YY/T 0918-2014 PDF English
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Test method for determining bacterial retention of membrane/ filter assembly utilized for infusion liquid filtration
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YY/T 0918-2014: PDF in English (YYT 0918-2014) YY/T 0918-2014
YY
PHARMACEUTICAL INDUSTRY STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
ICS 11.040.20
C 31
Test method for determining bacterial retention of
membrane / filter assembly utilized for infusion liquid
filtration
ISSUED ON: JUNE 17, 2014
IMPLEMENTED ON: JULY 01, 2015
Issued by: China Food and Drug Administration
Table of Contents
Foreword ... 3
Introduction ... 4
1 Scope ... 5
2 Normative references ... 5
3 Terms and definitions ... 5
4 Overview of test method ... 5
5 Significance and use ... 6
6 Apparatuses ... 6
7 Purity of reagents and materials ... 7
8 Reagents and materials ... 8
9 Preparation method of bacterial challenge stock solution ... 9
10 Identification of Brevundimonas diminuta ... 11
11 Preparation of bacterial challenge suspension ... 12
12 Sample and instrument preparation ... 13
13 Test steps ... 14
14 Result expression ... 15
Appendix A (Informative) Liquid filtration membrane/filter bacterial retention cycle
test plan ... 17
Bibliography ... 19
Test method for determining bacterial retention of
membrane / filter assembly utilized for infusion liquid
filtration
1 Scope
Test method specified in this Standard applies to the evaluation of bacterial retention of
sterilizing-grade membranes or filter assemblies utilized for infusion liquid filtration
for medical devices with a nominal pore size not exceeding 0.22 μm.
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 6682, Water for analytical laboratory use - Specification and test methods
YY/T 0929.1, Sterilizing-grade filters for medical infusion equipment - Part 1:
Integrity test for fluid filters
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
log reduction value; LRV
The 10-base logarithm of the ratio of the number of challenge microorganisms to the
number of filtrate microorganisms.
4 Overview of test method
Use a certain volume and concentration of Brevundimonas diminuta (ATCC 19146)
bacterial suspension – at a pressure difference of no more than 200 kPa on both sides
of the membrane and a flow rate of 2 mL/min ~ 4 mL/min per square centimeter of
effective filtration area (EFA) – to challenge the sterilized membrane or filter assembly
for testing, so that the final challenge level is not less than 107 CFU/cm2 EFA. After all
the filtrate is filtered through the analytical filter membrane, place the analytical filter
membrane on the solid culture medium for culture. Bacteria that are able to pass through
the test filter membrane or filter assembly will form visible colonies on the analytical
filter, which can be counted.
Note: When it is necessary to assess whether the filter membrane or filter assembly
can maintain its bacterial retention ability after long-term use, the test plan given
in Appendix A can be used.
5 Significance and use
5.1 This test method is designed to evaluate the bacterial retention ability of sterilizing-
grade filter membrane or filter assembly under clinical use conditions.
5.2 The challenge level per square centimeter of effective filtration area to withstand
107 of bacteria is much higher than the general sterilization filtration process. This
challenge level is selected to provide a high degree of safety guarantee that the filter
membrane or filter assembly can retain a large number of microorganisms.
6 Apparatuses
6.1 Stainless steel pressure vessel.
6.2 Air conditioner.
6.3 47 mm filter device, connected by hose.
6.4 Diaphragm-protected pressure gauge, with suitable measuring range.
6.5 Valve, resistant to high pressure steam, connected by hose.
6.6 Pipeline, resistant to high pressure steam, be capable of withstanding a pressure of
350 kPa.
6.7 Liquid flow meter.
6.8 Hose clamp.
6.9 Biochemical incubator, 30 ℃ ± 2 ℃.
6.10 Biological safety cabinet.
6.11 Ultra-clean workbench.
The instrument assembly is shown in Figure 1.
8.2.5 Tryptic soy broth
Prepare according to manufacturer’s instructions.
8.3 Analytical reagents and materials
8.3.1 M plate count agar
Prepare according to manufacturer’s instructions.
8.3.2 Peptone water (1 g/L)
Dissolve the peptone into water; distribute it into screw-top bottles in appropriate
volumes to prepare a 10-fold dilution; sterilize at 121 °C for 15 minutes.
8.4 Brevundimonas diminuta
Brevundimonas diminuta (ATCC 19146).
8.5 Analytical membrane
Diameter 47 mm, pore size 0.45 μm.
9 Preparation method of bacterial challenge stock solution
9.1 General
The following two methods have been widely used for the preparation of
Brevundimonas diminuta suspensions. These methods are not exclusive of other
equally effective methods. However, it is important that all Brevundimonas diminuta
challenge suspensions used are monodisperse and comply with the provisions of
Chapter 10.
9.2 Isolation and preservation of strains
Culture according to the instructions for Brevundimonas diminuta and check purity by
the streak plate. Check whether the colony morphology is consistent and identify single
colonies of Brevundimonas diminuta according to Chapter 10.
9.2.1 Stock cultures
Prepare the stock culture from the single colony isolated in 9.2. Inoculate tryptic soy
agar slant and culture at 30 ℃ ± 2 ℃ for 24 h. Use sterile liquid paraffin to cover the
slant and store at 4 °C. Check viability and purity weekly. Alternatively, tryptic soy
semi-solid agar stab can be used instead of slant culture.
9.2.2 Long-term storage of cultures
Freeze-dry or store in liquid nitrogen.
9.3 Preparation of challenge stocks in saline lactose broth
9.3.1 Inoculate stock culture (9.2.1) into10 mL of sterile tryptic soy broth and culture it
at 30 °C ± 2 °C for 24 hours.
9.3.2 Transfer 2 mL of the well-mixed broth culture solution into 1 L of sterile saline
lactose broth; vortex to mix; incubate at 30 °C ± 2 °C for 24 hours.
Note: The bacterial suspension in saline lactose broth can be stored at 4 °C before use,
for no more than 8 hours.
9.3.3 Determine the concentration of viable bacteria in the challenge bacterial
suspension according to Chapter 11 (general concentration 107 CFU/mL ~ 108
CFU/mL).
9.3.4 Identify Brevundimonas diminuta according to Chapter 10.
9.4 Preparation of Brevundimonas diminuta frozen bacteria paste
9.4.1 Inoculate stock culture (9.2.1) into 10 mL of sterile growth medium A (8.2.1) and
culture it at 30 °C ± 2 °C for 24 hours.
9.4.2 Transfer 10 mL of the bacterial suspension obtained in 9.4.1 into 500 mL of sterile
growth medium A, and incubate at 30 °C ± 2 °C for 24 hours.
9.4.3 Transfer 200 mL of the bacterial suspension obtained in 9.4.2 into 10 L of sterile
growth medium A; prepare 10 L of seed culture solution; culture at 30 °C ± 2 °C for 24
hours.
9.4.4 Inoculate the above 10 L of seed culture solution into 500 L of growth medium A.
Incubate aerobically at 30 ℃ ± 2 ℃. Monitor growth by spectrophotometric analysis
at 500 nm and draw a growth curve.
9.4.5 When the culture reaches the stationary growth phase, collect the bacterial cells
by continuous centrifugation.
9.4.6 Resuspend the bacterial cells in 2 ~ 3 times the volume of cold sterile buffer for
storage.
9.4.7 Centrifuge the bacterial cell suspension and resuspend the bacterial cells in an
equal volume of buffer for storage. Determine the bacterial concentration (viable
bacterial concentration is generally 1×1012 CFU/mL).
9.4.8 Transfer all the bacterial paste (such as 50 mL) into a sterile plastic centrifuge
tube, use dry ice acetone or liquid nitrogen to freeze it; store it at -60 °C.
9.5 Preparation of challenge stock solution by frozen strain paste
9.5.1 Use sterile forceps to clamp the test tube; immerse it in 80% (volume fraction)
alcohol for disinfection; ignite the alcohol until most of the alcohol is burned out.
9.5.2 Aseptically remove the test tube cap; under normal temperature conditions, put
the test tube into a conical flask containing a sterile magnetic stirring rod and stroke-
physiological saline solution – containing 0.001 mol/L ~ 0.002 mol/L magnesium
chloride (MgCl2) – of 20 times the volume of bacterial paste (for example, transfer 50
mL of frozen bacterial paste into 1 L of sterile solution).
Note: Magnesium chloride (MgCl2) needs to be dissolved in the solvent before adding
the frozen bacteria paste.
9.5.3 Place the flask on the magnetic stirrer and mix until the contents in the test tube
are evenly suspended (40 minutes).
9.5.4 Determine the viable bacterial concentration according to Chapter 11 (bacterial
suspension concentration is generally 1×1010 CFU/mL ~ 2×1010 CFU/mL).
9.5.5 Identify Brevundimonas diminuta according to Chapter 10.
10 Identification of Brevundimonas diminuta
10.1 Colony morphology
10.1.1 The colonies of Brevundimonas diminuta are beige, slightly raised, and
completely transparent.
10.1.2 After culturing at 30 °C (optimal growth temperature) for 24 hours, it grows to
the size of a needle tip, and the diameter will be 1 mm ~ 2 mm after cultivating for 36
h ~ 48 h.
10.2 Microscopic observation
10.2.1 Perform Gram staining.
10.2.1.1 Use a compound optical microscope equipped with a calibrated eyepiece
micrometer and an oil immersion lens of good resolution to examine the prepared
strains. Observe the size and distribution of microorganisms in several fields of view.
10.2.1.2 The strains prepared by staining shall be Gram-negative, short rod-shaped
bacteria with a size of (0.3 μm ~ 0.4 μm) × (0.6 μm ~ 1.0 μm), which mainly exists in
the form of single cells.
10.2.2 Perform flagella staining (optional). Brevundimonas diminuta is characterized
by polar single flagella.
10.3 Biochemical properties
lactose broth or sterile physiological saline, so that the test filter membrane per square
centimeter can withstand the challenge of at least 107 microorganisms; mix thoroughly.
11.3 Take samples from the prepared Brevundimonas diminuta challenge suspension
under sterile conditions.
11.4 Use 0.1% peptone water to dilute the bacterial suspension to a dilution of 10-6
under sterile conditions.
11.5 Use membrane filtration method or direct plate coating method to conduct two
parallel viable bacterial analyses.
11.5.1 For the membrane filtration method, use 1 mL of diluent of dilution 10-4 ~ 10-6
for filtration. Before adding 1.0 mL of ten-fold dilution, first add 50 mL of sterile
physiological saline solution to the funnel of the filter holder; after filtering, use 50 mL
of sterile physiological saline solution to rinse the funnel wall; remove the analytical
filter membrane from the funnel and place it on the agar medium.
11.5.2 For the direct plate coating method, use 0.1 mL of diluent of dilution 10-3, 10-4,
10-5 to directly coat the plate.
11.6 Incubate the membrane filter plate or directly coat the plate at 30 ℃ ± 2 ℃ for 48
hours.
11.7 Take a direct coating plate of 30 CFU ~ 300 CFU or a membrane filter plate of 20
CFU ~ 200 CFU to count the colonies and calculate the initial bacterial suspension
concentration (CFU/mL).
11.8 Compare the viable bacterial concentration with the direct microscopic count
results in 11.1. The number of viable bacteria shall not be less than 25% of the total
number of bacteria.
12 Sample and instrument preparation
12.1 When testing the filter membrane material, cut it into a round test sample of φ47
mm and install it into the filter device. When testing a sterilized fluid filter, take the
finished product directly for testing, and there is no need to sterilize it before testing.
12.2 Use sterilization paper to respectively wrap the inlet and outlet joints of the test
sample filter component (for filter membrane materials), negative control filter
component and positive control filter component; sterilize according to the
manufacturer’s instructions. Sterilization procedures should be confirmed using
biological indicators or thermocouples.
If necessary, perform an integrity test on the filter component of the test sample under
sterile conditions according to YY/T 0929.1.
13.1.9 Remove the analysis filter component; evacuate the downstream for 15 seconds;
remove all liquid; transfer the membrane from the filter component to the M plate count
agar under sterile conditions; incubate at 30 °C ± 2 °C; record the number of colonies
at the 72nd hour and on the 7th day.
13.2 Bacterial challenge test and positive control test
13.2.1 Add the required volume of bacterial suspension prepared in Chapter 11 (at least
500 mL for each branch) into the pressure vessel.
13.2.2 Close valve C and adjustable valves A, B, C and D, and open valves A and B.
13.2.3 Pressurize the pressure vessel to 200 kPa.
13.2.4 Slowly open the adjustable valve D to allow the challenge bacteria suspension
to fill the test sample filter assembly, the positive control filter assembly and their
downstream analysis filter assemblies. Exhaust air from each filter assembly into a
suitable disinfectant. When each filter assembly is filled with liquid, close its exhaust
valve.
13.2.5 Open adjustable valves A and B.
13.2.6 Open adjustable valve D, and use adjustable valves A and B to adjust the flow
rate to an effective filtration area of 2 mL/min ~ 4 mL/min per square centimeter of the
test filter membrane and the positive control filter membrane.
13.2.7 When all liquid filtration is completed, close valves A and B and adjustable
valves A and B.
13.2.8 Close the air regulator and release the pressure in the container.
13.2.9 Remove each branch analysis filter assembly; evacuate the downstream for 15
seconds; remove all liquid; transfer the membrane from the filter assembly to the M
plate count agar under sterile conditions; incubate at 30 °C ± 2 °C; record the number
of colonies at the 72nd hour and on the 7th day.
13.2.10 Identify whether each colony is Brevundimonas diminuta or contaminating
bacteria (Part 10).
13.3 Integrity test
After the challenge test is completed, if necessary, perform an integrity test on the test
sample filter component according to YY/T 0929.1.
14 Result expression
14.1 Identification of filters
Report the membrane/filter manufacturer’s name, nominal pore size, effective filtration
area and other relevant data.
14.2 Operating conditions
Report pressure, differential pressure, temperature, flow and other relevant parameters.
14.3 Challenge bacteria count
Report the bacterial concentration determined in step 11.7, and calculate and report the
total bacterial count in the challenge suspension.
14.4 Number of bacteria in filtrate
14.4.1 Control
Report the number of colonies observed on the positive and negative control analysis
filters.
14.4.2 Test sample
Report the number of colonies observed on the analysis filter membrane for the test
sample. Report each colony as Brevundimonas diminuta or contaminating bacteria.
14.5 Filter integrity
If applicable, report the parameters and results of the integrity test in 12.1 and 13.3;
indicate whether the filter can pass the integrity test; explain the basis for the judgment.
14.6 Filtration performance
Calculate and report the log reduction value.
14.7 Results evaluation
The test is invalid when bacterial colonies appear on the negative control analysis filter
or non-test microorganisms appear on the test analysis filter.
When the test sample analysis shows that no bacterial colonies appear on the filter
membrane and the positive control is positive, judge that the sterilization requirements
are met.
......
Source: Above contents are excerpted from the PDF -- translated/reviewed by: www.chinesestandard.net / Wayne Zheng et al.
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