Microbiologists have the continuing challenge of evaluating, validating, and implementing emerging testing technologies to enhance the performance of their laboratories. The now dated USP general informational chapter <1113> Microbial Characterization, Identification and Strain Typing endeavored to discuss a hierarchy of responses to microorganisms isolated from pharmaceutical manufacturing facilities, ingredients, and drug products based on the criticality of the testing. The objectives, which remain valid, are to evaluate microbial contamination risk associated with both non-sterile and sterile product products. As pharmaceutical manufacturers, we want to manage both the microbial count and exclude objectionable microorganisms from non-sterile products and exclude all microorganisms from sterile products. Microbial identification helps us to define the microbial contamination risk level with non-sterile products while the location of the source of the microorganisms is useful in product failure investigations designed to mitigate risk.
The available technologies are evolving. The question is how can we employ them in a useful, complaint, and cost-effective manner? One source of information that can guide us is the experience gained during the introduction of these technologies in the allied sub-disciplines of food and clinical microbiology. The goals in these two sub-disciplines differ from pharmaceutical microbiology, but are sufficiently similar to be useful. Clinical microbiologists work with infectious disease specialists to evaluate clinical specimens to confirm patient infection, identify the infectious agent, and recommend suitable antibiotics, anti-fungal agents or antivirals for successful treatment of the infectious disease. In contrast, food microbiologists monitor to confirm sanitary conditions in food harvesting and processing plants, control that number of microorganisms in foods to prevent spoilage, and enable their industry to harvest, process and distribute foods with sufficient controls, so they are less likely to contain foodborne pathogens. Pharmaceutical microbiologists control microorganisms in our manufacturing facilities, utilities, pharmaceutical ingredients, and process equipment, and exclude microorganisms from our drug products.
Adoption of Emerging Technologies
A major question asked by microbiologists and their management alike is when should our industry transition from traditional microbiological culture-based methods to modern microbiological methods that are frequently based on molecular methods using nucleic acids as targets. Often this transition will be directed to specific applications. The most compelling reasons to make this transition are improved quality of the results, better contamination control, reduced time to the result, greater testing throughput, reduced analyst time, and reduced overall testing cost. These tests could be included in New Drug Applications/Biologic License Applications regulatory filings or as supplements to filings for approved drug products, but our regulatory affairs colleagues actively discourage their inclusion as added complications. As with the introduction of new manufacturing technologies, it is uncertain that the business justification is strong enough to drive these innovations in a risk adverse industry (Mantle and Lee, 2020).
Testing Methods for Non-Sterile Drug Products
As a practicing pharmaceutical microbiologist, the author always felt the testing and release of non-sterile drug products was a greater challenge to microbiologists than sterile products were as sterilization processes are predictable, controllable, and validatable and thus are more the domain of the process engineer.
With product-release and shelf-life microbial testing three levels of non-sterile products testing are required: 1) Microbial enumeration, 2) testing for the absence of specified microorganisms, and 3) screening for objectionable microorganisms. The test methods and acceptance criteria are found in USP <61> Microbiological examination of nonsterile products: Microbial enumeration tests, <62> Microbiological examination of non-sterile products: Tests for specified microorganisms and <1111> Microbiological examination of non-sterile products: Acceptance criteria for pharmaceutical preparations and substances for pharmaceutical use.
In addition to the <62> absence of specified microorganism requirements, pharmaceutical manufacturers must comply with the Federal CGMP regulations 21 CFR 211.113 Control of microbiological contamination that states: “a) Appropriate written procedures, designed to prevent objectionable organisms in drug products not required to be sterile, shall be established and followed.”, and 21 CFR 211.165 Testing and release for distribution (b) that states: “There shall be appropriate laboratory testing, as necessary, of each batch of drug product required to be free of objectionable microorganisms”.
CFR 211.113 does not define the term objectionable microorganisms, but they can be broadly defined as: 1) Microorganisms that can proliferate in a product adversely affecting the chemical, physical, functional and therapeutic attributes of that pharmaceutical product, and 2) Microorganisms that due to their numbers in the product and their pathogenicity can cause infection in the patient in the route of administration when treated with that pharmaceutical product. As the result of the work of a broad-based industry task force, PDA TR No. 67 Exclusion of Objectionable Microorganisms from Non-Sterile Pharmaceuticals, Medical Devices and Cosmetics, published in October 2014, recommended modification of the <61> and <62> tests to screen for objectionable microorganisms. Is this recommendation enough?
Future options under discussion in the USP Microbiology Expert Committee include developing nucleic acid-based tests for the individual specified microorganisms that may be extended to screen an expanding range of objectionable microorganisms or eliminating specified microorganism screening altogether and replacing it with enumerating and isolating the predominant microorganisms in a drug product and employing modern microbiological methods to identify representative isolates, e.g., MALDI-TOF mass spectrometry supplemented with 16s rRNA base sequencing. A prominent microbiological contract-testing laboratory successfully promoted this latter identification strategy. The relative merits of these options and the opportunities to coordinate with the other two major pharmacopeias need definition.
A more radical step would be to de-emphasize species screening altogether and transition to genomic screening for virulence, antibioticresistance and biocide-resistance as indicators of microbial infection risk. An example of this approach is screening for extended-spectrum beta-lactamases and carbapenem-resistant Enterobacteriaceae and not the wider species (Gazin et al, 2012).
Multiplex PCR Screening
Multiple companies have developed instrumentation, sponsored evaluations, and are marketing clinical panels for bacterial, fungal, and parasitic pathogens found in sputum, stool, urine and blood based on multiplex PCR technologies. The best systems will be highly automated, give comparable results to current methods, have internal controls, be closed systems, and deliver results in less than two hours (Buss et al, 2015). The author believes this technology may be applied in the future to biofermenter contamination, objectionable microorganism and purified water screening.
Strain typing has been promoted as a tool for use in product failure investigations linking the source of the microbial contamination to pharmaceutical ingredients, processing equipment, operators or microbiologists conducting the product testing. The argument went as follows: if you found the same strain in the testing area but not the manufacturing facility, it would be strong evidence of a laboratory error. The author suspects that this application has been over hyped. The approach has been valuable in clinical trials for new antibiotics showing that infection subsequent to treatment was unique with a different strain responsible or demonstrating that extrinsic contamination during patient use and not intrinsic contamination during manufacturing was the cause of microbial contamination of the marketed product in the field. An example of the use of strain typing was in a national product-related Fusarium keratitis outbreak where the contact lens solution isolates were genotyped by multi-locus sequence typing (MST) confirming extrinsic fungal contamination due to the failure of the antimicrobial preservative system and biofilm formation in the lens case from different fungal strains and not fungal contamination during lens solution manufacturing (Chang et al, 2006). In epidemiological investigations of foodborne infection outbreaks, strain typing has traced the source of the infection to a single product, processing plant or farm, whole genomic sequencing (WGS) has proved very useful. For example, WGS of all U.S. Listeria monocytogenes isolates from patients, food, and the environment in September 2013 transformed the epidemiology of an outbreak investigation (Jackson et al., 2016).
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Microbial Testing for Sterile Drug Products
USP general informational chapter <1071> Rapid Sterility Testing of Short-lived Products: A Risk-based Approach identified five candidate technologies suitable to replace the traditional sterility tests described in USP <71> Sterility Tests for microbial contamination detection in short-lived products such as gene and cell therapies. The technologies were described generically as respiration, ATP bioluminescence, nucleic acid-based, flow and solid phase cytometric methods. Many of these technologies have a long history of use in clinical, food and pharmaceutical microbiology and details of their evaluation, validation, and implementation is published in the peer-reviewed technical literature, so it is the intent of the USP Microbiology Expert Committee to write official test chapters generically describing their application for sterility testing. For example, respiration-based sterility tests are the industry standard in cell therapy (England et al, 2019). As official compendial methods, these tests would not require method validation as alternative methods to USP <71> and could be implemented after demonstration of method suitability for the intended to be tested products. This would reduce the barrier for implementation, but regulatory approval to test and release a drug product using the technology would still be required.
A promising, emerging technology is Raman optical spectroscopy that could provide label-free bacterial detection, identification, and antibiotic susceptibility testing in a single step in clinical specimens like blood, urine, and sputum (Ho et al., 2019) and investigate medical biofilms (Beier et al. 2012). Opportunities directed towards pharmaceutical microbiology include purified water monitoring with the ability to enumerate, identify, and classify waterborne bacteria into planktonic and biofilm-derived organisms facilitating the timing of a purified water distribution system sanitization (Magalhaes et al, 2020) and microbial identification (Ho et al, 2019).
Next Generation Sequencing
To the non-specialist the difference between the terms Next Generation Sequencing (NGS) and Whole Genome Sequencing (WGS) is confusing, largely because they overlap and are time driven. Originally, DNA sequencing technology was dominated for around 30 years by procedures using the dideoxynucleotide chain termination method (Sanger sequencing). In the mid-2000’s, a number of new methods appeared termed next-generation sequencing methods that used different biochemical approaches and instrument analyzing long sequences, at lower cost, more rapidly, with greater levels of automation. With WGS, the entire genome is randomly fragmented and the bases from resulting DNA sequences are read and compared to sequence databases or to genome structures following assemble. This can be applied to a single bacterium, a mixture, or a meta-genome without separating the genomes or culturing the microorganisms (Goldberg et al., 2015; Deurenberg et al., 2016; Allard et al. 2018).
The advantages in clinical microbiology are more obvious than in food and pharmaceutical microbiology and include identification of hard to and slow to grow pathogens, identification of toxin-production, antibiotic-resistance, and virulence genes, and strain identification and typing. With food microbiology, these technologies are invaluable in outbreak investigations by state and federal agencies but their value for routine facility and product monitoring is debatable (Ferguson, 2020; Klijn et al., 2020)
As emerging technologies move from research to clinical laboratories, there are opportunities to selectively implement them in food and pharmaceutical microbiology. (See Table 1 for a simple comparison of the advantages and disadvantages of the different technologies). Given the capital cost of the instrumentation, reagent costs, and skill level required to support these technologies, smaller companies may choose to use contract testing laboratories for these tests, and larger companies set up centers of excellence to provide testing services to multiple sites within their company. It must be emphasized that microbiologists recommending these technologies must make a compelling business case for their implementation and meet regulatory expectations.
- Allard, M. et al. 2018 Genomics of foodborne pathogens for microbial food safety. Curr. Opin. Biotechnol. 49: 224-229
- Beier, B.D et al. 2012 Raman micro-spectroscopy for species identification and mapping within bacterial biofilms AMB Express 2:35-41
- Buss SN, Leber A, Chapin K, et al. 2015 Multicenter evaluation of the BioFire FilmArray gastrointestinal panel for etiologic diagnosis of infectious gastroenteritis. J Clin Microbiol. 53(3):915-925. doi:10.1128/JCM.02674-14
- Chang, D.C. et al 2006 Multistate outbreak of Fusarium keratitis associated with use of a contact lens solution. JAMA 296(8):953-963
- Deurenberg, R.H. et al 2017 Application of next generation sequencing in clinical microbiology and infection prevention. J. Biotechnol. 243: 16-24
- England, MR., Stock, F., Gebo, JET., Frank, KM., Lau, AF. (2019). Comprehensive Evaluation of Compendial USP<71>, BacT/Alert Dual-T, and Bactec FX for Detection of Product Sterility Testing Contaminants. J. Clin. Microbiol. 57:1548-18
- Ferguson, B. 2020 Adoption of WGS: What is going on? August/September 2020
- Gazin, AU, M, Paasch F, Goossens H, Malhotra-Kumar S, MOSAR WP2 and SATURN WP1 Study Teams SO 2012 Current trends in culture-based and molecular detection of extendedspectrum- β-lactamase-harboring and carbapenem-resistant Enterobacteriaceae. J Clin Microbiol. (4):1140-1146
- Goldberg, B. et al 2015 Making the leap from research laboratory to clinic: Challenges and opportunities for next-generation sequencing in infectious disease diagnostics. mBio 6(6) e01888-15
- Ho, C-S et al 2019 Rapid identification of pathogenic bacteria using Raman spectroscopy and deep learning Nature Communications | https://doi.org/10.1038/s41467-019-12898-9
- Jackson, D.C. et al 2016. Implementation of nationwide realtime whole-genome sequencing to enhance Listeriosis outbreak detection and investigation. Clin. Infect. Dis. 63(3):380–6
- Klijn, A. et al 2020 he benefits and barriers of whole-genome sequencing for pathogen source tracking: A food industry perspective. Food Safety Magazine June/July 2020
- Mantle, J.L. and K. H. Lee 2020 NIIMBL-facilitated Active Listening Meeting between industry and FDA identifies common challenges for adoption of new biopharmaceutical manufacturing technologies. PDA J. Pharm. Sci. & Technol.
- 10.5731/pdajpst.2019.011049 doi:10.5731/pdajpst.2019.011049
- Maruthamuthu , M et al 2020 Raman spectra-based deep learning – A tool to identify microbial contamination in the pharmaceutical industry Authorea. June 22, 2020.
- DOI: 10.22541/au.159285368.84034189
- Magalhaes, A., Goldberg Oppenheimer, P., Overton, T., and Wright, K.: Real-Time Monitoring of Industrial Biofilms using Confocal Raman Microscopy and Multivariate Analysis, biofilms 9 conference, Karlsruhe, Germany, 29 September–1 Oct 2020, biofilms9-35, https://doi.org/10.5194/biofi lms9-35, 2020