Microbial Limit Test of Chinese Pharmacopeiawp.chp.org.cn/upload/userfiles/20181205/...2018/12/05 · Chinese Pharmacopoeia of 2015 Edition Microbiological Examination of

Microbial Limit Test of Chinese Pharmacopeia

Ma ShihongNational Institutes for Food and Drug Control

Date: Oct. 30, 2018, Beijing

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Historical Revision1

Similarities and differences with ICH protocol2

Microbial Limit Test of Chinese Pharmacopeia

Challenges and developments3

NIFDC1. Historical Revision

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Chinese Pharmacopoeia Major events

1995 Edition The microbial limit test was firstly included

2000 Edition The microbial limit standard was firstly included

2005 Edition The requirement for methodology verification was firstly included

2010 Edition The requirement for culture medium suitability test was firstly included

2015 Edition Generally in line with ICH Microbial Test Method

In 1978, the Ministry of Health issued the first Tentative Standard of Drug and Healthcare

In 1986, the Ministry of Health issued Standard of Drug and Healthcare

1. ChP Historical Revision

NIFDCHarmonization(USP/EP/JP)

The USP and the EP Microbial Limits Tests are in the final stages of harmonization. They were signed off to Stage6A at the November,2005 meeting of the PDG held in Chicago, IL USA (USP 2006a).Stage6A:REGIONAL ADOPTION

USP31 General Information/<1196> Pharmacopeial Harmonization: The following policy statement was approved by the PDG at its September 2002 meeting.Table2. Status of Harmonization-General ChaptersTests for specified microorganisms EP Stage4Microbial Enumeration EP Stage4Microbial Attributes EP Stage4Stage4:Official Inquiry

Harmonization Timing/IssuesEP:2007/01 USP:2009/05 JP:2007/10

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Pharmacopeia USP/EP/JP/ChP2015 Edition of Chinese Pharmacopoeia before 2010

Classification basis Classification as per nutrition conditions Classification as per microbial species

Quality control classification Aerobes and anaerobes Bacteria and fungi

Testing system Directly corresponding to the culture conditions

Selecting culture or distinguish by macroscopic inspection

Evaluation Simple and strict Easily causing missed detection

——Systemic difference between microbial test of Chinese Pharmacopeia and foreign Pharmacopeia


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Pharmacopeia Chp,2015 Edition Chp,2010 Edition

Classification Nutrition conditions Microbial species

Item 1Total count of aerobes, 3-5dPeptone agar/broth(TSA/B)

Bacterial count, 3dNutrient agar （NA）

Item 2Total count of mould and yeast, 5-7d

Sabouraud dextrose agar(SDA)Count of mould and yeast, 5d

Sodium rose bengal agar（RA）


Testing items and culture medium

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Testing items and culture medium

No. Chp,2015 Edition Chp,2010 Edition

1* Cholate tolerant Gram negative bacteria Coli group

2* Escherichia coli Escherichia coli

3* Salmonella Salmonella

4* Pseudomonas aeruginosa Pseudomonas aeruginosa

5* Staphylococcus aureus Staphylococcus aureus

6 Clostridium Clostridium

7 Candida albicans Candida albicans

*: There is obvious change in the culture medium system.


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Standard for microbial counting:

The retest is not compulsory, but the accuracy of the results is analyzed via the

laboratory investigation.

The limit standard is represented by exponential method, and maximum acceptable

limits follow the 2X standard:

101cfu: acceptable maximum limit is 20;

102cfu: acceptable maximum limit is 200; and so on.

Basic Characteristics of Pharmacopeia of 2015 Edition


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Result interpretation of control bacteria:

The retest is not compulsory, but investigator and analyze the accuracy of the results via

the laboratory.

Fewer chemical tests or other appropriate methods used for further identification.

(Microscopic test, biochemistry, chromatography & spectrum technology and

molecular biology, etc.)



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Requirements for laboratory environment:

(1105), (1106)

(9203) not lower than Grade B unidirectional flow against Grade D background


Be comprehensively in line with international standard;

Serving for drug microbes from final product test to process control.


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USP35 Chinese Pharmacopoeia of 2015 Edition

<61>Microbiological Examination of NonsterileProducts: Microbial Enumeration Tests

(1105) Microbial limit test for non-sterile products: microbial count method

<62>Microbiological Examination of NonsterileProducts: Tests for Specified Microorganisms

(1106) Microbial limit test for non-sterile products: control bacteria test method

<1111>Microbiological Attributes of NonsterilePharmaceutical Product

(1107) Microbial limit standard for non-sterile drugs

2. Differences and Similarities with ICH Protocol

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Formulation type TAMC TYMC Control bacteria

Solid/liquid via oral administration

103

102

102

101

Escherichia coli, no salmonella should be detected in chemical drug substance and organ extract

Via oral mucosa/gingiva/nasal cavity 102 101 Escherichia coli, staphylococcus aureus,

pseudomonas aeruginosa

Via ear/skin 102 101 Staphylococcus aureus, pseudomonas aeruginosa

Inhalation via respiratory tract 102 101

Escherichia coli, staphylococcus aureus, pseudomonas aeruginosa, cholate tolerant Gram-negative bacteria

Vagina/urinary tract 102 101 Staphylococcus aureus, pseudomonas aeruginosa, candida albicans, clostridium (TCM）

Solid/liquid via rectal medication

103

102

102

102 Staphylococcus aureus, pseudomonas aeruginosa

Other local medication 102 102 Staphylococcus aureus, pseudomonas aeruginosa

2. Differences and Similarities with ICH protocol

ChP compulsory standard and USP recommended standard！

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USP<1111>Microbi

al test method

for non-sterile

products:

Microbial

standard for

reagents and

medicinal

materials


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Formulation type TAMC TYMC Control bacteria

Raw medicinal materials and excipients 103 102 *(Not specified in a unified

manner)

TCM extracts 103 102 *(Not specified in a unified manner)

TCM decoction slices *(Not specified in a unified manner)

*(Not specified in a unified manner)

Absence of salmonella in 10 g, and cholate tolerant Gram-negative bacteria should be lower than 104 cfu/g


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Escherichia coli〔CMCC(B) 44102〕 Bacillus subtilis 〔CMCC(B) 63501〕 ——ATCC 6633 Staphylococcus aureus〔CMCC(B) 26003〕——ATCC 6538P Salmonella paratyphi B〔CMCC(B) 50094〕 Pseudomonas aeruginosa〔CMCC(B) 10104〕 Clostridium sporogenes〔CMCC(B) 64941〕 Candida albicans [CMCC(F) 98001] ——ATCC10231 Aspergillus niger 〔CMCC(F) 98003〕


Staphylococcus aureus〔Such as ATCC6538,NCIMB9518,CIP4.83, or NBRC13276〕Pseudomonas aeruginosa〔Such as ATCC9027, NCIMB8626, CIP82.118, or NBRC13275〕Escherichia coli〔Such as ATCC8739,NCIMB8545,CIP53.126, or NBRC3972〕

Salmonella enterica subsp. enterica serovar Typhimurium or, as an alternative, 〔Such as ATCC 14028〕Salmonella enteria〔Such as NBRC100797,NCTC6017, or CIP80.39〕Clostridium sporogenes such as ATCC 11437 (NBRC 14293, NCIMB 12343, CIP 100651) or ATCC 19404 (NCTC 532 or CIP 79.3).Candida albicans〔Such as ATCC10231,NCPF3179,IP48.72, or NBRC1594〕Aspergillus brasiliensis 〔Such as ATCC 16404，IMI149007, IP 1431.83, or NBRC 9455 〕

Differences in Standard Bacteria Strains

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Properties of test sample:1. Water solubility or oiliness2. Antibacterial activity3. Strict standard

1. Pore size of the membrane: not greater than 0.45μm.

2. Diameter: 50/75 mm.

Membrane

material

Total rinsing volume not greater than 1000 mL

Membrane-filter Method

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二级过滤的75mm薄膜过滤器专利

Membrane-filter Method

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19

SDA and Sodium Rose Bengal Agar Culture Medium

coli aeruginosa aureus subtilis

Culture in nutritive agar culture medium at 35℃ for 48 hrs 75 81 91 79

Culture in sodium rose bengal agar culture medium at 35℃ for 48 hrs 57 47 0 0

Culture in sodium rose bengal agar culture medium at 25℃ for 72 hrs 45 41 0 0

Table. Results of specific experiment of sodium rose bengal agar culture medium

When there is severe bacterial contamination, the sodium rose bengal

agar culture medium can inhibit a portion of Gram positive bacteria.

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Result interpretation of control bacteria testSeparation analysis for detected microbes: biochemical tests or other appropriate methods used for further identification. For example: Application of triple sugar iron agar in salmonella test


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www.themegallery.com

Guarantee Concept for Systematic Experiment Results

9203（13 items） WHO GPML（12 items）1 Personnel Personnel2 Media Reagents and culture media3 Reagents Reagents and culture media4 Species Reference materials and reference cultures5 Environment Environment6 Equipment Equipment7 Sample Sampling，Sample handling and identification8 Test method Validation of test methods9 Disposal of contaminated waste Disposal of contaminated waste10 Quality (result and test) Quality assurance of results and performance，Testing

procedures11 Experiment records12 Result interpretation and report Test reports13 Document /

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How to control the quality of test system?USP/EP/ Chinese Pharmacopoeia 2010/2015 Edition——Culture medium suitability test

Culture Media

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USP38：<61>Microbiological Examination of Nonsterile Products: Microbial

Enumeration Tests：Growth Pormotion of the Media。<62> Microbiological

Examination of Nonsterile Products: Tests for Specified Microorganisms：Growth

Promotion and Inhibitory Properties of the Media. Test each batch of ready-prepared

medium and each batch of medium prepared either from dehydrated medium or from

ingredients. For a freshly prepared inoculum, growth of the microorganisms

comparable to that previously obtained with a previously tested and approved

batch of medium occurs.

Culture Medium Suitability Test

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24

USP: compare to that previously obtained with a previously tested and approved batch of medium occurs.

The Drug Microbial Laboratories in China are small and dispersed, and thousands of enterprises

and basic-level drug test institutions can hardly effectively carry out reference culture medium

verification and evaluation;

In the inspection, it is difficult to evaluate the verification of reference culture medium by the

microbial laboratory of an enterprise.

Culture Medium Suitability Test

ChP: control culture medium

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Quality control of culture medium:

USP—Previously verified culture medium

Chinese Pharmacopoeia —Control culture medium

Ma Shihong, Yang Meiqin, Liu Peng, Cao Limei, Hu Changqin (National Institutes for Food and Drug Control, Beijing, 100050)Development and application of control culture medium in Chinese Pharmacopoeia of 2010 version, Chinese Pharmaceutical Affairs, 2012（8）

Control culture medium

•National Institutes for Food and Drug Control studied the supply according to national standard material procedurePatent number of single-dose separate package free from weighing: ZL200920108836.8

NIFDCControl Culture

Medium

WWW.nifdc.org.cnStandard substance

query

Standard substance supply

NIFDC3.Challenges and Developments

TCM accounts for a large portion in the constitution of Chinese Drugs, and its

microbial characteristics bring huge challenges to the ICH protocol.

NIFDCCasesCase: The standard bacteria count for radix isatidis granules should not be greater than 1000cfu/g, and that of a certain radix isatidis granules tested by a drug testing institution was 1200cfu/g, which was submitted to National Institutes for Food and Drug Control for arbitration.

Note: Method suitability

Qualified Unqualified

Plating method (1: 10 test solution) 1 mL/plate 0.5 mL/plate 0.2 mL/plate

Counting result (cfu/g) 650 910 1380

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Vigorous SDA bacterial growth brings challenges to result interpretation.

3.Challenges and Development

If bacterial growth makes the TYMC result not conforming to the

requirements of microbial limit, “SDA + antibiotics” can be used for

re-counting.

TYMC＞TAMC

What to do if SDA first count exceeds TAMCstandard？

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Data rounding, full data comparison, and uncertainties of microbial results

3.Challenges and Development

No. Batch No. Original test results Retest results Retest conclusion1 171006 2400 20 Conformed2 171005 260 Lower than 10 Conformed3 180303 1700 120 Conformed4 171101 6200 21000 Non-conformed5 171101 2300 Lower than 10 Conformed6 180203 770 1100 Non-conformed7 180201 1900 2200 Non-conformed8 180304 3400 4600 Non-conformed9 180203 3700 9500 Non-conformed

Table Test results of 9 batches of retest samples of an oral solution (standard: the total count of aerobes should not be greater than 200 cfu/mL)

According to the Clause 26, Chapter 4, drug surveillance market [2006] No. 379

Management Rules for Drug Quality Spot Check, “(II) The samples obviously non-

uniform or lower than the amount needed for test” will not be accepted for retest.

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Control bacteria test results led to the evaluation and

assessment of large amount of objectionable microorganism

3. Challenges and Development

In 2018, microbial limit test was performed for 298 batches of a pediatric coughing and asthma TCM granules produced by 61 enterprises, in which the total count of aerobes, and total count of mould and yeast all conformed to the requirements; after the test of E. coli was qualified, suspected bacterial colonies was detected in the control bacteria test of 15 samples, in which A. baumanii was detected in 12 samples, and pantoeawas detected in 3 samples, and both are human conditioned pathogen, in which A. baumanii is an important pathogen in hospital infection.

Objectionable Microorganisms！

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Whether it is pathogenic bacteria? Various domestic and foreign

standards Authoritative textbook and brochure Authoritative literature report. FDA: Bad Bug Book

NIFDCMicrobial Limit Test of Chinese Pharmacopeia

ChP2015ICH

EvaluationUSP40 EP JP17

（1105）Microbial limit test for non-sterile products: Microbial Enumeration Tests

<61>Microbiological examination of nonsterile products: Microbial Enumeration Tests

2.6.12 4.05

Basically consistent: There are differences only in some verified bacterial strains, control culture medium and operation details, etc.; however, due to relatively high load of TCM microbes, there is quit a challenge in the implementation of ChP.

（1106）Microbial limit test for non-sterile products: Control Bacteria Test Method

<62>Microbiological Examination of nonsterile products: Tests for Specifiedmicroorganisms

2.6.13 4.05

Basically consistent: There are differences only in some verified bacterial strains, control culture medium,operation details and result interpretation, etc.; however, due to the characteristics of TCM contamination, there is quit a challenge in the implementation of ChP.

（1107）Microbial limit standard for non-sterile drugs

<1111>Microbiological Examination ofNon-sterile products: acceptance criteriafor pharmaceutical preparations andsubstances for pharmaceutical use

5.1.4 5.

Partially consistent: There are significant differences in the practical scope, salmonella test requirements, microbial limit standard for small and micro dose, such as patch, TCM (plant drug) standard, etc.

On the whole, the ChP Microbial Limit Test of 2015 edition is basically consistent with the ICH protocol in the test method, test procedure, experiment requirements, standard characterization method, etc.; due to the specificity of TCM, the ChP of 2015 edition has relatively high challenge in the implementation, and the TCM standard should be fully studied in the future, and further coordination with ICH protocol should be further studied and discussed.

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Microbiology Laboratory of China Institute for Food and Drug Control (Old Address of Temple of Heaven )Institute for Chemical Drug ControlNational Institutes for Food and Drug ControlMicrobiology Lab Add: No.2 Tiantan Xili, Beijing, 10050 P.R.China

NIFDC Temple of Heaven (Old Address)

NIFDC Da Xing (New Address)

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Thank you！

Ma ShihongE-mail: [email protected]: 010-67095990Fax: 010-67095148

Documents

Microbial Limit Test of Chinese Pharmacopeiawp.chp.org.cn/upload/userfiles/20181205/...2018/12/05 · Chinese Pharmacopoeia of 2015 Edition Microbiological Examination of