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Janvier 2024

La Vague n°80

Microbiologie, Développement durable

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The alternatives and rapid Microbiological methods & Pharmacopeias world

Alternatives Rapid Microbiological Methods Pharmacopeias World Bonnevay La Vague 80 2024

1. Introduction & the different main pharmacopeias

The main pharmacopoeias have different structures, internal organization, and mode of operation. In summary:

European Pharmacopeia (Ph. Eur.)

is a multinational Pharmacopoeia that acts as an umbrella and integrates the main components of several national compendia still maintained in Europe. It is well recognized as a reference in many other countries and regions. It is edited and published by the European Directorate for Quality of Medicines (EDQM) which belongs to the Council of Europe. Ph.Eur. was founded in 1964 and is valid for the EU and many additional states. Every 3 years a new edition is published in both English and French. Ph. Eur. covers General Chapters (General notices, Methods of analysis, material for containers & containers, reagents, and general texts), General Monographs (e.g., on Dosage forms, vaccines for human & veterinary use or herbal drugs and herbal drug preparations), and Specific Monographs. New texts and revisions are published in Pharmeuropa, an online journal designed to gather public feedbacks.

The United States Pharmacopeias (USP).

USP–NF is a combination of two compendia, the United States Pharmacopeia (USP) and the National Formulary (NF). It sets standards for chemical and biological drug substances, dosage forms, compounded preparations, excipients, medical devices, and dietary supplements manufactured, distributed, and consumed worldwide. USP has been founded in 1820. USP is a national Pharmacopoeia that is recognized reference in many other countries and regions. It is annually updated, along with two supplements, and is published in English. USP-NF is edited and published by U.S. Pharmacopeial Convention, a scientific nonprofit organization independent from the US FDA. USP is the only national pharmacopoeia that is not part of HAs. USP covers Monographs for drug substances, dosage forms, and compounded preparations. Monographs for dietary supplements and ingredients appear in a separate section of the USP. Excipient monographs are part of the NF. Public input and interactions are vital to the development of these standards. Pharmacopeial Forum (PF) is the bimonthly online journal through which USP develops and revises standards for the USP–NF by a process of public review and comment.

The Japanese Pharmacopeia (JP)

is the national Pharmacopoeia of Japan. It was founded in 1886 and is now edited and published by the Ministry of Health, Labor & Welfare (MHLW). A new edition is published every 5 years since JP9. JP is published in Japanese and English languages. For Legal Status JP is define by the Act on Securing Quality, Efficacy and Safety of Pharmaceuticals, Medical Devices, Regenerative and Cellular Therapy Products, Gene Therapy Products, and Cosmetics. It covers standards for Chemical Drugs (APIs and Preparations), Biological Drugs (APIs and Preparations), and Excipients, including requirements for packagingmaterials.ThecreationofJPmonographsismanagedby Pharmaceuticals and Medical Devices Agency (PMDA) in interaction with pharmaceutical industries and other public stakeholders. Expert Committees review the drafts prior to public consultation via the Japanese Pharmacopoeial Forum, a Japanese and English journal for publication of draft texts.

Chinese Pharmacopeia (ChP)

is a national Pharmacopoeia of the People’s Republic of China. It was founded in 1953 but it has several thousand-year compendial roots and due to this has a special focus on traditional Chinese herbal medicines. ChP is edited and published by the Pharmacopoeia Commission of China National Medical Products Administration (NMPA). The last version was published in 2020 and the official languages are Chinese and English. ChP covers the standards for Chemical Drugs, Biological products and excipients including requirements for packaging materials. ChP also covers standards for traditional Chinese medicines addressing, for example, Chinese Materia Medica and Prepared Slices of Chinese Crude Drugs, Oil, Fats and Extractives, as well as Traditional Chinese Patent Medicines and Single Herb Preparations. Other part covers Western Medicines such as chemical drugs, antibiotics, biochemical preparations and Radiopharmaceuticals, excipients for pharmaceutical use and biologics. Since the 2015 edition, ChP has a separate volume on general monographs and excipients.

2. Main compendial microbiology chapters & Pharmacopeias

In 1989, the Pharmacopeial Discussion Group (PDG) was formed by the EDQM of the Council of Europe, the United States Pharmacopeial Convention Incorporated, and the Japanese Pharmacopoeia of the MHLW. Since October 2023, the Indian Pharmacopoeia Commission (IPC) officially joined as a member in the PDG and the World Health Organization (WHO) has participated in the PDG as an observer since May 2001. The PDG generally meet twice a year (either face-to-face or by videoconference) to work on pharmacopeial harmonization topics. The purpose of the PDG is to harmonize pharmacopeial standards (excipient monographs and selected general chapters) in three major regions of the world. Harmonization reduces manufacturers’ burden of having to perform analytical procedures in different ways, using different acceptance criteria, in order to satisfy pharmacopeial requirements that differ between regions. The ultimate aim is to arrive at interchangeable methods or requirements so that demonstration of compliance using a general chapter from one of the 3 pharmacopoeias implies that the same results would be obtained using the general chapter of either of the other pharmacopoeias.

To help regulatory authorities and other users recognize the interchangeability of selected harmonized general chapters, the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) has issued topic-specific annexes with information about a limited number of these texts to facilitate their implementation.

For Microbiology methods topics, the oldest and most widely used general microbiology methods, i.e., the sterility test, microbial enumeration method, specified microorganisms detection, and bacterial endotoxin test, have been harmonized between USP, JP and Ph. Eur. pharmacopoeias for over 10 years now:

  • STERILITY TEST, through ICH Q4B annex 8. This chapter has been revised to indicate its status within the context of harmonization, Chapter Ph. Eur. 2.6.1., USP <71>, and JP 4.06 are harmonized.
  • MICROBIAL ENUMERATION TEST, through ICH Q4B annex 4A. This chapter has been revised to indicate its pharmacopeial harmonization, Chapter Ph. Eur. 2.6.12., USP <61>, and JP 4.05 are harmonized.
  • TEST FOR SPECIFIED MICROORGANISMS, through ICH Q4B annex 4B. Harmonization Chapter Ph. Eur. 2.6.13., USP <62>, and JP 4.05 are harmonized.
  • MICROBIOLOGICAL QUALITY OF NON-STERILE PHARMACEUTICAL PREPARATIONS AND SUBSTANCES FOR PHARMACEUTICAL USE: This chapter has been revised to indicate its status within the context of harmonization, Ph. Eur. chapter 5.1.4., USP <1111>, and JP General Information 12 are harmonized.
  • BACTERIAL ENDOTOXIN TEST, through ICH Q4B annex 14 is harmonized, Ph. Eur. Chapter 2.6.14., USP <85>, and JP 4.01 are harmonized.

3. Alternative Microbiological methods & Pharmacopeias

Traditional microbiological test methods have served industrial microbiologists well over the last 100 years. For example, the first pharmacopeial sterility test appears in the British pharmacopeia in 1932. Despite their limitations, the compendial microbiological assay are still a regulatory requirement for many pharmaceutical products releases.

In the 80s and 90s, one of the main issues with the traditional microbiological methods was the time to results. More rapid results were needed because a bioburden assay was at least 5 to 7 days, a sterility test 14 days, a Mycoplasma assay 28 days, and finally a Mycobacteria detection test 56 days.

Then the term Alternative methods appeared in the 90s and 2000s because issues it became increasingly clear that traditional methods were not only associated with long time to results but also with accuracy, precision, specificity, LOD and LOQ, robustness, etc. The expression Alternative and Rapid Microbiological Methods (ARMM) came into use, including both novel and automated traditional (and thus also “alternative”) methods. The expression Modern Microbiological Methods is also used.

Toward the end of the 90s, a number of specific and dedicated conferences, congresses, and trainings on Rapid, Modern, Alternative microbiological methods appeared.
In the 90s impedancemetry, epifluorescence and bioluminescence were mainly used. Then in the 2000s Nucleic Acid Test based on PCR, and then qPCR, started to be evaluated and implemented. Pharmacopoeias may appear to be lagging in the adoption of these alternative methods and to be still based solely on traditional methods, but this is not entirely correct. For more than 20 years, pharmacopoeias have been evolving, offering guides and recent compendial methods based on science and adapted to specific needs.

In May 2003, over 20 years ago, an international symposium on Microbiological control methods in European pharmacopeia present and future was organized by EDQM in Copenhagen, Denmark. During that symposium, a session dedicated to alternative or new microbiological methods took place. Presentation on validation and registration of rapid microbiological methods (Dr P. Newby from GSK and Dr R. Dabbah from USP), testing of water with solid phase cytometry (Dr S. Guyomard from Aventis), detection of Mycoplasma by PCR (Dr. L. Mallet from Aventis Pasteur and Dr. T. Hammerle from Baxter), and microbial identification with 16S rRNA methods (Dr T. Sasaki from National Institute of infection Diseases from Japan) were presented. Following this 2003 symposium, European pharmacopeia agreed on the need of recognition from both European regulatory authorities as well as European Pharmacopeia and created a specific subgroup to work on alternative microbiological methods. This led to the chapter 5.1.6. published in 2006 and the specific Working Party on Mycoplasma (WP MYC). In the meantime, USP was already working on a guide on the validation of alternative methods with a compendial approach, the chapter <1223>, also published in USP 29 in 2006.

In the general notices from the different pharmacopeias, there is specific sections about alternative analytical procedures. With the agreement of the competent authority, alternative procedures may be used for control purposes, provided that they enable an unequivocal decision to be made as to whether compliance with the standards of the monographs would be achieved if the official procedures were used. In the event of doubt or dispute, the analytical procedures of the Ph. Eur. are to be alone authoritative. (See chapter 1.1.2.5. from General notices Ph. Eur.).

3.1 Alternative and rapid Microbiological methods & European Pharmacopeia

An important chapter is chapter 5.1.6. Alternative methods for control of microbiological quality (last version applicable since July 2017). The first version of this chapter was written by a dedicated subgroup from group 1 (Microbiology), following May 2003 EDQM symposium in Copenhagen. Published in 2006, it was designed to provide both information and guideline. In 2016, in the supplement 9.2 from European pharmacopoeia, this chapter was entirely revised to take into account technological developments in alternative microbiological methods. The introduction and the sections concerning the 3 major types of microbiological tests have been reworded and expanded. In addition, information on the use of alternative methods for process analytical technology (PAT) was also added. The objective of this chapter is to facilitate the implementation and use of alternative microbiological methods where this can lead to cost-effective microbiological control and improved quality assurance for pharmaceutical products. These alternative methods may also find a place in environmental monitoring. The principle of detection, enumeration, isolation, and identification of the methods that have successfully been used in the quality control (QC) of pharmaceuticals are described and a guidance on how to validate alternative microbiological methods is provided. Finally, 3 examples of validation protocols of alternative microbiological methods as per chapter 5.1.6 are presented, including a sterility test based on ATP, a bioburden test based on solid phase cytometry, and a microbial identification test based on rDNA 16S sequencing.

Since 2023, the group 1 (Microbiology) from EDQM started to work on the update of this chapter (Solène Le Maux, Conference Pharmalab oral presentation, 21st November 2023 in Dusseldorf, the revision of chapter 5.1.6. Ph. Eur.). The chapter will be updated both to reflect the techniques currently in use and to update the validation guidance.

In parallel, the guidance chapter 5.1.9 GUIDELINES FOR USING THE TEST FOR STERILITY will also be updated and will include indication on the possibility to use the official method, 2.6.1 Sterility, or an alternative method in accordance with the principles provided in Chapter 5.1.6 Alternative methods for control of microbiological quality.

For sterility testing of cell-based preparations, the chapter 2.6.27 MICROBIOLOGICAL EXAMINATION OF CELL-BASED PREPARATIONS was first published in June 2006 and then updated in 2016 and 2021. This chapter suggests that automated growth-based methods include more flexibility for the incubation temperatures and provide examples of temperature settings where the test volume allows 2 incubation conditions. In addition, it includes an updated list of micro-organisms used for method validation. Information about the sensitivity to be achieved during validation was also added. The chapter considers the characteristics and limitations of these cell-based preparations, in particular their shelf-life, which may not always allow for completion of conventional microbiological examination tests before administration to the patient. Also, it discusses the amounts available for testing and sampling-related issues (with the proposed 1% of total amount of product to be tested).

In the introduction of chapter 2.6.12 MICROBIAL ENUMERATION TEST and 2.6.13 Test for SPECIFIFED MICROORGANISMS, there are the mention that alternative microbiological procedures, including automated methods, may be used, provided that their equivalence to Pharmacopoeia methods has been demonstrated.

In Chapter 2.6.7. MYCOPLAMAS, since January 2008 this chapter describes the two classical methods (i.e., the microbiological culture method and the indicator cell culture method) along with the Nucleic Acid amplification Techniques (NAT ) may be used as an alternative after suitable validation. The chapter describes the guideline for such NAT validation, the LOD to be reached and the comparability protocol. At the end of 2018, a WP MYC was assembled to update this chapter. After reviewing comments received and after discussions with the relevant Groups of Experts/Working Parties, this chapter has been amended and will be republished for public consultation in Pharmeuropa 36.1 (January 2024). For more details, see presentation from Dr Thuy Bourgeois, EDQM, in the pre-Conference workshop Pharmalab 2023, 20 Nov 2023 entitled “International Mycoplasma qPCR testing User Day”.

The general chapter 2.6.21. NUCLEIC ACID AMPLIFICATION TECHNIQUES was published in July 2014. In this general chapter, the PCR method is described as the reference technique. Alternative methods may be used if they satisfy the quality requirements described. This section establishes the requirements for sample preparation, in vitro amplification of DNA sequences, and detection of the specific PCR products. With the aid of PCR, defined DNA sequences can be detected. RNA sequences can also be detected following reverse transcription of the RNA to complementary DNA (cDNA) and subsequent amplification. A specific section is dedicated to Quality Assurance and two examples of validation of NAT in plasma pools (one for HCV and the other for the B19 virus) are discussed.

Since 2018, it is mentioned in chapter 2.6.16. TESTS FOR EXTRANEOUS AGENTS IN VIRAL VACCINES FOR HUMAN USE, that sensitive molecular methods with broad detection capabilities are available. These new approaches include high-throughput sequencing (HTS) methods, NAT (e.g., polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), product-enhanced reverse transcriptase (PERT) assays) for whole virus families or random-priming methods (associated or not with sequencing), hybridization to oligonucleotide arrays, and mass spectrometry with broad-spectrum PCR. These methods may be used either as an alternative to in vivo tests and specific NAT or as a supplement/alternative to in vitro culture tests, based on the risk assessment and with the agreement of the competent authority. For Spiroplasmas and Mycobacteria detection, it is specified that NAT (see above chapter 2.6.21) may be used as an alternative, provided such assay is validated and shown to be comparable to the culture method.

Likewise, in the guidance chapter 5.2.3 CELL SUBSTRATES FOR THE PRODUCTION OF VACCINES FOR HUMAN USE, NAT methods are also mentioned as potential alternatives (following adequate validation). In addition, new sensitive molecular methods with broad detection capabilities are referred to, including novel high-throughput sequencing (HTS) methods.

A future chapter under construction, chapter 2.6.41. “High Throughput Sequencing for the detection of viral extraneous agents“, will describe the technology, methods, and workflow of the HTS methods, as well as guidelines for their validation. This future chapter is being prepared by the HTS WP (Ph. Eur. group of Expert: regulators, OMCLs and industry from Europe, US, Canada) and should be published in Pharmeuropa 2024.

In the field of pyrogens and endotoxins assay, European pharmacopoeia is at the forefront with the chapter 2.6.30. Monocyte activation test, first applicable in 2010 and updated in 2016. The monocyte- activation test (MAT) is used to detect or quantify substances that activate human monocytes or monocytic cells to release endogenous mediators such as pro-inflammatory cytokines such as tumour necrosis factor alpha (TNFα), interleukin-1 beta (IL-1β), and interleukin-6 (IL- 6). These cytokines are known to have a role in fever pathogenesis.

Consequently, the MAT will detect the presence of pyrogens in the test sample. The MAT is suitable, after a product-specific validation, as a replacement for the classical rabbit pyrogen test. Since 2018, in chapter 2.6.8. describing Pyrogens test in Rabbit (RPT), it is mentioned that wherever possible and after product-specific validation, the pyrogen test ought to be replaced by the monocyte-activation test (2.6.30). The EDQM is working for complete removal of chapter 2.6.8. in all related texts (60 texts) and a new guideline on Pyrogenicity (future guideline 5.1.13.) prepared by the WP BET has been finalized and published in PHARMEUROPA 34.2 for public comments. Finally, a new update and the 3rd version of chapter 2.6.30. MAT will be published in 2024. For more information on Pyrogens, see Dr Gwenaël Ciréfice (EDQM) presentation during track 3, Pharmalab congress November 2023.

On the topic of Endotoxins, the new chapter 2.6.32 TEST FOR BACTERIAL ENDOTOXINS USING RECOMBINANT FACTOR C was published in July 2020 and has been applicable since January 2021. The EDQM was the first pharmacopeia to introduce in compendia recombinant Factor C (rFC) reagent for endotoxins testing. The rFC reagent was added to the BET WP work program at the 131st session of Ph. Eur. Commission (June 2008). In 2016, the guideline chapter 5.1.10. Guideline for BET opens the way for the use of rFC and states: “the use of alternative reagents such as recombinant factor C (rFC) as a replacement to the amoebocyte lysate eliminates the use of a reagent extracted from live animals”. Chapter 2.6.32. describes a BET that uses a rFC based on the horseshoe crab gene sequence, and a fluorimetry end-point detection method. This chapter is no longer a standalone chapter and, since Pharmeuropa 34.3. (July 2022), the EDQM has published updated versions Water for injections (0169) and Purified water (0008), two fundamental Ph. Eur. monographs. Both texts have been revised to allow the use of rFC to test for bacterial endotoxins, as described in general chapter 2.6.32. These revisions have been adopted, the revised monographs published in supplement 11.4, and the implementation date is 1 April 2024. For users, update mean that they can select the test described in 2.6.32 directly when testing pharmaceutical waters, that is, without the requirement of a side-by-side comparison against the tests described in general chapter 2.6.14 Bacterial endotoxins.

3.2 Alternative and rapid Microbiological methods & USP

In parallel to chapter 5.1.6. on alternative microbiological methods, the UPS chapter <1223> VALIDATION OF ALTERNATIVE MICROBIOLOGICAL METHODS provides guidance on the selection, evaluation, and use of microbiological methods as alternatives to compendial methods. To properly implement alternative methods, one must consider a number of important issues before selecting the analytical technology and qualifying that method with the actual product. These issues include, but are not limited to, identification of suitable alternative methodology, development of user specifications for equipment selection, demonstration of the applicability of the method as a replacement for a standard compendial method, and qualification of the method in the work environment it is meant to be used. The limitations of the use of CFU (Colony Forming Unit) as a standard signal for microbiological methods are also described. This chapter has been updated in May 2018 but was first made official in the 2nd Supplement of USP 29-NF 24 in 2006, at about the same time as the Ph. Eur. chapter 5.1.6. (Applicable in 2006.)

A new guideline chapter <1071> Rapid Sterility Testing of Short- Life Products: A Risk-Based Approach was published June 2019 and became official in December 2019. USP created an expert panel in 2015 called “Modern Microbiological Methods (MMM) Expert Panel” to work on this chapter and a stimuli article was published in PF 43.5, (September-October 2017). In the chapter’s introduction, it is mentioned that the use of rapid microbial tests (RMTs) should be risk-based so the stakeholder can select the preferred technology for their intended use and balance user requirement specifications (URS) including time to result, specificity, limit of detection (LOD), sample size, and product attributes. This general informational chapter discusses the needs of those who manufacture/prepare and test products with a short shelf life and the associated URS and includes a brief discussion of suitable methods for risk-based rapid microbial testing for the release of short shelf-life sterile products (hereafter referred to in this chapter as “short-life products”).

After the publication of this new chapter Guideline <1071>, two new chapters were published in 2021 but are not yet applicable: chapter <72>: RESPIRATION-BASED RAPID MICROBIAL METHODS FOR THE RELEASE OF SHORT SHELF-LIFE PRODUCTS and chapter <73>: ATP BIOLUMINESCENCE-BASED RAPID MICROBIAL METHODS FOR THE RELEASE OF SHORT SHELF-LIFE PRODUCTS. Still at the stage of public comment, another new chapter was published in PF 48(5) in September 2022 and is entitled chapter <74> Solid Phase Cytometry-Based Rapid Microbial Methods for the Detection of Contamination in Short Shelf-Life Products. Finally, as part of future work from 2024 onwards, USP announced two chapters centered on the theme of rapid sterility testing for short shelf-life products: chapter <75>: NUCLEIC ACID AMPLIFICATION METHODS and chapter <76>: FLOW CYTOMETRY METHODS.
On Mycoplasma, a new chapter published in September 2022 and now at the stage of public comment, is entitled chapter<77> MYCOPLASMA NUCLEIC ACID AMPLIFICATION TESTS. This new chapter describes criteria for selecting a NAT to detect mycoplasma that is comparable to either the compendial methods from Mycoplasma Tests chapter <63>: the agar and broth media procedure (method A) or the indicator cell culture procedure (method B). The new USP MEC (Microbiology Expert Committee) will address the comments and work on that chapter in 2024.
Also related to the use of NAT based methods, it is of note that USP will work on a NUCLEIC ACID AMPLIFICATION TEST (NAT) for Burkholderia cepacia detection.
Finally on the topic of pyrogens and endotoxins, USP released in 2023 a new chapter <86> “Bacterial Endotoxins Test Using Recombinant Reagents”. It has entered the stage of public comment in PF 49(6), 1st November 2023. The deadline for comments is January 31st, 2024. This new chapter provides additional techniques using non-animal derived reagents to complement the Bacterial Endotoxins Test <85>. This test uses a reagent containing the recombinant Factor C (rFC) protein or a recombinant cascade reagent (rCR) containing recombinant Factor C, recombinant Factor B, and recombinant pro-clotting enzyme. The main difference with Ph. Eur. chapter 2.6.32. is that the current draft chapter <86> proposed the rCR reagent. For Pyrogens, USP has not yet planned a chapter even in a guideline mode for MAT to replace RPT.

3.3 Alternative and rapid Microbiological methods and Japanese Pharmacopoeia

Like in Ph. Eur. chapter 5.1.6. and USP chapter <1223>, the JP includes a chapter on ARMMs: chapter <G4-6-170> RMM Rapid Microbiological Methods. In the introduction to this chapter, it is mentioned that, since the 1980s, it has become clear that most bacteria in the natural environment have low growth ability in conventional culture media and that the detection, enumeration, and identification of these bacteria are difficult by means of culture methods alone. Compared to the conventional methods, these new methods are not necessarily superior in every respect, but they usually offer a shorter time to result, greater accuracy and can also applied to fungi and viruses. The chapter describes all detection targets and principles; it is divided in 2 parts distinguishing direct methods such as solid phase and flow cytometry and indirect methods. Then, a specific paragraph is dedicated to the validation part and, finally, there is an overview of different possible applications such as quality control of water, sterility, microbial limit test, antimicrobial and preservatives effectiveness, raw materials, etc. The JP offers some considerations on RMMs, mentioning that RMMs rely on principles that differ from those of the conventional methods and, as such, are based on other signals than CFU. The JP also states that correlation between RMMs and conventional methods is not always required and that these methods can be applied to quality control via the PAT.

Overall, these novel methods should improve both accuracy and time to results compared to conventional method. Moreover, approaches that use phylogenic analyses based on high-throughput sequencing offer the possibility to glimpse at the real microbial world in pharmaceutical manufacturing facilities.

Since JP15, the Mycoplasma testing for cell substrates used for the production of biotechnological/biological products, chapter <G3- 14-170> describes NAT mycoplasma applications. The two classical tests are described, that is, the culture method and the indicator cell culture method. However, it also includes a method C based on NAT. It underlines that several NAT methods are available without prescribing any particular ones. Overall, the selected NAT method should be validated for adequate sensitivity (ie, detection limit) and specificity, as well as for robustness (ie, results unaffected by small variations in extraction method parameters or in composition of the reaction mix). Any NAT method can be used as long as it’s specificity and sensitivity have properly been validated, as described in the chapter.

A chapter entitled rapid counting of microbes using fluorescent staining chapter <G4-8-152> has been part of JP since JP15 Supplement 2. This chapter overviews rapid methods using fluorescence staining for the quantitative estimation of viable microorganisms. It first underlines that incubation on an agar medium has been widely used for quantitative estimation of viable microorganisms, but that several environmental microorganisms of interest are not easily grown under usual conditions. To overcome this problem, new microbial detection methods based on fluorescence or luminescence have been developed. Two methods are described in the chapter, the CFDA-DAPI double staining method and the microcolony method. These rapid and accurate methods tend to give higher counts than the other techniques as they provide quantitative estimates of viable microorganisms based on a very specific definition of viability, which may be different from that implicit in other methods.

For microbial identification, chapter <G4-7-160> Rapid identification of microorganisms based on Molecular Biological Methods has been included since JP 16. This chapter describes methods to identify or enumerate microorganisms (bacteria and fungi), found in in-process control tests or lot release tests of pharmaceutical products, at the species or genus level, based on their DNA sequence homology. The identification of isolates found in the sterility test or aseptic processing can be helpful for investigating the causes of contamination. Furthermore, information on microorganisms found in raw materials used for pharmaceutical products, processing areas of pharmaceutical products, and so on, is useful in designing measurements to control the microbiological quality of drugs.

For endotoxins test, a new guideline was published in September 2019, chapter <G4-4-180> “Bacterial Endotoxins Test and alternative methods using recombinant protein reagents for endotoxin assay”. In the § 4 of this new guideline, the measurement by alternative methods using recombinant protein-reagents for endotoxin assay and points is considered. As of now, this chapter is still a guideline and recombinant reagents are still considered by JP as alternative methods. If these reagents for endotoxin assay are used as an alternative method, users need to demonstrate that accuracy, precision, sensitivity, specificity, etc. are equal or better compared to Bacterial Endotoxins Test <4.01> using lysate reagents.

3.4 Alternative and rapid Microbiological methods & Chinese Pharmacopoeia

The main traditional and conventional methods (sterility, endotoxins, microbial enumeration tests, etc.) described in the Chinese Pharmacopoeia are not yet fully harmonized with Ph. Eur., USP and JP. However, since 2020, the ChP has moved closer to these other pharmacopeias although there are still slight differences.

Since ChP 2020, a chapter is dedicated to ARMMs: chapter 9201 Guidelines for Validation of Alternative Microbiological Methods for Pharmaceutical products. The objective of this chapter is to provide guidelines for validating methods to be used as alternatives to classical culture-based microbiological methods for pharmaceutical products. With the rapid development of microbiological analytical technology, and in order to meet the demands of in-process control of pharmaceutical manufacture, some new technologies for pharmaceutical microbiological control have been introduced. Compared with traditional methods, the new technologies are rapid and allow real-time or near-real-time monitoring. The chapter divides technologies in 3 types: (1) growth-based detection methods, where a detectable signal such as bioluminescence, electrochemical methods and turbidimetry, is usually achieved after a period of subculture ; (2) direct detection measurement of viable microorganisms, such as solid phase cytometry and flow cytometry ; (3) specific cell component analysis such as fatty acid analysis, NATs, and genetic fingerprinting. The types and Validation Parameters of Microbiological Tests are described with a table including validation Parameters by Type of Microbiological Test (qualitative or quantitative).

For endotoxins testing, a new guideline was also introduce in ChP 2020, under chapter 9251 Guidelines for Application of Bacterial Endotoxin Test Methods. The guidelines further explain the content and application of bacterial endotoxin test methods. At the end of this chapter, the rFC method is described as an attachment. Of note, Factor C is a protein that is sensitive to bacterial endotoxin in Tachypleus Amebocyte Lysate and is capable of selectively recognizing that endotoxin. According to the testing principle, this method does not have the G-factor bypass interference and is highly specific. It is thus suitable for testing sample containing β-glucan interference. The reagent used in this method does not contain factor B, coagulase or coagulogen, etc., therefore, a sample containing an inhibitory or potentiating effect on the above substances is suitable for use in the rFC method. However, according to the ChP, rFC reagent is still considered as an alternative method compared to traditional TAL/LAL reagents.

4. Conclusion

Overall, the interest for ARMMs is still high in the pharmaceutical industry. This is mainly due to the over 20 years of experiences and feedback on some methods and to the appearance of a number of ARMMs specifically designed for use in pharmaceutical sector. However, the registration and full implementation of ARMMs still remains quite low despite a slight increase observed since 2020.

The conventional and traditional microbiological methods used for detection, enumeration and identification of microorganisms can hardly be qualified as modern. These methods have been highly successful, remained both quite cheap and simple to perform. However, they are also labor intensive, take between 5 (bioburden) up to 56 days (Mycobacteria detection) to generate results, and require expert interpretation that, in some contexts, may raise questions of data integrity. Moreover, they remain difficult to automate and will never provide real-time data for accurate and responsive process control.

In contrast, ARMMs methods could provide rapid results, in some cases in real time or in near real time as some used in-line or on-line PAT applications. These applications tend to be easier to automate, to be labor efficient, and show outstanding data integrity compliance. There is a real need for the introduction, validation, and implementation of these ARMMs in pharmaceutical industries as they could help address the requirements for a highly technological and competitive twenty first century industry!

As things stand today, it would be wrong to claim that pharmacopeias are old-fashioned and rather conservative with regards to analytical methods. Pharmacopeias have their own pace but keep very close to contemporary science.

For over 20 years now, pharmacopoeias have helped and supported the pharmaceutical industry by working to develop alternative microbiological methods into guidelines, at least, or into the best compendial alternative microbiological methods. This helped introduce novel methods and guided future users toward the validation and implementation of their approach, until acceptability by competent health authorities.

The Author Thierry Bonnevay is a Sanofi employee and may hold shares and/or stock options in the company. Thierry Bonnevay would like to thank Jean Sebastien Bolduc (Global Scientific & Medical Publications Manager from SANOFI) for proofreading this article and for his comments.

Thierry Bonnevay La Vague 80 A3P

Thierry BONNEVAY – SANOFI VACCINE

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  • ECA, 11th Pharmalab 2023 conference, Dusseldorf, 20-22 November 2023. Solène Le Maux, EDQM, oral presentation “Revision of Ph. Eur. chapters 5.1.6 Alternative methods for control of microbiological quality”/ Thuy Bourgeois, EDQM, oral presentation “Current revision of Ph. Eur. chapter 2.6.7 Mycoplasmas and its impact on other Ph. Eur. texts” / Gwenaël Ciréfice, EDQM, oral presentation “Towards animal free pyrogen tests in the Ph. Eur: latest progress” / Paul Newby, Gilberto Dalmaso, Bryan Riley, Peter Cooney, and Kim Tyndall. The Introduction of Qualitative Rapid Microbiological Methods for Drug-Product Testing. Pharmaceutical Technology PROCESS ANALYTICAL TECHNOLOGY 2004.
  • British Pharmacopeia. 1932. B. test for sterility.
  • Moldenhauer, J. The Rush to Rapid Microbiological Methods – Or Not. Eur. Pharm. Rev. 2017, Issue 2. Am. Pharm. Rev. (accessed Oct. 28, 2021).
  • Miller, M. The Encyclopedia of Rapid Microbiological Methods: The New Fourth Volume Discusses Technologies, Regulatory Acceptance and Validation Case Studies. Eur. Pharm. Rev. 2013, Issue 3.

Glossary

✻
  • ARMM – Alternative and Rapid Microbiological Methods Adenosine Triphosphate
  • ATP – Adenosine Triphosphate
  • BET – Bacterial Endotoxin Test
  • ChP – Chinese Pharmacopeia
  • EDQM – European Directorate for Quality of Medicines
  • EU – Europe
  • FDA – U.S. Food and Drug Administration
  • HAs – Health Authorities
  • HTS – High Throughput Sequencing
  • ICH – International Council for Harmonization
  • IPC – Indian Pharmacopeia Commission
  • JP – Japanese Pharmacopeia
  • LAL – Limulus Amebocyte Lysate
  • LOD – Limit Of Detection
  • LOQ – Limit of Quantification
  • MHLW – Ministry of Health, Labor & Welfare, Japan
  • NAT – Nucleic Acid Tests
  • NF – National Formulary
  • NMPA – National Medical Products Administration, China
  • OMCLs – Official Medicines Control Laboratories
  • PAT – Process Analytical Technology
  • PCR – Polymerase Chain Reaction
  • PDG – Pharmacopeial Discussion Group
  • PF – Pharmacopeial Forum
  • Ph. Eur. – European Pharmacopeia
  • PMDA – Pharmaceuticals and Medical Devices Agency, Japan Quality Control
  • QC – Quality Control
  • qPCR – quantitative Polymerase Chain Reaction
  • rFC – recombinant Factor C
  • RMM – Rapid Microbiological Methods
  • rRNA – Ribosomal Ribonucleic Acid
  • USP – United States Pharmacopeia
  • WHO – World Health Organization

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