Streamline Your Microbial Quality Control Testing

APPLICATION NOTE Credible leads to Incredible® Page 1 Quinn Osgood, BSE; Jyoti K Jha, PhD; Rahul Tevatia, PhD; Cara Wilder, PhD; Nilay Chakraborty, PhD, MBA ATCC, Manassas VA ABSTRACT Microbial quality control testing is a cornerstone of pharmaceutical product development and essential for the safety, efficacy, and purity of biological products. To ensure the accuracy of these tests, pharmaceutical manufacturers need full confidence that their microbial controls are authenticated, low passage, and precisely quantitated. Leveraging innovative preservation technologies, ATCC has developed our best-in-class microbial quality control strains in a rapidly rehydrating pelleted format that delivers consistent quantitation. In this study, we demonstrate the performance of this novel suite of products in streamlining microbial quality control testing. INTRODUCTION Microbial contamination of pharmaceutical products is one of the leading causes of product recalls in the pharmaceutical industry.¹,² These recalls can lead to extensive financial losses, regulatory consequences, brand erosion, and reputational damage.¹,² More importantly, microbial contamination can significantly affect patient health and safety, potentially resulting in permanent disabilities or life-threatening conditions. To ensure product safety and integrity, pharmaceutical manufacturers are required by their respective government agencies to perform robust quality control procedures that, when properly followed, can help identify microbial contamination prior to product release. Pharmaceutical manufacturers in the United States perform tests such as antimicrobial effectiveness testing, environmental monitoring, growth promotion testing, bioburden testing, sterility testing, and suitability testing as recommended by the United States Pharmacopeia (USP).³-⁷ To ensure the accuracy of these tests, the regulatory guidance recommends the use of test strains that are maintained by reputable culture collections like ATCC. While ATCC has consistently met the needs for microbial quality control testing by providing authenticated reference materials, the laboratories performing microbial quality control testing often face infrastructure and resource limitation challenges when preparing control strains for testing. For instance, many testing laboratories choose to develop and maintain their own internal control banks of reference materials. Creation and maintenance of the control banks requires complex expansion protocols and extensive laboratory space and equipment. These processes can be error prone. Precise quantitation of the reference materials is also a challenge. Several quality control tests require reference materials to be prepared in either a low-titer or high-titer format. Meeting these specifications is traditionally obtained by preparing a culture of the required strain, which can require a significant investment in time and resources. Additionally, accurate quantification and passage of the microbial strains require highly skilled microbiologists to be involved. All these factors can make the process of microbial quality control testing a costly, time-intensive, and often inconvenient affair. DEVELOPMENT AND PERFORMANCE EVALUATION OF MICROQUANT™ – QUANTITATIVE, RAPIDLY REHYDRATING REFERENCE MATERIALS FOR STREAMLINED MICROBIAL QUALITY CONTROL TESTING Page 2 Order online at www.atcc.org, call 800.638.6597, 703.365.2700, or contact your local distributor. To address these challenges and to help streamline microbial quality control testing in pharmaceutical manufacturing, ATCC has developed MicroQuant™. This novel product suite leverages an innovative preservation technology to deliver precisely quantitated microbial reference materials in a single-use format that rapidly rehydrates and is convenient to store at 2-8°C. This product suite allows use of authenticated reference microbial strains from ATCC in an easy-to-use format. In this study, we demonstrate the precise quantitation and rapid rehydration of MicroQuant™ controls and showcase their performance against similar products on the market. MATERIAL S AND METHODS DEVELOPMENT OF MICROQUANT™ CONTROLS In this study, we targeted microbial strains listed in the microbial quality control tests as referenced in USP chapters <51> and <61> (Table 1);³,⁵ each of these chapters details assays that require a specific quantity of microorganisms. The selected microorganisms were cultured in tightly controlled conditions using methods and media outlined in the specific product sheets for each strain. Following microbial culture, the strains were then processed using a proprietary preservation process to create a user-friendly microbial pellet format (Table 2). Table 1: ATCC® microbial strains used in the study ATCC® No. Species Strain Designation USP <51> USP <61> 16404™ Aspergillus brasiliensis WLRI 034(120) ✓ ✓ 6633™ Bacillus spizizenii (Formerly Bacillus subtilis) NRS 231 ✓ 10231™ Candida albicans 3147 ✓ ✓ 8739™ Escherichia coli Crooks ✓ 9027™ Pseudomonas paraeruginosa (Formerly Pseudomonas aeruginosa) R. Hugh 813 ✓ ✓ 6538™ Staphylococcus aureus subsp. aureus FDA 209 ✓ ✓ Table 2: ATCC® MicroQuant™ products ATCC® No. Description Quantitation (CFU/vial) 16404-LQ-PACK™ MicroQuant™ Aspergillus brasiliensis, low CFU 100-1,000 16404-HQ-PACK™ MicroQuant™ Aspergillus brasiliensis, high CFU 10⁷-10⁸ 6633-LQ-PACK™ MicroQuant™ Bacillus spizizenii, low CFU 100-1,000 6633-HQ-PACK™ MicroQuant™ Bacillus spizizenii, high CFU 10⁷-10⁸ 10231-LQ-PACK™ MicroQuant™ Candida albicans, low CFU 100-1,000 10231-HQ-PACK™ MicroQuant™ Candida albicans, high CFU 10⁷-10⁸ 8739-LQ-PACK™ MicroQuant™ Escherichia coli, low CFU 100-1,000 8739-HQ-PACK™ MicroQuant™ Escherichia coli, high CFU 10⁷-10⁸ 9027-LQ-PACK™ MicroQuant™ Pseudomonas paraeruginosa, low CFU 100-1,000 9027-HQ-PACK™ MicroQuant™ Pseudomonas paraeruginosa, high CFU 10⁷-10⁸ 6538-LQ-PACK™ MicroQuant™ Staphylococcus aureus subsp. aureus, low CFU 100-1,000 6538-HQ-PACK™ MicroQuant™ Staphylococcus aureus subsp. aureus, high CFU 10⁷-10⁸ EVALUATING THE PROCESSING TIME AND QUANTITATION OF MICROQUANT™ CONTROLS MicroQuant™ controls were evaluated for their performance in the following two areas: the time required to process the pellet and the quantitation of the product following recovery of the material. Both attributes were also evaluated against comparable products from other vendors. To evaluate preparation time, MicroQuant™ products and comparable vendor products were removed from the appropriate storage unit. Vendor products that were designed to be stored at -20°C were thawed according to instructions provided on the respective instructions for use (IFU). MicroQuant™ products and vendor products that were designed to be stored at 2-8°C were rehydrated according to instructions provided on the respective IFU. The processing time for each product was evaluated in units of minutes. To evaluate quantitation following recovery, MicroQuant™ products and comparable vendor products were prepared as stated above and were plated on non-selective media. Following an appropriate growth period, CFUs were enumerated. Order online at www.atcc.org, call 800.638.6597, 703.365.2700, or contact your local distributor. Page 3 RESULT S MICROQUANT™ PRODUCTS RAPIDLY REHYDRATE WITH CONSISTENT PROCESSING TIMES The time required to process MicroQuant™ products was assessed and compared to that of other vendor products with similar applications. In this analysis, we followed ATCC’s IFU for MicroQuant™ products and the respective IFUs for vendor supplied products to process the strains. Both the IFU-described processing time and maximum observed processing time required by users were reported (Figure 1). Here, MicroQuant™ products consistently rehydrated in approximately 1 minute with minor user-to-user variations. When compared to vendor products, the overall processing time for MicroQuant™ was much shorter than lyophilized products (Company A and B) and similar to frozen products (Company C and D). Figure 1: Evaluating the processing time of MicroQuant™ products in comparison to similar vendor products following removal from storage. This is a compilation of observations including a minimum of 6 units from each product for each vendor. No differences in processing time were seen between species or titer range. 70 60 50 40 30 20 5 4 3 2 1 0 Storage 2-8°C Storage -20°C Company D Company C Company B Company A ATCC Time (min) Maximum Observed Processing Time Vendor Reported Processing Time MicroQuant™ delivers precise quantitation that is consistent for low and high CFU targets We then assessed the recoverable CFU from both the low and high CFU preparations for each of the MicroQuant™ strains following rehydration with the respective proprietary buffers. Our data demonstrate that MicroQuant™ products can consistently achieve the indicated target ranges for all tested organisms (Figure 2). P. paraeruginosa A. brasiliensis B. spizizenii C. albicans E. coli S. aureus Recoverable CFU Target Range MicroQuant™ Low CFU Products 10⁴ 10³ 10² 10¹ P. paraeruginosa A. brasiliensis B. spizizenii C. albicans E. coli S. aureus Recoverable CFU Target Range MicroQuant™ High CFU Products 10⁹ 10⁸ 10⁷ 10⁶ Figure 2: Evaluating MicroQuant™ quantitation following recovery. Recoverable CFU for low and high CFU MicroQuant™ products. The target range was 100-1,000 CFU for low CFU and 10⁷-10⁸ CFU for high CFU MicroQuant™. Each bar represents 60 data points collected by a minimum of three separate biologists. Error bars show standard deviation. When evaluating the quantitation of MicroQuant™ products against comparable vendor products that target the same titer ranges, we found that MicroQuant™ is the only single-use control that is completely within the acceptable range for all six cultures for both the low (Figure 3) and high (Figure 4) CFU formats. At the time of our analysis, only two vendors had inventory to ship all six species at both high and low concentrations. Page 4 Order online at www.atcc.org, call 800.638.6597, 703.365.2700, or contact your local distributor. 2000 1500 1000 500 0 Company D Company C Company B Company A ATCC CFU/Unit Format A. brasiliensis 2000 1500 1000 500 0 Company D Company C Company B Company A ATCC CFU/Unit Format E. coli 2000 1500 1000 500 0 Company D Company C Company B Company A ATCC CFU/Unit Format B. spizizenii 2000 1500 1000 500 0 Company D Company C Company B Company A ATCC CFU/Unit Format P. paraeruginosa 2000 1500 1000 500 0 Company D Company C Company B Company A ATCC CFU/Unit Format C. albicans 2000 1500 1000 500 0 Company D Company C Company B Company A ATCC CFU/Unit Format S. aureus Figure 3: Comparison of low CFU product quantitation (100 to 1000 CFU per unit) of MicroQuant™ and comparable vendor products. Each bar represents a minimum of 12 data points, error bars show standard deviation. 10¹⁰ 10⁹ 10⁸ 10⁷ 10⁶ Company D Company C Company B Company A ATCC CFU/Unit Format E. coli N.A. 10¹⁰ 10⁹ 10⁸ 10⁷ 10⁶ Company D Company C Company B Company A ATCC CFU/Unit Format P. paraeruginosa N.A. 10¹⁰ 10⁹ 10⁸ 10⁷ 10⁶ Company D Company C Company B Company A ATCC CFU/Unit Format S. aureus N.A. 10¹⁰ 10⁹ 10⁸ 10⁷ 10⁶ Company D Company C Company B Company A ATCC CFU/Unit Format A. brasiliensis N.A. N.A. 10¹⁰ 10⁹ 10⁸ 10⁷ 10⁶ Company D Company C Company B Company A ATCC CFU/Unit Format B. spizizenii N.A. N.A. 10¹⁰ 10⁹ 10⁸ 10⁷ 10⁶ Company D Company C Company B Company A ATCC CFU/Unit Format C. albicans N.A. Figure 4: Comparison of high CFU product quantitation (10⁷ to 10⁸ CFU per unit) of MicroQuant™ and comparable vendor products. Each bar represents a minimum of 12 data points, error bars show standard deviation. N.A. = Not Available. 10801 University Boulevard Manassas, Virginia 20110-2209 703.365.2700 703.365.2701 sales@atcc.org www.atcc.org AP-082025-v03 ©2025 American Type Culture Collection. The ATCC trademark and trade name, and any other trademarks listed in this publication are trademarks owned by the American Type Culture Collection unless indicated otherwise. These products are for laboratory use only. Not for human or diagnostic use. ATCC products may not be resold, modified for resale, used to provide commercial services or to manufacture commercial products without prior ATCC written approval. CONCLUSION Our data show that we have successfully created a convenient, quantitative microbial product format that can consistently provide a precise range of CFU for a suite of organisms in accordance with the specifications described in the USP <51> and <61> compendial assays. When assessed against comparable vendor products on the market, MicroQuant™ was the only single-use control that demonstrated a precise level of quantitation that was completely within both the low and high CFU target ranges for all six cultures. We also demonstrated that this product format can be rapidly processed in approximately a minute, minimizing the amount of hands-on time required to prepare materials for quality control testing. When compared to similar single-use products that are also stored at 2-8°C, the processing time for MicroQuant™ was considerably shorter. Overall, this novel suite of products provides best-in-class, original source strains from ATCC in a ready-to-use, rapidly rehydrating format that delivers consistent quantitation, enabling testing labs to minimize handling and streamline microbial quality control testing. PRECISION IN EVERY PELLET, TRUST IN EVERY TEST ƒ Sourced from reference strain: Products were created from the authenticated ATCC strains mentioned in regulatory guidelines ƒ Precisely quantitated: Products are available in high-titer (10⁷ to 10⁸ CFU per vial) and low-titer (100 to 1,000 CFU per vial; 10 assays) formats to meet USP General Chapter requirements. Our precise quantitation across batches ensures lot-to-lot consistency. ƒ Single-use format: Our highly convenient ready-to-use format enables fast assay setup while minimizing handling. Reduces the need for special handling knowledge and the maintenance of expansive microbial cultures. ƒ Immediate rehydration at room temperature: With a processing time of a minute, you can reduce your hands-on time while improving efficiency. ƒ Convenient storage at 2-8°C: MicroQuant™ products are stable in refrigerator temperatures for 6-12 months (see individual products for expiry), making them easy to store and ready to use anytime—no need to thaw. ƒ Reliable: Each product is manufactured by ATCC under ISO 17034, so you can trust that you are receiving high-quality original source material. REFERENCES 1. Hock SC, et al. Contamination Trends & Proposed Solutions. Pharmaceutical Engineering April 2023. Accessed online: https://ispe.org/ pharmaceutical-engineering/march-april-2023/contamination-trends-proposed-solutions 2. atel R, et al. A retrospective regulatory analysis of FDA recalls carried out by pharmaceutical companies from 2012 to 2023. Drug Discov Today 29(6): 103993, 2024. 3. USP General Chapters. <51> Antimicrobial Effectiveness Testing. In: USP-NF. Rockville, MD: United States Pharmacopeia. DOI: https:// doi.usp.org/USPNF/USPNF_M98790_03_01.html 4. USP General Chapters. <60> Microbiological Examination of Nonsterile Products Tests for Burkholderia Cepacia Complex. In: USP-NF. Rockville, MD: United States Pharmacopeia. DOI: >https://doi.usp.org/USPNF/USPNF_M12455_02_01.html 5. USP General Chapters. <61> Microbial Examination of Nonsterile Products: Microbial Enumeration Tests. In: USP-NF. Rockville, MD: United States Pharmacopeia. DOI: https://doi.usp.org/USPNF/USPNF_M98800_01_01.html 6. USP General Chapters. <62> Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms. In: USP-NF. Rockville, MD: United States Pharmacopeia. DOI: https://doi.usp.org/USPNF/USPNF_M98802_01_01.html 7. USP General Chapters. <71> Sterility Tests. In: USP-NF. Rockville, MD: United States Pharmacopeia. DOI: https://doi.usp.org/USPNF/ USPNF_M98810_01_01.html