RMM's
Enumeration & Presence/Absence:
Real-Time PCR Detection of Burkholderia cepacia in Pharmaceutical Prod-ucts Contaminated with Low Levels of Bacterial Contamination
Jimenez L, Jashari T, Vasquez J, et al. (2018). PDA J Pharm Sci and Tech, vol. 72, pp. 73-80.
A real-time polymerase chain reaction (RT-PCR) assay was developed to detect Burkholderia cepacia in pharmaceutical products contaminated with low levels of bacteria. Different pharmaceutical suspen-sions were artificially contaminated with B. cepacia, Escherichia coli, Staphylococcus aureus, and Bacillus megaterium. After a 24 h incubation in trypticase soy broth with Tween 20, samples were streaked on mannitol salt, phenyl ethyl alcohol, eosin methylene blue, MacConkey, and pseudomonas isolation agar. Microbial DNA was extracted from each sample by using a Tris-EDTA, proteinase K, Tween 20 buffer. Regular PCR targeting the 1.5 kilobases 16S rRNA eubacterial gene and cloning showed the predominant DNA in the extracted mix belonged to E. coli. Selective media isolation of bacterial contamination showed B. cepacia only detected on pseudomonas isolation while eosin meth-ylene blue and MacConkey detected only E. coli. RT-PCR using primers PSL1 and PSR1 amplified a 209 bp 16S rRNA fragment using a Roche LightCycler 96® system with SYBR green I, a common double-stranded binding dye. The cycle at which fluorescence from amplification exceeds the back-ground fluorescence was referred to as quantification cycle.
Method Verification Requirements for an Advanced Imaging System for Microbial Plate Count Enumeration
Jones D, Cundell T, (2018). PDA J Pharm Sci and Tech, vol. 72 pp. 199-212.
The Growth Direct™ System that automates the incubation and reading of membrane filtration micro-bial counts on soybean-casein digest, Sabouraud dextrose, and R2A agar differs only from the tradi-tional method in that micro-colonies on the membrane are counted using an advanced imaging system up to 50% earlier in the incubation. Based on the recommendations in USP _1223_ Validation of New Microbiological Testing Methods, the system may be implemented in a microbiology laboratory after simple method verification and not a full method validation.
Use of a real-time microbial air sampler for operational cleanroom monitoring.
Eaton T, Wardle C, Whyte W, (2014). PDA J Pharm Sci Technol, vol. 68(2), pp. 172-84.
A sampler that detects and counts viable particles in the air of cleanrooms in real-time was studied. It was found that when the sampler was used to monitor airborne particles dispersed from a number of materials used in cleanrooms, including garments, gloves, and skin, the number of viable particles dispersed from these materials was greater than anticipated. It was concluded that a substantial proportion of these viables were of a non-microbiological origin. When the sampler was used to monitor a non-unidirectional airflow cleanroom occupied by personnel wearing cleanroom garments, it was found that the airborne viable concentrations were unrealistically high and variable in compar-ison to microbe-carrying particles simultaneously measured with efficient microbial air samplers. These results confirmed previously reported ones obtained from a different real-time sampler. When the real-time sampler was used in a workstation within the same cleanroom, the recorded viables gave results that suggest that the sampler may provide an effective airborne monitoring method, but more investigations are required.
https://www.ncbi.nlm.nih.gov/pubmed/24668604
Application of rapid microbiological methods for the risk assess-ment of controlled biopharmaceutical environments.
Sandle T, Leavy C, Jindal H, Rhodes R, (2014). J Appl Microbiol, vol. 116(6), pp. 1495-505.
AIMS:
To assess the different operational states within a biopharmaceutical grade clean room, using a rapid microbiological method. The method was a novel system, based on spectrometry, designed for sam-pling, discriminating, and enumerating airborne particles. Central to the study was the aim to deter-mine the microbiological levels as a clean room went from standard use through maintenance and shutdown, disinfection, and then back to standard use. The objective was to evaluate whether a rap-id method could replace conventional environmental monitoring using growth-based media.
METHODS AND RESULTS:
The instrument evacuated was a BioVigilant IMD-A(®) System, which is a real-time and continuous monitoring technology based on optical spectroscopy that can differentiate between biological parti-cles and inert ones (biological particles expressed as bio-counts based on the detection of microbial metabolites). The results indicated that certain activities lead to a high generation of biological parti-cles and in showing an increase over the baseline, would be regarded as presenting a microbiological risk to the cleanroom. These activities include removing HEPA filter grilles, turning off an air handing unit, and tasks which requires an active personnel presence, such as cleaning and disinfection.
CONCLUSIONS:
The optical instrument can be used to process sufficient information, so that clean rooms can be returned to use following a period of unexpected downtime or following maintenance without the need to wait for the results from growth-based methods. As such, this type of rapid microbiological method is worth exploring further for clean room air monitoring.
SIGNIFICANCE AND IMPACT OF THE STUDY:
Few studies have been undertaken which examine air-monitoring devices that can both enumerate and discriminate particulates, in a volume of air as 'inert' or 'biological'. This study extends this lim-ited field. Furthermore, the data collected in relation to cleanrooms is of interest in helping microbi-ologists understand that risks posed by different activities in relation to clean air-handling systems and personnel particle shedding.
http://www.ncbi.nlm.nih.gov/pubmed/24575809
Alternative to Ph. Eur. pour-plate method for detection of microbial contamination in non-sterile pharmaceutical preparations
Palicz A, Paul A, Hofmann A, Denzel K, (2014).
The SimPlate method was developed and validated for determining microbial count in order to rec-ord possible microbial contamination in non-sterile pharmaceutical preparations according to the European Pharmacopoeia (Ph. Eur.). In blank solutions, the validation results showed that the per-formance of the SimPlate method was in a similar range (between 1 cell to over 106 cells) or better than that obtained with the European Pharmacopoeia pour-plate method. According to the data, the SimPlate method was sufficiently accurate, specific, linear, repeatable and robust to determine the total aerobic microbial count (TAMC, e.g. Pseudomonas aeruginosa, Staphylococcus aureus) and total combined yeast/mould count (TYMC, e.g. Candida albicans, Aspergillus brasiliensis) in the solutions compared to the pour-plate method. Further development demonstrated the interchangeability of the SimPlate method and the pharmacopoeial pour-plate method for the determination of TAMC and TYMC in non-sterile pharmaceutical preparations. A dilution of 1:100 of Mycobacterium phlei e volumine cellulae in NaCl-peptone buffer showed comparable results between the SimPlate method and the pour-plate method for the detection of TAMC and TYMC with a detection limit of 100 CFU/g. Optimal incubation time was found to be between 24-28 h for TAMC and 3 days for TYMC. The mi-crobial count of samples with and without Mycobacterium phlei e volumine cellulae differed by not more than a factor of 2 in accordance with the European Pharmacopoeia. Compared with the phar-macopoeial pour-plate method, the recovery of micro-organisms with the SimPlate method was mostly higher, and never lower (from factor 1 to factor 3). The selected method for the determina-tion of microbial counts was suitable to record possible microbial contamination in Mycobacterium phlei e volumine cellulae extract and showed good correlation with the European Pharmacopoeia pour-plate method.
http://www.gba-group.de/fileadmin/media/Newsletter1603_Pharma/SN2014-01.pdf
Application of flow cytometry for rapid bioburden screening in vac-cine virus production.
Bhusari PK, Tabor DE, Yamagata R, Galinski MS, (2012). PDA J Pharm Sci Technol, vol. 66(5), pp. 445-52.
Sensitive and timely detection of bioburden in presterile filtration product in aseptic processing of biologics is a critical parameter for microbial control and assurance of final product sterility. An appli-cation of automated flow cytometry system was developed for rapid microbial assessment and in-process control in vaccine virus production. In order to minimize the background signal caused by the components of the chicken egg substrate sample matrix, a sample processing method to clear somat-ic cell debris was included. The sample processing and the automated analysis take approximately 5 to 7 min per test sample and the method provides objective results in real time, enabling uninter-rupted processing. The flow cytometry method was compared with the standard aerobic plate count method using tryptic soy agar in a parallel study of 1566 independent production-scale samples. The method was further characterized by spike recovery of five model bacterial organisms in representa-tive sample matrix. In comparison to the culture method, the flow cytometry method was shown to be 96.2% sensitive and 98.2% specific for the detection of bioburden at a level of sensitivity suitable for the process stage requirement with the advantage of a nearly instantaneous time to result.
https://www.ncbi.nlm.nih.gov/pubmed/23035028
Validation of the BacT/ALERT 3D System for Rapid Sterility Testing of Biopharmaceutical Samples.
Jimenez L, Rana N, Amalraj J, Walker K, Travers K, (2012). PDA J Pharm Sci and Tech, vol. 66, pp. 38-54.
ABSTRACT: The BacT/ALERT 3D system was validated to determine the sterility of different types of biopharmaceutical samples such as water for injection, unprocessed bulk, and finished bulk. The installation, operation, and performance qualification were completed and verified under good manufacturing practices. During the installation and operation validation stages, the functionality and security of the system and software were completed and verified.
For the performance qualification, 11 microorganisms were evaluated, six compendial (Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Candida albicans, Aspergillus niger, Clostridium sporogenes), one representing the number one microbial species in sterile product recalls (Burkholderia cepacia), and four environmental isolates (Kocuria rhizophila, Staphylococcus haemolyticus, Methylobacterium radiotolerans, and Penicillium spp.). Nine of the microorganisms were spiked into three different types of biopharmaceutical samples by three different analysts on different days to ascertain the equivalence, ruggedness, sensitivity, time of detection, and repeatability. In all samples, the BacT/ALERT exhibited equivalent or better detection than the standard test. With the exception of M. radiotolerans, all 11 microorganisms were detected within 2.5 days using the BacT/ALERT system and the standard test. The detection times for M. radiotolerans in the three sample types averaged 5.77 days. The minimum detectable level of cells for all the microorganisms tested was found to be within 1 to 2 CFU. The system optimized sterility testing by the simultaneous on-line, non-destructive incubation and detection of microbial growth.
http://journal.pda.org/content/66/1/38.abstract
Evaluation of growth based rapid microbiological methods for sterility testing of vaccines and other biological products.
Parveen S, Kaur S, Wilson-David SA, et al. (2011). Vaccine, Issue 29, pp. 8012-23.
Most biological products, including vaccines, administered by the parenteral route are required to be tested for sterility at the final container and also at various stages during manufacture. The sterility testing method described in the Code of Federal Regulations (21 CFR 610.12) and the United States Pharmacopoeia (USP, Chapter <71 >) is based on the observation of turbidity in liquid culture media due to growth of potential contaminants. We evaluated rapid microbiological methods (RMM) based on detection of growth 1) by adenosine triphosphate (ATP) bioluminescence technology (Rapid Milliflex® Detection System [RMDS]), and 2) by CO2 monitoring technologies (BacT/Alert and the BACTEC systems), as alternate sterility methods. Microorganisms representing Gram negative, Gram positive, aerobic, anaerobic, spore forming, slow growing bacteria, yeast, and fungi were prepared in aliquots of Fluid A or a biological matrix (including inactivated influenza vaccines) to contain approximately 0.1, 1, 10 and 100 colony forming units (CFU) in an inoculum of 10 ml. These preparations were inoculated to the specific media required for the various methods: 1) fluid thioglycollate medium (FTM) and tryptic soy broth (TSB) of the compendial sterility method (both membrane filtration and direct inoculation); 2) tryptic soy agar (TSA), Sabouraud dextrose agar (SDA) and Schaedler blood agar (SBA) of the RMDS; 3) iASTand iNST media of the BacT/Alert system and 4) Standard 10 Aerobic/F and Standard Anaerobic/F media of the BACTEC system. RMDS was significantly more sensitive in detecting various microorganisms at 0.1 CFU than the compendial methods (p < 0.05), whereas the compendial membrane filtration method was significantly more sensitive than the BACTEC and BacT/Alert methods (p < 0.05). RMDS detected all microorganisms significantly faster than the compendial method (p < 0.05). BacT/Alert and BACTEC methods detected most microorganisms significantly faster than the compendial method (p < 0.05), but took almost the same time to detect the slow growing microorganism P. acnes, compared to the compendial method. RMDS using SBA detected all test microorganisms in the presence of a matrix containing preservative 0.01% thimerosal, whereas the BacT/Alert and BACTEC systems did not consistently detect all the test microorganisms in the presence of 0.01% thimerosal. RMDS was compatible with inactivated influenza vaccines and aluminum phosphate or aluminum hydroxide adjuvants at up to 8 mg/ml without any interference in bioluminescence. RMDS was shown to be acceptable as an alternate sterility method taking 5 days as compared to the 14 days required of the compendial method. Isolation of microorganisms from the RMDS was accomplished by re-incubation of membranes with fresh SBA medium and microbial identification was confirmed using the MicroSEQ Identification System. BacT/Alert and BACTEC systems may be applicable as alternate methods to the compendial direct inoculation sterility method for products that do not contain preservatives or anti-microbial agents.
http://cat.inist.fr/?aModele=afficheN&cpsidt=24627047
Determination of indicator bacteria in pharmaceutical samples by multiplex PCR.
Faranjnia S, Hassan M, et al. (2009). Journal of Rapid Methods & Automation in Microbiology, vol. 17(3), pp. 328-338.
Rapid and sensitive detection techniques for indicator pathogens are important in pharmaceutical industry. However, common detection methods rely on bacterial culture in combination with biochemical tests, a process that typically takes 5-6 days to complete. Thus, the aim of this study was to develop a multiplex polymerase chain reaction (mPCR) assay for simultaneous detection and identification of four indicator pathogenic bacteria in a single reaction. Specific primers for indicator bacteria, namely Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella, were applied to allow simultaneous detection of them, and the sensitivity and specificity of each primer pairs were determined. In the mPCR with mixed DNA samples, specific bands for corresponding bacteria were simultaneously detected. Agarose gel electrophoresis of PCR products revealed 100% specificity of mPCR with single bands in the expected sizes. Low levels of microbial contamination less than 10 cfu per milliliter or gram of product were detected using mPCR assay. The detection of all four indicator pathogenic bacteria were completed in less than 8 h with this novel mPCR method, whereas the conventional United States Pharmacopeia methods and uniplex PCR required 5-6 days and 27 h for completion, respectively. Using mPCR assay, the microbial quality control of non-sterile pharmaceutical products can be performed in a cost-effective and timely manner in pharmaceutical industry. Detection of pathogenic indicators of Escherichia coli, Staphylococcus aureus, Salmonella and Pseudomonas aeruginosa is one of the mandatory tests in microbial quality of non-sterile pharmaceutical products; therefore, rapid and sensitive detection of the contaminations is of great importance for product release. According to the results of the present study, simultaneous detection of low levels of four major potential pathogenic bacteria in pharmaceutical finished products can be performed using mPCR in a cost-effective and timely manner, and upon these properties of the mPCR assay it could have potential applications in pharmaceutical industry.
http://onlinelibrary.wiley.com/doi/10.1111/j.1745-4581.2009.00154.x/abstract
Description and validation of a rapid (1 h) flow cytometry test for enumerating thermophilic bacteria in milk powders
Flint S, Walker K, et al. (2007). Journal of Applied Microbiology, vol. 102(4), pp. 909-915.
Aims: The aim of this study was to develop a rapid assay for enumerating thermophilic bacteria in milk powder.
Methods and Results: The BactiFlowTM flow cytometer was used to count bacteria based on esterase activity in viable bacterial cells. A protocol for total viable bacteria was modified by heat-treating the sample to selectively label thermophilic bacteria. Samples of milk powder dissolved in 0·1% peptone were treated with 0·8% ethylenediaminetetraacetic acid to reduce background interference because of denatured milk proteins. Either thermophilic bacteria were added to the dissolved milk powder or milk powder solutions were incubated at 55°C for 2–3 h to enrich the natural thermophile population for testing. Results from the BactiFlow were compared with traditional plate count results.
Conclusions: Thermophilic bacteria in milk powder can be enumerated within 1 h using the BactiFlow flow cytometer.
Significance and Impact of the Study: Microbiological test results obtained within 1 h can potentially be used to monitor manufacturing processes, effectively trace problems and provide confidence in the manufacture of product
http://www.ncbi.nlm.nih.gov/pubmed/17381733
Flow cytometric analysis of bacteria- and virus-like particles in lake sediments.
Duhamel S, Jacquet S, (2006). Journal of Microbiological Methods, vol. 64, pp. 316-332.
Flow cytometry (FCM) was successfully used to analyze freshwater bacteria and viruses in lake sediments after relatively simple sample treatment and optimization of dilution/fixation/staining procedures. Biological particles from Lakes Geneva and Bourget were first separated from the sediments by using both Sodium Pyrophosphate (0.01 M final concentration) and Polyoxyethylene-Sorbitan Monooleate (10% final concentration) and sonicating for 3 min in a water bath. The best results (based on FCM signature and the highest virus and bacterial yields from the sediments) were obtained by formaldehyde fixation carried out within less than one hour (2% final concentration, vs. no fixation or using glutaraldehyde at different concentrations), SYBR-Green II staining (×1 / 20,000 stock solution concentration, vs. use of SYBR-Gold and SYBR-Green I dyes at different concentrations). There was a considerable loss of particles after only a few days of storage at either 4 or - 22 °C. For FCM analysis, the samples were diluted in Tris–EDTA buffer (pH 8) and heated for 10 min at 75 °C after incubating for 5 min in the dark. The bacterial and viral counts paralleled those obtained using epifluorescence microscopy (EFM), but EFM always gave lower counts than FCM. Analysis of the distribution of the viruses in the water column and in the sediments of Lakes Bourget revealed a marked gradient, with larger quantities in the top layer of the sediment than in the water above it. These results are discussed, as well as the possible novel application of flow cytometry in the study of aquatic viral ecology.
http://www.sciencedirect.com/science/article/pii/S016770120500151X
Raman spectroscopy and chemical imaging for quantification of filtered waterborne bacteria.
Escoriza MF, van Briesen JM, et al. (2006). Journal of Microbiological Methods, vol. 66, pp. 63-72.
Rapid and reliable assessment of pathogenic microbial contamination in water is critically important. In the present work we evaluated the suitability of Raman Spectroscopy and Chemical Imaging as enumeration techniques for waterborne pathogens. The prominent C–H stretching band observed between 2800–3000 cm-1 of the spectrum is used for quantification purposes. This band provides the highest intensity of the bacterial-spectrum bands facilitating the detection of low number of microorganisms. The intensity of the Raman response correlates with number of cells present in drops of sample water on aluminum-coated slides. However, concentration of pathogens in drinking and recreational water is low, requiring a concentration step, i.e., filtering. Subsequent evaluation of filtering approaches for water sampling for Raman detection showed significant background signal from alumina and silver membranes that reduces method sensitivity. Samples concentrated by filtration show good correlation between Raman spectroscopy and other quantification methods including turbidity (R2=0.92), plate counts (R2=0.87) and dry weight (R2=0.97). Background interferences did not allow for evaluation of this relationship at low cell concentrations.
http://www.sciencedirect.com/science/article/pii/S0167701205003325
Rapid and Simple Quantification of Bacterial Cells by Using a Microfluidic Device.
Sakamoto C, Yamaguchi N, Nasu M, (2005). Applied and Environmental Microbiology, vol. 71(2), pp. 1117–1121.
This study investigated a microfluidic chip-based system (on-chip flow cytometry) for quantification of bacteria both in culture and in environmental samples. Bacterial numbers determined by this technique were similar to those obtained by direct microscopic count. The time required for this on-chip flow cytometry was only 30 min per 6 samples.
http://aem.asm.org/content/71/2/1117.full
Pfizer Case Study: Rapid Microbial Methods For Manufacturing Recovery After Hurricane María
Montenegro-Alvarado JM, Salvas J, Weber J, Mejías S, Arroyo R
Good manufacturing practices (GMPs) are a prerequisite for commercial production in the pharmaceu-tical industry. They are a basic set of requirements to ensure patient safety. For different reasons, GMP conditions may be lost, including, for example, as part of a planned shutdown for maintenance or con-struction or due to an unplanned disruptive event. This article shares a case study in which rapid mi-crobiological methods (RMMs) were used to evaluate risk and expedite recovery of GMP conditions after the devastation of Hurricane María in Puerto Rico.
www.pharmaceuticalonline.com
In-process microbial testing: statistical properties of a rapid alterna-tive to compendial enumeration methods.
Friedman EM, Warner M, Shum SC, Adair F, (2015). PDA J Pharm Sci Technol, vol. 69(2), pp. 264-9.
In-process tests are used between manufacturing steps to avoid the cost of further processing mate-rial that is apt to fail its final tests. Rapid microbiological methods that return simple negative or posi-tive results are attractive in this context because they are faster than the compendial methods used at product release. However, using a single such test will not reliably detect barely unacceptable ma-terial (sensitivity) without generating an undesirable number of false rejections (poor specificity). We quantify how to achieve a balance between the risks of false acceptance and false rejection by per-forming multiple rapid microbiological methods and applying an acceptance rule. We show how the end user can use a simple (and novel) graph to choose a sample size, the number of samples, and an acceptance rule that yield a good balance between the two risks while taking cost (number of tests) into account.
https://www.ncbi.nlm.nih.gov/pubmed/25868992
Electrochemical detection of bacterial cells using synthetic polymer “foot printing”- MICROPRINT
Padmanabhan S, Kelly M, Eswara LB, Seddon B, Hayes J, O’Reilly N, Dempsey E
MICROPRINT BIO-CARD is a bacterial detection system based on cell-enzyme profiling. The device encompasses a cell-capture membrane and a complement of electrodes which quantifies the elec-trons derived from enzyme reactions. This technology intends to replace current practice for the quantification of total bio-burden or individual species which is both labour intensive and time-consuming; testing typically requires 18-24 hours for a confirmatory result.Here we present a selective polymer “foot-printing” based bio-assay, involving imprinting of cells on a polyurethane film. Electrochemical method is used for the detection of enzymes expressed from the captured bacteria. Escherichia coli (a gram-negative bacterium) was selected as a model organ-ism and a range of enzymes arising from major metabolic pathways were targeted and screened as candidates for detection.
http://www.micra.ie/UserFiles/frJose/file/MICROPRINT.pdf
Gas Consumption
Sterility Testing of Injectable Products: Evaluation of the Growth-based BacT/ALERT ® 3D™ Dual T Culture System
Kaiser SJ, Mutters NT, Backhaus J, et al. (2016). PDA J Pharm Sci and Tech, vol. 70, pp. 568-576.
Sterility testing as described in the European Pharmacopoeia Chapter 2.6.1 as well as the United States Pharmacopeia Chapter 71 requires a 14 day incubation period of the test product in two different me-dia and at two different temperatures. Because of extensive personnel requirements for test perfor-mance and quality assurance, alternative and partially automated methods for product sterility testing are of interest. The study objective was to evaluate the applicability of the BacT/ALERT® 3D™ Dual T system (Biomérieux, Nürtingen, Germany) for detection of microbial contaminants according to current pharmacopoeia standards. In addition, we compared the BacT/ALERT® 3D™ Dual T system to conventional pharmacopoeia sterility testing using the direct inoculation method. The results showed no significant disadvantages of sterility testing by BacT/ALERT® 3D™ Dual T compared to the direct inoculation method regarding the ability to detect microbial contamination. Furthermore, product testing using the BacT/ALERT® 3D™ Dual T system met the compendia requirements for method qualification. Altogether, our data provide evidence that the BacT/ALERT® 3D™ Dual T system is a promising alternative for sterility testing of injectable products of sample volume below 10 mL and without antimicrobial activity.
https://www.ncbi.nlm.nih.gov/pubmed/27325593
Validation of the BacT/ALERT 3D System for Rapid Sterility Testing of Biopharmaceutical Samples.
Jimenez L, Rana N, Amalraj J, Walker K, Travers K, (2012). PDA J Pharm Sci and Tech, vol. 66, pp. 38-54.
The BacT/ALERT® 3D system was validated to determine the sterility of different types of biopharmaceutical samples such as water for injection, unprocessed bulk, and finished bulk. The installation, operation, and performance qualification were completed and verified under good manufacturing practices. During the installation and operation validation stages, the functionality and security of the system and software were completed and verified. For the performance qualification, 11 microorganisms were evaluated, six compendial (Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Candida albicans, Aspergillus niger, Clostridium sporogenes), one representing the number one microbial species in sterile product recalls (Burkholderia cepacia), and four environmental isolates (Kocuria rhizophila, Staphylococcus haemolyticus, Methylobacterium radiotolerans, and Penicillium spp.). Nine of the microorganisms were spiked into three different types of biopharmaceutical samples by three different analysts on different days to ascertain the equivalence, ruggedness, sensitivity, time of detection, and repeatability. In all samples, the BacT/ALERT® exhibited equivalent or better detection than the standard test. With the exception of M. radiotolerans, all 11 microorganisms were detected within 2.5 days using the BacT/ALERT® system and the standard test. The detection times for M. radiotolerans in the three sample types averaged 5.77 days. The minimum detectable level of cells for all the microorganisms tested was found to be within 1 to 2 CFU. The system optimized sterility testing by the simultaneous on-line, non-destructive incubation and detection of microbial growth.
https://www.ncbi.nlm.nih.gov/pubmed/22307661
Evaluation of growth based rapid microbiological methods for sterility testing of vaccines and other biological products.
Parveen S, Kaur S, Wilson-David SA, et al. (2011). Vaccine, Issue 29, pp. 8012-23.
Most biological products, including vaccines, administered by the parenteral route are required to be tested for sterility at the final container and also at various stages during manufacture. The sterility testing method described in the Code of Federal Regulations (21 CFR 610.12) and the United States Pharmacopoeia (USP, Chapter <71 >) is based on the observation of turbidity in liquid culture media due to growth of potential contaminants. We evaluated rapid microbiological methods (RMM) based on detection of growth 1) by adenosine triphosphate (ATP) bioluminescence technology (Rapid Milliflex® Detection System [RMDS]), and 2) by CO2 monitoring technologies (BacT/Alert and the BACTEC systems), as alternate sterility methods. Microorganisms representing Gram negative, Gram positive, aerobic, anaerobic, spore forming, slow growing bacteria, yeast, and fungi were prepared in aliquots of Fluid A or a biological matrix (including inactivated influenza vaccines) to contain approximately 0.1, 1, 10 and 100 colony forming units (CFU) in an inoculum of 10 ml. These preparations were inoculated to the specific media required for the various methods: 1) fluid thioglycollate medium (FTM) and tryptic soy broth (TSB) of the compendial sterility method (both membrane filtration and direct inoculation); 2) tryptic soy agar (TSA), Sabouraud dextrose agar (SDA) and Schaedler blood agar (SBA) of the RMDS; 3) iASTand iNST media of the BacT/Alert system and 4) Standard 10 Aerobic/F and Standard Anaerobic/F media of the BACTEC system. RMDS was significantly more sensitive in detecting various microorganisms at 0.1 CFU than the compendial methods (p < 0.05), whereas the compendial membrane filtration method was significantly more sensitive than the BACTEC and BacT/Alert methods (p < 0.05). RMDS detected all microorganisms significantly faster than the compendial method (p < 0.05). BacT/Alert and BACTEC methods detected most microorganisms significantly faster than the compendial method (p < 0.05), but took almost the same time to detect the slow growing microorganism P. acnes, compared to the compendial method. RMDS using SBA detected all test microorganisms in the presence of a matrix containing preservative 0.01% thimerosal, whereas the BacT/Alert and BACTEC systems did not consistently detect all the test microorganisms in the presence of 0.01% thimerosal. RMDS was compatible with inactivated influenza vaccines and aluminum phosphate or aluminum hydroxide adjuvants at up to 8 mg/ml without any interference in bioluminescence. RMDS was shown to be acceptable as an alternate sterility method taking 5 days as compared to the 14 days required of the compendial method. Isolation of microorganisms from the RMDS was accomplished by re-incubation of membranes with fresh SBA medium and microbial identification was confirmed using the MicroSEQ Identification System. BacT/Alert and BACTEC systems may be applicable as alternate methods to the compendial direct inoculation sterility method for products that do not contain preservatives or anti-microbial agents.
http://cat.inist.fr/?aModele=afficheN&cpsidt=24627047
Bioluminescence:
Evaluation of an ATP-Bioluminescence Rapid Microbial Screening Method for In-Process Biologics.
Spaeth S, Tran O, Liu Z, (2018). PDA J Pharm Sci and Tech, vol. 72, pp. 574-583.
This study compared an adenosine triphosphate (ATP)-based bioluminescence rapid microbial method (RMM) with a conventional sterility method for biologics sample testing. The RMM is based on a comparison of ATP levels in inoculated and uninoculated microbiological growth medium samples following growth enrichment incubation. The biologics samples qualified in this study were recombi-nant monoclonal antibodies and hybridoma cell culture supernatants. Initially, the lot-to-lot variation in background ATP of these samples posed significant challenges. Two strategies to increase the sig-nal-to-noise ratio (positive result/background ATP) were evaluated: enzyme-based signal amplifica-tion and reduction of the broth-based noise through broth selection. Following qualification of the RMM for antibody and cell culture samples, the RMM was also utilized for rapid screening of several sources of purified water. This ATP-based RMM has proved invaluable in routine testing of diverse biologics samples at our discovery research site and plays a key role in the investigation of contami-nated samples.
https://www.ncbi.nlm.nih.gov/pubmed/29954921
Evaluation of an ATP-Bioluminescence Rapid Microbial Screening Method for In-Process Biologics.
Spaeth S, Tran O, Liu Z, (2018). PDA J Pharm Sci and Tech, vol. 72, pp. 574-583.
This study compared an adenosine triphosphate (ATP)-based bioluminescence rapid microbial method (RMM) with a conventional sterility method for biologics sample testing. The RMM is based on a comparison of ATP levels in inoculated and uninoculated microbiological growth medium samples following growth enrichment incubation. The biologics samples qualified in this study were recombi-nant monoclonal antibodies and hybridoma cell culture supernatants. Initially, the lot-to-lot variation in background ATP of these samples posed significant challenges. Two strategies to increase the sig-nal-to-noise ratio (positive result/background ATP) were evaluated: enzyme-based signal amplifica-tion and reduction of the broth-based noise through broth selection. Following qualification of the RMM for antibody and cell culture samples, the RMM was also utilized for rapid screening of several sources of purified water. This ATP-based RMM has proved invaluable in routine testing of diverse biologics samples at our discovery research site and plays a key role in the investigation of contami-nated samples.
https://www.ncbi.nlm.nih.gov/pubmed/29954921
Validation of the BacT/ALERT 3D System for Rapid Sterility Testing of Biopharmaceutical Samples.
Jimenez L, Rana N, Amalraj J, Walker K, Travers K, (2012). PDA J Pharm Sci and Tech, vol. 66, pp. 38-54.
The BacT/ALERT® 3D system was validated to determine the sterility of different types of biopharmaceutical samples such as water for injection, unprocessed bulk, and finished bulk. The installation, operation, and performance qualification were completed and verified under good manufacturing practices. During the installation and operation validation stages, the functionality and security of the system and software were completed and verified. For the performance qualification, 11 microorganisms were evaluated, six compendial (Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Candida albicans, Aspergillus niger, Clostridium sporogenes), one representing the number one microbial species in sterile product recalls (Burkholderia cepacia), and four environmental isolates (Kocuria rhizophila, Staphylococcus haemolyticus, Methylobacterium radiotolerans, and Penicillium spp.). Nine of the microorganisms were spiked into three different types of biopharmaceutical samples by three different analysts on different days to ascertain the equivalence, ruggedness, sensitivity, time of detection, and repeatability. In all samples, the BacT/ALERT® exhibited equivalent or better detection than the standard test. With the exception of M. radiotolerans, all 11 microorganisms were detected within 2.5 days using the BacT/ALERT® system and the standard test. The detection times for M. radiotolerans in the three sample types averaged 5.77 days. The minimum detectable level of cells for all the microorganisms tested was found to be within 1 to 2 CFU. The system optimized sterility testing by the simultaneous on-line, non-destructive incubation and detection of microbial growth.
https://www.ncbi.nlm.nih.gov/pubmed/22307661
Evaluation of growth based rapid microbiological methods for sterility testing of vaccines and other biological products.
Parveen S, Kaur S, et al. (2011). Vaccine, Issue 29, pp. 8012-23.
Most biological products, including vaccines, administered by the parenteral route are required to be tested for sterility at the final container and also at various stages during manufacture. The sterility testing method described in the Code of Federal Regulations (21 CFR 610.12) and the United States Pharmacopoeia (USP, Chapter <71 >) is based on the observation of turbidity in liquid culture media due to growth of potential contaminants. We evaluated rapid microbiological methods (RMM) based on detection of growth 1) by adenosine triphosphate (ATP) bioluminescence technology (Rapid Milliflex® Detection System [RMDS]), and 2) by CO2 monitoring technologies (BacT/Alert and the BACTEC systems), as alternate sterility methods. Microorganisms representing Gram negative, Gram positive, aerobic, anaerobic, spore forming, slow growing bacteria, yeast, and fungi were prepared in aliquots of Fluid A or a biological matrix (including inactivated influenza vaccines) to contain approximately 0.1, 1, 10 and 100 colony forming units (CFU) in an inoculum of 10 ml. These preparations were inoculated to the specific media required for the various methods: 1) fluid thioglycollate medium (FTM) and tryptic soy broth (TSB) of the compendial sterility method (both membrane filtration and direct inoculation); 2) tryptic soy agar (TSA), Sabouraud dextrose agar (SDA) and Schaedler blood agar (SBA) of the RMDS; 3) iASTand iNST media of the BacT/Alert system and 4) Standard 10 Aerobic/F and Standard Anaerobic/F media of the BACTEC system. RMDS was significantly more sensitive in detecting various microorganisms at 0.1 CFU than the compendial methods (p < 0.05), whereas the compendial membrane filtration method was significantly more sensitive than the BACTEC and BacT/Alert methods (p < 0.05). RMDS detected all microorganisms significantly faster than the compendial method (p < 0.05). BacT/Alert and BACTEC methods detected most microorganisms significantly faster than the compendial method (p < 0.05), but took almost the same time to detect the slow growing microorganism P. acnes, compared to the compendial method. RMDS using SBA detected all test microorganisms in the presence of a matrix containing preservative 0.01% thimerosal, whereas the BacT/Alert and BACTEC systems did not consistently detect all the test microorganisms in the presence of 0.01% thimerosal. RMDS was compatible with inactivated influenza vaccines and aluminum phosphate or aluminum hydroxide adjuvants at up to 8 mg/ml without any interference in bioluminescence. RMDS was shown to be acceptable as an alternate sterility method taking 5 days as compared to the 14 days required of the compendial method. Isolation of microorganisms from the RMDS was accomplished by re-incubation of membranes with fresh SBA medium and microbial identification was confirmed using the MicroSEQ Identification System. BacT/Alert and BACTEC systems may be applicable as alternate methods to the compendial direct inoculation sterility method for products that do not contain preservatives or anti-microbial agents.
http://cat.inist.fr/?aModele=afficheN&cpsidt=24627047
Flow Cytometry
Description and validation of a rapid (1 h) flow cytometry test for enumerating thermophilic bacteria in milk powders.
Flint S, Walker K, et al. (2007). Journal of Applied Microbiology, vol. 102(4), pp. 909-915.
Aims: The aim of this study was to develop a rapid assay for enumerating thermophilic bacteria in milk powder.
Methods and Results: The BactiFlowTM flow cytometer was used to count bacteria based on esterase activity in viable bacterial cells. A protocol for total viable bacteria was modified by heat-treating the sample to selectively label thermophilic bacteria. Samples of milk powder dissolved in 0·1% peptone were treated with 0·8% ethylenediaminetetraacetic acid to reduce background interference because of denatured milk proteins. Either thermophilic bacteria were added to the dissolved milk powder or milk powder solutions were incubated at 55°C for 2–3 h to enrich the natural thermophile population for testing. Results from the BactiFlow were compared with traditional plate count results.
Conclusions: Thermophilic bacteria in milk powder can be enumerated within 1 h using the BactiFlow flow cytometer.
Significance and Impact of the Study: Microbiological test results obtained within 1 h can potentially be used to monitor manufacturing processes, effectively trace problems and provide confidence in the manufacture of product
http://www.ncbi.nlm.nih.gov/pubmed/17381733
Flow cytometric analysis of bacteria- and virus-like particles in lake sediments.
Duhamel S, Jacquet S, (2006). Journal of Microbiological Methods, vol. 64, pp. 316-332.
Flow cytometry (FCM) was successfully used to analyze freshwater bacteria and viruses in lake sediments after relatively simple sample treatment and optimization of dilution/fixation/staining procedures. Biological particles from Lakes Geneva and Bourget were first separated from the sediments by using both Sodium Pyrophosphate (0.01 M final concentration) and Polyoxyethylene-Sorbitan Monooleate (10% final concentration) and sonicating for 3 min in a water bath. The best results (based on FCM signature and the highest virus and bacterial yields from the sediments) were obtained by formaldehyde fixation carried out within less than one hour (2% final concentration, vs. no fixation or using glutaraldehyde at different concentrations), SYBR-Green II staining (×1 / 20,000 stock solution concentration, vs. use of SYBR-Gold and SYBR-Green I dyes at different concentrations). There was a considerable loss of particles after only a few days of storage at either 4 or - 22 °C. For FCM analysis, the samples were diluted in Tris–EDTA buffer (pH 8) and heated for 10 min at 75 °C after incubating for 5 min in the dark. The bacterial and viral counts paralleled those obtained using epifluorescence microscopy (EFM), but EFM always gave lower counts than FCM. Analysis of the distribution of the viruses in the water column and in the sediments of Lakes Bourget revealed a marked gradient, with larger quantities in the top layer of the sediment than in the water above it. These results are discussed, as well as the possible novel application of flow cytometry in the study of aquatic viral ecology.
http://www.sciencedirect.com/science/article/pii/S016770120500151X
Rapid and Simple Quantification of Bacterial Cells by Using a Microfluidic Device.
Sakamoto C, Yamaguchi N, Nasu M, (2005). Applied and Environmental Microbiology, vol. 71(2), pp. 1117–1121.
This study investigated a microfluidic chip-based system (on-chip flow cytometry) for quantification of bacteria both in culture and in environmental samples. Bacterial numbers determined by this technique were similar to those obtained by direct microscopic count. The time required for this on-chip flow cytometry was only 30 min per 6 samples.
http://aem.asm.org/content/71/2/1117.full
Spectroscopy
Raman spectroscopy and chemical imaging for quantification of filtered waterborne bacteria.
Escoriza MF, VanBriesen JM, et al. (2006). Journal of Microbiological Methods, vol. 66, pp. 63-72.
Rapid and reliable assessment of pathogenic microbial contamination in water is critically important. In the present work we evaluated the suitability of Raman Spectroscopy and Chemical Imaging as enumeration techniques for waterborne pathogens. The prominent C–H stretching band observed between 2800–3000 cm-1 of the spectrum is used for quantification purposes. This band provides the highest intensity of the bacterial-spectrum bands facilitating the detection of low number of microorganisms. The intensity of the Raman response correlates with number of cells present in drops of sample water on aluminum-coated slides. However, concentration of pathogens in drinking and recreational water is low, requiring a concentration step, i.e., filtering. Subsequent evaluation of filtering approaches for water sampling for Raman detection showed significant background signal from alumina and silver membranes that reduces method sensitivity. Samples concentrated by filtration show good correlation between Raman spectroscopy and other quantification methods including turbidity (R2=0.92), plate counts (R2=0.87) and dry weight (R2=0.97). Background interferences did not allow for evaluation of this relationship at low cell concentrations.
http://www.sciencedirect.com/science/article/pii/S0167701205003325
Nucleic Acid Amplification techniques (NAT)
Validation of a NAT-based Mycoplasma assay according European Phar-macopoiea
Deutschmann SM, Kavermann H, Knack Y, (2010). Biologicals, vol. 38, pp. 238–248.
Eucaryotic expression systems are widely used to produce biologicals for human use, e.g. vaccines, recombinant proteins and monoclonal antibodies. As part of the safety testing the current U.S. Food and Drug Administration (FDA) regulatory guidelines as well as several European Pharmacopoiea monographs requests the demonstration of the absence of Mycoplasma in the cell culture in the biore-actors prior to harvest and further downstream processing. In recent years progress has been made in the development of a sensitive NAT-based method for the detection of Mycoplasma species in CHO cells, e.g. Eldering et al. This method is based on a nucleic acid amplification technique using a very sensitive touch-down PCR-profile. The presence of mollicutes DNA in the test specimens is deter-mined by an approx. 450 bp target sequence which is amplified and this amplicon is finally detected by polyacrylamide gel electrophoresis. Based on this method a ready-to-use test kit was developed. In this report the validation of both method variants according the European Pharmacopoiea monograph 2.6.7 "Mycoplasmas" is described. The validation demonstrated the robustness and precision as well as a sufficient specificity of both assay formats. The validated sensitivity fulfills the requirements of the European Pharmacopoiea for a PCR-based method proposed as an alternative to the time consum-ing indicator cell culture and the culture method for the detection of Mollicutes (requested sensitivity of at least 10 colony-forming-units/mL).
https://www.ncbi.nlm.nih.gov/pubmed/20207553
Determination of indicator bacteria in pharmaceutical samples by multiplex PCR.
Faranjnia S, Hassan M, et al. (2009). Journal of Rapid Methods & Automation in Microbiology, vol. 17(3), pp. 328-338.
Rapid and sensitive detection techniques for indicator pathogens are important in pharmaceutical industry. However, common detection methods rely on bacterial culture in combination with biochemical tests, a process that typically takes 5-6 days to complete. Thus, the aim of this study was to develop a multiplex polymerase chain reaction (mPCR) assay for simultaneous detection and identification of four indicator pathogenic bacteria in a single reaction. Specific primers for indicator bacteria, namely Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella, were applied to allow simultaneous detection of them, and the sensitivity and specificity of each primer pairs were determined. In the mPCR with mixed DNA samples, specific bands for corresponding bacteria were simultaneously detected. Agarose gel electrophoresis of PCR products revealed 100% specificity of mPCR with single bands in the expected sizes. Low levels of microbial contamination less than 10 cfu per milliliter or gram of product were detected using mPCR assay. The detection of all four indicator pathogenic bacteria were completed in less than 8 h with this novel mPCR method, whereas the conventional United States Pharmacopeia methods and uniplex PCR required 5-6 days and 27 h for completion, respectively. Using mPCR assay, the microbial quality control of non-sterile pharmaceutical products can be performed in a cost-effective and timely manner in pharmaceutical industry. Detection of pathogenic indicators of Escherichia coli, Staphylococcus aureus, Salmonella and Pseudomonas aeruginosa is one of the mandatory tests in microbial quality of non-sterile pharmaceutical products; therefore, rapid and sensitive detection of the contaminations is of great importance for product release. According to the results of the present study, simultaneous detection of low levels of four major potential pathogenic bacteria in pharmaceutical finished products can be performed using mPCR in a cost-effective and timely manner, and upon these properties of the mPCR assay it could have potential applications in pharmaceutical industry.
http://onlinelibrary.wiley.com/doi/10.1111/j.1745-4581.2009.00154.x/abstract
Genetic Fingerprinting
Improvement of the cDNA-AFLP method using fluorescent primers for transcription analysis in bacteria.
Decorosi F, Viti C, et al. (2005). Journal of Microbiological Methods, vol. 63(2), pp. 211-215.
Here the cDNA-fluorescent amplified fragment length polymorphism (cDNA-FAFLP) technique, an improvement of cDNA-AFLP method, was used in order to analyse expression profiling in bacteria. The obtained results were validated by real-time PCR. This is the first report that validates the foreseen transcription pattern by cDNA-FAFLP with the application of real-time PCR in bacteria. This new protocol offers the possibility of quick and reliable analysis of transcription profiles, also avoids the problem linked with the use of radioisotopes, and allows a quick identification of genes differentially expressed in bacteria.
http://www.ncbi.nlm.nih.gov/pubmed/15939496
Review & Regulation
Total Laboratory Automation in Clinical Microbiology: a Micro-Comic Strip
McAdam AJ, (2018). Journal of Clinical Microbiology, vol. 56(4)
Laboratory automation in clinical microbiology has the potential to revolutionize laboratory operations (1, 2). A number of clinical microbiology laboratories have automated part or most of their work and found that testing can be performed accurately, with reduced turnaround times, improvements in la-boratory efficiency, and increased flexibility in the level of skill required to perform work in the labor-atory (3–7). Even highly complex tasks such as visual interpretation of Gram stains, of culture results, and of susceptibility tests can be automated (4, 8–14). Use of total laboratory automation has the po-tential to allow staff to perform more-complex tasks that will take advantage of their expertise (1, 15). It also has the potential to affect laboratory needs for expert technologists. How might clinical tech-nologists view the possible effects of total laboratory automation? Read the comic strip to find out.
https://www.ncbi.nlm.nih.gov/pubmed/29581314
Ensuring Data Integrity by Automated Microbiology Testing
Gay S, Samson Y, McDowall RD, (2017). Pharmaceutical Microbiology White Paper Series
Microbiology testing in pharmaceutical development and manufacture is used for environmental monitoring, sterility testing and detection and identification of microorganisms and the applicable regulations for this work are Good Manufacturing Practices (GMP) [Refs 1, 2]. Currently, data integri-ty is a major issue in the pharmaceutical industry and citations from FDA warning letters for microbi-ology laboratories can be classified as:
• Failure to perform actual testing
• Falsification of data e.g. reporting failed tests as passes or modifying records
The reasons are that microbiological testing is often manual and data often relies on observation that can be manipulated, often without photographs of the plates. Alternatively, if blank paper worksheets are used they can be photocopied and passing results substituted. Some typical warning letter citations can be seen in Table 1.
Microbiological testing can take between 3 – 7 days for environmental monitoring and 14 days for pharmacopoeial sterility testing. Data integrity could be compromised if there is a need to release a batch before testing is completed but also if there are large numbers of EM tests conducted manually.
Towards a Rapid Sterility Test?
Sandle T, (2015). Journal of Microbial & Biochemical Technology, vol. 7(4), pp. 216-217.
Sterility test is an established method for detecting the presence of viable forms of microorganisms in or on finished pharmaceutical products. Sterility, in this sense, means that a product is free from viable microorganisms (although not necessarily metabolic by-products or toxins). The classic form sterility test examines a pharmaceutical product in contact a culture medium, as a way of detecting the possible presence of viable microorganisms. The test is mandatory for all aseptically filled prod-ucts. In recent years a number of new technology platforms have emerged. This has been facilitated by a change in policy by the U.S. Food and Drug Administration (FDA), opening the door to alternatives to the pharmacopeia methods. This short review assesses some of these technologies.
http://www.omicsonline.org/open-access/towards-a-rapid-sterility-test-1948-5948-1000209.pdf
Rapid methods update: revisions to a United States Pharmacope-ia chapter
Miller MJ, (2015). PhD Microbiology Consultants
From 2010 to 2013, European Pharmaceutical Review published a very successful series on rapid methods (RMM) that included hot topics such as the European Medicines Agency’s and US Food and Drug Administration’s expectations, implementation strategies, scientific principles behind the tech-nologies and validation. The final article of the 2012 series introduced the United States Pharmaco-peia’s (USP’s) plan to revise informational chapter <1223>, Validation of Alternative Microbiological Methods. On June 1, 2015, a substantially modified chapter <1223> was published in the second supplement to USP38/NF33 with an official date of 1st December 2015. Because the original USP chapter was published almost 10 years ago, this article will review the most notable changes and compare them with what is recommended in the Parenteral Drug Association (PDA) Technical Report Number 33 and the proposed revision to European Pharmacopoeia (Ph. Eur.) chapter 5.1.6.
http://www.europeanpharmaceuticalreview.com/34483/topics/microbiology-rmm/rapid-methods-update-revisions-to-a-united-states-pharmacopeia-chapter/
A Fresh Look at USP <1223> Validation of Alternative Microbiologi-cal Methods and How the Revised Chapter Compares with PDA TR33 and the Proposed Revision to Ph. Eur. 5.1.6
Miller MJ, (2015).
The validation and implementation of rapid and alternative microbiological methods has gained sig-nificant momentum over the past decade, with multinational firms validating new technologies for a wide range of applications including finished product release testing (e.g., sterility), environmental monitoring, in-process control, Wfi analysis and microbial identification. When applicable, companies have submitted validation data to regulatory agencies and received approval to implement these same technologies for essential quality control uses. For example, Novartis obtained regulatory ap-proval from more than 50 different countries for releasing their vaccine products using a rapid ATP bioluminescence sterility test.
http://www.americanpharmaceuticalreview.com/Featured-Articles/177873-A-Fresh-Look-at-USP-1223-Validation-of-Alternative-Microbiological-Methods-and-How-the-Revised-Chapter-Compares-with-PDA-TR33-and-the-Proposed-Revision-to-Ph-Eur-5-1-6/
Overview of rapid microbiological methods evaluated, validated and implemented for microbiological quality control
Gordon O, Gray JC, Anders HJ, Staerk A, Schlaefli O, (2011). Novartis Pharma Stein AG, Gunther Neuhaus, University of Freiburg
The risk for patients through spoiled or otherwise adulterated pharmaceuticals has been acknowl-edged for many centuries and led to the establishment of Good Manufacturing Practice (GMP) and pharmacopoeial guidelines. Besides chemical purity, pharmaceuticals also have to meet microbiolog-ical standards, the latter primarily depending on the administration route. Drug products which are injected directly into blood vessels or tissues or that are applied directly into eyes and ears represent a greater infection risk than products which are administered orally or onto intact healthy skin. While parenteral drug products are required to be free from any viable microorganism (USP <71>, Ph. Eur. 2.6.1), oral and topical products are not required to be sterile, but are subject to strict guidelines limiting the number and types of acceptable microorganisms (USP <61> and <62>, Ph. Eur. 2.6.12 and 2.6.13).
http://cdn2.hubspot.net/hub/54227/file-14536665-pdf/docs/2011-novartisrmm-epr-trimmedandcroped-final.pdf
Guidance for IndustryComparability Protocols — Chemistry, Manufacturing, and Controls Information
U.S. Department of Health and Human Services, Food and Drug AdministrationCenter for Drug Evaluation and Research (CDER) Center for Biologics Evaluation and Research (CBER) Center for Veterinary Medicine (CVM) (2003).
This guidance provides recommendations to applicants on preparing and using comparability proto-cols for postapproval changes in chemistry, manufacturing, and controls (CMC). The guidance applies to comparability protocols that would be submitted in new drug applications (NDAs), abbreviated new drug applications (ANDAs), new animal drug applications (NADAs), abbreviated new animal drug applications (ANADAs), or supplements to these applications, except for applications for protein products. Well-characterized synthetic peptides submitted in these applications are included within the scope of this guidance. This guidance also applies to comparability protocols submitted in drug master files (DMFs) and veterinary master files (VMFs) that are referenced in these applications. The FDA is providing this guidance in response to requests from those interested in using comparability protocols.
http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm070545.pdf
Guidance for IndustryFormal Meetings between the FDA and Sponsors or Applicants of PDUFA Products
U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evalu-ation and Research (CDER) Center for Biologics Evaluation and Research (CBER)
This guidance provides recommendations to industry on formal meetings between the Food and Drug Administration (FDA) and sponsors or applicants relating to the development and review of drug or biological drug products (hereafter products) regulated by the Center for Drug Evaluation and Research (CDER) and the Center for Biologics Evaluation and Research (CBER). This guidance does not apply to abbreviated new drug applications, applications for biosimilar biological products, or sub-missions for medical devices. For the purposes of this guidance, formal meeting includes any meeting that is requested by a sponsor or applicant (hereafter requester(s)) following the request procedures provided in this guidance and includes meetings conducted in any format (i.e., face to face, telecon-ference, videoconference, or written response).
http://www.fda.gov/downloads/drugs/guidancecomplianceregulatoryinformation/guidances/ucm437431.pdf
