1561-2017_English: PDF (YYT1561-2017)
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YY/T 1561-2017English165 Add to Cart 0--3 minutes. Auto-delivered. Tissue engineering medical device products - Remnant α-Gal antigen determination in scaffold materials utilizing animal tissues and their derivatives YY/T 1561-2017 Valid YY/T 1561-2017

BASIC DATA
Standard ID YY/T 1561-2017 (YY/T1561-2017)
Description (Translated English) Tissue engineering medical device products--Remnant ��-Gal antigen determination in scaffold materials utilizing animal tissues and their derivatives
Sector / Industry Medical Device & Pharmaceutical Industry Standard (Recommended)
Classification of Chinese Standard C45
Classification of International Standard 11.040.40
Word Count Estimation 10,150
Date of Issue 2017-03-28
Date of Implementation 2018-04-01
Drafting Organization China Institute of Food and Drug Test, the Fourth Military Medical University of Chinese People's Liberation Army, crown Hao Biological Technology Co., Ltd.
Administrative Organization National Surgical Implants and Orthopedic Instruments Standardization Technical Committee Tissue Engineering Medical Devices Sub-Technical Committee (SAC/TC 110/SC 3)
Proposing organization China Food and Drug Administration
Issuing agency(ies) State Food and Drug Administration

YY/T 1561-2017
PHARMACEUTICAL INDUSTRY STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
ICS 11.040.40
C 45
Tissue engineering medical device products - Remnant α-Gal
antigen determination in scaffold materials utilizing animal
tissues and their derivatives
ISSUED ON: MARCH 28, 2017
IMPLEMENTED ON: APRIL 01, 2018
Issued by: State Food and Drug Administration
Table of Contents
Foreword ... 3 
Introduction ... 4 
1 Scope ... 5 
2 Normative references ... 5 
3 Terms and definitions... 5 
4 Reagents and instruments ... 5 
5 Test procedures ... 7 
6 Calculation of antigen content ... 9 
7 Test acceptance criteria ... 11 
8 Test report ... 11 
References ... 12 
Tissue engineering medical device products - Remnant α-Gal
antigen determination in scaffold materials utilizing animal
tissues and their derivatives
1 Scope
This Standard provides a quantitative determination method for remnant α-Gal antigen
in biological materials utilizing animal tissues and their derivatives used in the
preparation of scaffold materials for tissue engineering medical device products.
This Standard applies to the α-Gal antigen determination in various biological materials
utilizing animal tissues and their derivatives used for the preparation of scaffold
materials for tissue engineering medical device 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/T S10993-20:2006, IDT)
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
3 Terms and definitions
Terms and definitions determined by GB/T 16886.20 and YY/T 0606.25 are applicable
to this document.
4 Reagents and instruments
4.1 Reagents
Reagents include:
a) phosphate buffered saline (PBS, pH 7.4);
5 Test procedures
5.1 Sample preparation
5.1.1 Test sample
Accurately weigh the test sample (2 mg ~ 10 mg) and place it in a 2.0 mL or 5 mL
sterile centrifuge tube. If it is a solid block or sheet test sample, use sterile ophthalmic
scissors to cut the sample into small pieces; for liquid or powder test sample, no special
treatment is required. Add the lysis solution (which can be a commercially available
product) to prepare the sample concentration of 2 mg/mL ~ 10 mg/mL [add 1% (volume
fraction) 1 mmol/L PMSF before use, which, for the convenience of operation, is
recommended to be 2 mL of lysis solution for each sample]; homogenize the sample at
low temperature for 2 min ~ 10 min, until all the samples are crushed to form a uniform
tissue homogenate; place at room temperature (25 °C ± 5 °C) for 30 min ~ 3 h until the
sample is completely lysed (that is, no obvious solid matter is observed with the naked
eye); centrifuge (at 3 000 r/min ~ 5 000 r/min) and take the supernatant for later use.
Note 1: For each sample, set three parallel samples, and take the mean ± SD as the final
determination result.
Note 2: According to the requirements of the entrusting party, take 3 samples of
different batch numbers or the same batch number to evaluate the process
stability of different batches or the same batch.
5.1.2 Standard curve samples
Accurately weigh 2 mg of Gal antigen negative biological material reference
(lyophilized powder) sample; place it in a 2 mL or 5 mL sterile centrifuge tube; add the
lysis solution [add 1% (volume fraction) 1 mmol/L PMSF before use] to prepare a
sample concentration of 2 mg/mL, containing Gal α 1-3gal-BSA. Place at room
temperature (25 °C ± 5 °C) for 30 min ~ 3 h until all samples are lysed; centrifuge (at
3 000 r/min ~ 5 000 r/min); then, take the supernatant; use the lysis solution for multi-
fold concentration gradient dilution, to obtain at least 5 serial dilutions of Gal α 1-3gal-
BSA, which are used to make the standard curve. In order to obtain the minimum
detectable concentration of each test, it shall be diluted to a concentration whose
detection OD value is equal to or similar to the detection OD value of the Gal antigen
negative reference, so as to determine its previous concentration as the minimum
detectable concentration of this test.
Note: It is advisable to determine the concentration range of Gal α 1-3gal-BSA through
pre-experiment according to the content of Gal antigen in the sample to be tested,
so as to ensure that the detection OD value of the sample to be tested is within
the range of the standard curve.
5.1.3 Gal antigen positive and Gal antigen negative reference samples
Accurately weigh 2 mg of Gal antigen positive and negative reference substances
(lyophilized powder), respectively; put them in the 2 mL or 5 mL sterile centrifuge tubes;
add the lysis solution to prepare 2 mg/mL [add 1% (volume fraction) 1 mmol/L PMSF
before use]; place at room temperature (25 °C ± 5 °C) for 30 min ~ 3 h until the samples
are completely lysed; centrifuge the lysed samples and take the supernatant for later use.
5.1.4 Control well samples
Set 1 sample of M86/Gal antigen negative biological material reference lysis solution
reaction sample as the 100% reaction value, and 1 sample of the lysis solution reaction
sample as the background value.
5.2 M86 antibody incubation
Take 200 μL of each sample in 5.1 and place them in the 1 mL centrifuge tubes; label
them; add an equal volume (200 μL) of M86 antibody solution with a dilution ratio of
1:100 ~ 1:200 to each tube (it shall be ensured that the M86 antibody is in excess, which
can be confirmed by preliminary experiments). After mixing, react for 2 h at room
temperature (25 °C + 5 °C) with gentle shaking (e.g., 100 r/min); then, incubate at 4 °C
overnight. The next day, collect the supernatant after centrifugation at 14 000 g and 4 °C
for 30 min.
Note: Since M86 is a non-purified antibody, there may be differences in activity
between batches. It is recommended to confirm whether the antibody activity is
different from the previous batch before using the new reagent, and re-verify the
rationality of the antibody dilution ratio if necessary.
5.3 Determination of remnant M86 antibody in supernatant by ELISA inhibition
method
5.3.1 Preparation of solid-phase antigen-coated plates
First, use deionized water to dilute Gal α 1-3gal-BSA (e.g., 500 μg/mL Gal-BSA) by a
certain number of times (e.g., 25 times); then, use carbonate buffer of pH 9.5 to dilute
it again (e.g., 10 times), to prepare a 2 μg/mL Galα1-3gal-BSA dilution solution. Take
100 μL/well; add it to the microtiter plate; mix well and shake gently at room
temperature for 2 hours; then, incubate at 4 °C overnight for coating. The next day, use
the washing liquor (0.05% Tween-20/PBS) to wash the plate at least 3 times (with gentle
shaking, 100 r/min); pat dry on absorbent paper. Add 200 μL/well of 1% (mass
concentration) serum albumin for sealing; place at 37 °C for 2 h with gentle shaking.
Then, wash and pat dry again.
Note 1: The Galα1-3gal-BSA-coated plate prepared once can be sealed and stored at
4 °C and used within one week.
a) Substitute the M86 binding inhibition rate of the test sample into the exponential
curve equation of the standard curve in 6.2, and calculate the mass concentration
(X, μg/mL) of the standard curve sample Gal-BSA corresponding to the test
sample.
b) Calculate the Gal antigenic epitopes per unit mass of the test sample.
Where:
Nt – the number of Gal antigenic epitopes per unit mass of the test sample, in units per
milligram or units per microgram (units/mg or units/μg);
X – the mass concentration of the standard curve sample Gal-BSA, that is, X in the
exponential curve equation of the standard curve in 6.2, in micrograms per
milliliter (μg/mL);
V – reaction volume of the test sample, in milliliters (mL);
NS – the number of Gal antigenic epitopes per unit of Gal-BSA, 1.82×1014/μg;
Mt – the mass of the test sample (equal to the mass concentration of the test sample in
the reaction system multiplied by the reaction volume), in micrograms or
milligrams (μg or mg).
Note 1: The mass concentration here refers to the final concentration when the sample
reacts with M86 in the reaction system; the volume refers to the volume when
added to the 96-well plate.
Note 2: As per Gal-BSA (Gal α 1-3gal-BSA) mass ≈ BSA content (which has been
verified by protein determination), BSA molecular mass: M = 66.33 kD, BSA
unit mass fraction: n = 9.08 × 1018/g, so, BSA molecules per unit mass of Gal-
BSA: 9.08 × 1018/g. Since the number of Gal antigenic residues contained in
BSA molecule in each Gal-BSA molecule is about 20, therefore, the number
of Gal antigenic residues per unit mass of Gal-BSA = 1.82 × 1014/μg.
If the α-Gal antigen in the test sample is in trace or trace amount and is difficult to
quantify, the minimum detectable concentration of this test shall be given, to indicate a
level lower than the minimum detectable concentration. The samples before the
decellularization or antigen removal process can be compared and detected at the same
time, as a reference to investigate the effectiveness of the process.
6.4 Determination of the minimum detectable concentration:
By finding the concentration at which the detection OD value of the lowest dilution
sample of the standard curve is equal to or similar to the detection OD value of the Gal