Microbial contamination of drugs is an important factor affecting drug quality and safety. In order to improve the quality of drugs and reduce drug use risks, drug regulatory authorities in China, the United States, and the European Union, as well as international organizations such as ICH, PIC/S, and WHO, all propose requirements for the contamination monitoring and quality control throughout the lifecycle management of drugs. These requirements also present new challenges for the updating of microbiological testing technologies and the construction of standard systems for drugs.

After more than 70 years of continuous development since its first publication in 1953, the Chinese Pharmacopoeia (ChP) has made significant progress in microbiological testing standards for drugs. The ChP 1953 Edition included "sterilization test methods for drugs" for the first time． The Hygienic Standards for Drugs initially issued in 1978 systematically included microbiological test methods. The second addendum to the ChP 1990 Edition formally included "microbial limit test" and preliminarily established the microbial acceptance criteria for drugs. The ChP 2000 Edition first comprehensively included microbial acceptance criteria for drugs, marking the establishment of standardized microbial quality control practices for pharmaceuticals in China. Since the start of the 21st century, the formulation and revision of microbiological standards for pharmaceuticals have entered a rapid development stage. The ChP 2005 Edition addressed the method suitability for microbiological tests; the ChP 2010 Edition standardized the quality control system for microbiological testing; the ChP 2015 Edition started the process of international harmonization. The ChP 2020 Edition emphasized process control requirements and innovatively introduced molecular techniques, basically creating a microbiological standard system for drugs with Chinese characteristics.

Along with the social and economic development and scientific and technological progress in China, the microbiological testing technologies and standards for drugs have also been continuously updated and improved. This article systematically analyzes the formulation and revision principles, main characteristics and development trends of microbiological-related general chapters and guidelines in the ChP 2025 Edition (Volume IV), focuses on the innovative concepts and development directions of microbiological-related standards, and deeply discusses the latest requirements in lifecycle management of drugs and risk-based drug quality control to provide a theoretical basis and reference for improving the microbiological standard system in the ChP.

1   Overview of revisions to microbiological-related standards

1.1   Revision of test methods and acceptance criteria

Based on the comprehensive evaluation of relevant standards in the ChP 2020 Edition by the Chinese Pharmacopoeia Commission, the formulation, revision, and improvement of standards have been conducted in terms of the applicability, standardization, harmonization, and implementation of the standards. The ChP 2025 Edition (Volume IV) has included a total of 26 microbiological-related standards, i.e., 10 general chapters and 16 guidelines (see Table 1). The ChP 2025 Edition contains 12 standards revised from the 2020 Edition, accounting for 46% (12/26) of the total number of standards, and 7 new standards, accounting for 27% (7/26). A total of 19 standards are revised or newly drafted, accounting for 73% (19/26) of the total number of microbiological-related standards. In addition, in the ChP 2025 Edition, microbiological-related content in 0251 Pharmaceutical Excipients and 0261 Water for Pharmaceutical Purposes has also been revised.

1.2   International harmonization of microbiological-related standards

During the establishment of microbiological-related standards in the ChP 2025 Edition, efforts have been made to actively draw on international experience while considering the national condition of China to steadily propel international harmonization with ICH. By directly and partially adopting relevant ICH standards, the ChP 2025 Edition has generally achieved the harmonization with internationally standards in microbiological testing methods. Except the requirement on mutual recognition of reference strains with ICH, Sterility Tests (1101), Microbial Enumeration Tests (1105), Tests for Specified Microorganisms (1106), and Microbiological Acceptance Criteria of Non-sterile Pharmaceutical Products (1107) have generally been harmonized with ICH, achieving a balance between the implementation of universal standards and the retention of individualized requirements. The improvements in microbiological testing methods and criteria in the ChP 2025 Edition not only reflect China's positivity in the internationalization of drug standards but also lay a solid foundation for the in-depth integration and development of the global pharmaceutical industry and the improvement of the scientificity and accuracy of standards.

1.2.1   Sterility tests With regard to the sterility test method, the ChP 2025 Edition has adjusted the quality control requirements for culture media, improved the strain selection for method suitability tests, and modified the special requirements for the culture of biological products. In the meanwhile, it is pointed out that the laboratory should comprehensively assess the necessity, frequency, and requirements of positive controls in routine test based on quality risk management requirements.

Table 1.Summary of microbiological-related standards in the ChP 2025 Edition

1.2.2   Microbial limit tests   In the microbial enumeration test, adjustments have been made in terms of microbial suspension preparation, growth promotion of the media, and microbial count reporting rules, and same as the sterility test, the requirement for positive control tests has been added. In addition, the pharmacopoeia also straightens out and specifies the acceptance criteria for preparations for inhalation use, rectal use, and other preparations for topical administration among non-sterile products. The assessment of raw materials and excipients has been separately listed so that the criteria for microbiological examinations are more scientific and accurate.

1.3   Improvement of relevant standards for the drug development stage

Based on the concept of quality by design (QbD), elements for the control of microbial contamination risks should be systematically considered in contemporary drug development. As the ChP is continually improved, standards in microbiological control have also proposed requirements with regard to antimicrobial effectiveness testing and water activity control during drug formulation screening and process development.

1.3.1   Antimicrobial effectiveness testing

The ChP 2010 Edition first included the guideline for antimicrobial effectiveness testing, which was later renamed Antimicrobial Effectiveness Testing (1121). The ChP 2025 Edition has further improved the original standard by introducing the concept of hurdle factor, emphasizing characteristics of drug products with antimicrobial agents and specifying medium suitability tests, the assessment and qualification of test strain concentrations, parallel determinations in membrane filtration method, and the calculation of enumeration results, improving the practicability of the method.

1.3.2   Application of water activity

The ChP 2025 Edition has newly included the Guideline for Application of Water Activity in Microbial Control for Non-sterile Pharmaceutical Products (9211), providing guidance for pharmaceutical industries in optimizing the formulation design, controlling the manufacturing process and setting reasonable microbial criteria for drugs from three aspects, i.e., water activity required for microbial growth, water activity and microbial control strategies for non-sterile products, and methods for the determination of water activity, so as to promote the implementation of the risk-based microbial control concept.

2   Risk-based quality management and standard system

2.1   Microbial risk assessment requirements

2.1.1   Innovation of laboratory quality management system The Guideline for the Standardization of Pharmaceutical Microbiological Laboratories (XIX Q) has undergone continuous optimization for 10 years since it was first included in the compendial system by the ChP 2010 Edition[8]. The Guideline for Quality Management of Microbiology Laboratory for Pharmaceutical Products (9203) in the 2025 Edition has been further harmonized with international pharmacopoeias and contemporary quality management systems, marking the entry of quality management of pharmaceutical microbiological laboratories in China into a new stage. Firstly, the quality risk management system and concept have been systematically improved by integrating ICH Q9 core elements; secondly, a supplier access assessment system has been established for the first time with reference to international standards such as ISO/IEC 17205, so as to ensure the suitability of products and services provided externally in laboratory activities; thirdly, the elements of record and data management have been improved based on the requirements of WHO and NMPA for data integrity, so as to improve the reliability of test data.

2.1.2   Upgrading of microbiological standards for pharmaceutical excipients Pharmaceutical excipients play such core roles as forming agents, carrier conveyors, and stabilizers in drug products. Following the implementation of generic drug consistency evaluation and bundling review and approval system, the system for the management of pharmaceutical excipients and packaging materials in China has been changed from an isolated regulatory system into a full-lifecycle management system with drug products as the core. The revision of General Chapter 0251 Pharmaceutical Excipients in the new edition has refined the provisions for microbial control, systematically identified microbial risks in pharmaceutical excipients using scientific assessment tools and assessment methods, and established scientific and reasonable criteria. Considering the diversity of pharmaceutical excipients, the new standard emphasizes that when general microbiological testing methods are not applicable, method suitability should be verified based on their characteristics, and scientific and effective testing protocols should be developed through systematic studies.

2.2   Standard system based on the process control concept

The Chinese Pharmacopoeia Commission attaches great importance to the construction of a microbiological quality control system in drug manufacturing. Based on the monitoring and control of bioburden, pharmaceutical manufacturers and regulatory authorities are provided with systematic, clear, and operable standards for microbiological process control of drugs in such fields as water for pharmaceutical purposes, water activity, disinfectant efficacy evaluation, sterilization, biological indicators, etc.

2.2.1   Monitoring and control of microorganisms in water for pharmaceutical purposes Microbial contamination is a key concern in the monitoring of the pharmaceutical water system. The ChP 2025 Edition has newly included the Guideline for Microbiological Monitoring and Control of Water for Pharmaceutical Purposes (9209), systematically constructing the technical specification covering water quality monitoring as well as risk management and control. The standard analyzes the key points in the control of microbiological-related metrics in the pharmaceutical water system. The microbial contamination control strategy for water for pharmaceutical purposes is described in terms of design, construction, maintenance, and daily management. The guideline coordinates and links with the general chapter on Water for Pharmaceutical Purposes (0261) in terms of sample quantity, operation methods, culture media, culture duration, etc., and provides guidance on the establishment of water system monitoring protocols and criteria. Besides, rapid microbial detection techniques for water and their application are introduced, enhancing the systematic and prospective quality assurance of water for pharmaceutical purposes.

2.2.2   Refinement of the sterilization and disinfection standard system In the ChP 2025 Edition, part content of "vapor phase sterilization" in the Methods of Sterilization (1421) has been adjusted, the term has been changed to "vaporization sterilization", and the concept of surface sterilization has been introduced. Considering the huge differences between domestic and foreign manufacturers in the control principle, process development, and validation of vaporization sterilization technology, the standard, based on the principle of seeking common ground while maintaining differences, re-iterates crucial technical requirements such as the definition, application scenarios, monitoring, and control of vaporization sterilization, refines key points including the influencing factors, process development and validation, and daily monitoring of the vaporization surface sterilization process, and proposes that users evaluate the risks of different application scenarios and select process parameters based on historical experience or develop appropriate new processes.

infectant efficacy assessment is a critical step to ensure microbial control in pharmaceutical manufacturing and testing environments. To meet the requirements for the qualification and periodic monitoring of the disinfection effect of disinfectants in Guidelines 9203 and 9205 of the ChP, the Guideline for Evaluation of Efficacy of Disinfectants in Pharmaceutical Microbiological Laboratories (9210) has been newly added. The content of the standard covers types, concentrations and mechanisms of action of disinfectants, medium suitability test, neutralizer validation, and specific methods and result judgment for evaluating the efficacy of bacterial strain and disinfectant, providing guidance on efficacy evaluation tests for disinfectants used in pharmaceutical environments, facilities, equipment, and laboratories.

3   Novel tools and novel methods with Chinese characteristics

3.1   Microbial identification and tracking techniques

In order to enhance the application of microbial identification and tracing techniques in the full lifecycle quality management of drugs, the pharmacopoeia has fully absorbed practical experience in the implementation of standards since the molecular testing standard for microorganisms was officially included in the ChP 2020 Edition, and was systematically added or revised with relevant standards. So far, six relevant general chapters and guidelines have successively come into effect, taking a leading position in the international pharmacopoeia system.

The Guideline for Microbial Characterization, Identification, and Strain Typing (9204) has supplemented the requirements for the identification of indistinguishable close-related species based on other validated characteristic gene sequences or whole genome sequences and introduced the MALDI-TOF MS identification technique, focusing on the refinement of the technical path and implementation specification of microbial tracking and investigation. At present, the Chinese Pharmacopoeia Commission is preparing the guideline for identification of microorganisms using MALDI-TOF MS to significantly improve the timeliness of microbial identification and simplify daily strain identification task. In the meanwhile, the ChP 2025 Edition has included the Guideline for microbial Whole-Genome Sequencing technology (9110) for the first time in the world, standardizing the requirements on the technical implementation from such perspectives as laboratory requirements, technical metrics, technical processes, factors influencing results, and methodological investigation and clarifying the important role of whole-genome sequencing technology at key links such as precise identification of microorganisms, tracking analysis, and risk assessment in the whole chain of pharmaceutical production.

The ChP 2025 Edition has successfully constructed a systematic and multi-dimensional technical standard system for microbial identification and tracking, laying a solid technical foundation for bioburden control, monitoring of objectionable microorganisms in non-sterile products, and risk-based whole-process control of microorganisms in drugs.

3.2   Monitoring and evaluation of objectionable microorganisms

Clinical study data show that 82.6% of pathogenic microorganisms responsible for nosocomial infection caused by non-sterile drugs were outside the scope of specified microorganisms in the ChP. In view of this, it is imperative to establish pertinent testing and control strategies for microorganisms with potential hazards (i.e., objectionable microorganisms) based on critical parameters such as product characteristics, clinical use, and manufacturing process. The ChP 2025 Edition has newly included the Guideline for Risk Control of Objectionable Microorganisms in Non-sterile Products (9212), which has clarified the definition of objectionable microorganisms and solved such problems in the industry as unclear typing of objectionable microorganisms and absence of risk control measures, providing pharmaceutical manufacturers and regulatory authorities with whole-process, standardized, and operable guidance for the identification and control of microbial risks and effectively promoting the implementation of the concept of control of objectionable microorganisms in non-sterile products in China.

Additionally, water-based products are susceptible to contamination by Burkholderia cepacia complex (BCC). BCC may account for up to 22% of all drug recalls due to microbial contamination. In view of this, the ChP 2025 Edition has timely included the Test for Burkholderia Cepacia Complex (1109) to specify the testing procedure for BCC in non-sterile drug products as well as their raw materials and excipients. The technical details include the preparation of the product to be examined, culture for enrichment, culture for selection and isolation, and result interpretation. This general chapter is the first testing technique for objectionable microorganisms included in the pharmacopoeia in China, expanding the scope of microbiological testing.

3.3   Systematic evaluation of microbiological methods

Reliable analysis methods are the cornerstone for getting accurate test results. The relevant standards for the development, evaluation, and use of methodologies in the prior domestic and foreign pharmacopoeia systems mainly focused on physicochemical testing techniques, while no suitable evaluation system for microbiological methodologies has been established. The ChP 2025 Edition has newly included the Guideline for Validation, Verification, and Transfer of Microbiological Analysis Methods (9213), which clarifies the terms and definitions related to the evaluation of microbiological analysis methods and standardizes requirements for different application scenarios, sample selection, parameter setting, and statistical analysis of data. This standard introduces for the first time the evaluation system of novel statistical tools and analytical methods applicable to microbiology for comprehensively guiding the establishment and use of microbiological analysis methods for drugs. Moreover, the Guideline for Validation of Alternative Microbiological Methods for Pharmaceutical Products (9201) in the pharmacopoeia has been concurrently revised to provide scientific guidance for the use of rapid detection techniques and automated systems in process control and final product release and vigorously promote the continuous improvement of quality risk management capabilities of novel biopharmaceutical companies.

4 Outlook

The issuance of the ChP 2025 Edition marks another significant step in the construction of China's drug standard systems. Adhering to the philosophy of "technology leads, standards pioneer", this revision has systematically constructed a microbiological technical standard system that meets the development needs of China's pharmaceutical industry. Three important breakthroughs have been made in the development of microbiological standards in the ChP 2025 Edition: i. Technical standards for microbiological testing in line with international standards have been established; ii. Risk assessment and process control requirements based on the full lifecycle quality control requirement for drugs have been enhanced; iii. It has taken the lead in promoting the implementation of the concept of evaluation and control of objectionable microorganisms in non-sterile products, contributing to the application of advanced and mature testing technologies in China. The revision of microbiological-related standards in the ChP 2025 Edition has successfully achieved the task goal of the Edition Outline of the ChP 2025 Edition.

To cooperate with the implementation of the microbiological-related standards in the ChP 2025 Edition, comprehensive and systematic industrial training under the unified guidance by the Chinese Pharmacopoeia Commission is required to enhance practitioners' understanding of novel technologies, novel requirements, and novel concepts. Besides, the leading entity for each standard should strengthen the collection and follow-up of related problems in the implementation of current standards and do well in the maintenance and interpretation of standards. Also, it is needed to review novel tools, novel methods, and novel directions from a more open, more distant, and more scientific perspective and continue to enhance the applicability and advanced nature of microbiological standards, so as to lay a solid technical support foundation for the high-quality development of China's pharmaceutical industry.

(Chinese Pharmacopoeia Commission)