Sterility Testing Of Products Research Articles

Microbial Control and Monitoring Strategies for Cleanroom Environments and Cellular Therapies

A well-validated and holistic program that incorporates robust gowning, cleaning, environmental monitoring, and personnel monitoring measures is critical for minimizing the microbial bioburden in cellular therapy manufacturing suites and the corresponding testing laboratories to ensure that the facilities are operating in a state of control. Ensuring product safety via quality control measures, such as sterility testing, is a regulatory requirement for both minimally manipulated (section 361) and more than minimally manipulated (section 351) human cells, tissues, and cellular and tissue-based products (HCT/Ps). In this video, we provide a stepwise guide for how to develop and incorporate the best aseptic practices for operating in a cleanroom environment, including gowning, cleaning, staging of materials, environmental monitoring, process monitoring, and product sterility testing using direct inoculation, provided by the United States Pharmacopeia (USP<71>) and the National Institutes of Health (NIH) Alternative Sterility Testing Method. This protocol is intended as a reference guide for establishments expected to meet current good tissue practices (cGTP) and current good manufacturing practices (cGMP).

Investigation of Bacterial Infections Among Patients Treated With Umbilical Cord Blood–Derived Products Marketed as Stem Cell Therapies

The number of clinics marketing stem cell products for joint diseases, chronic pain, and most recently, COVID-19, has increased despite warnings from the US Food and Drug Administration that stem cell products for these and other indications have not been proven safe or effective. To examine bacterial infections in 20 patients who received umbilical cord blood-derived products marketed as stem cell treatment. This case series is a national public health investigation including case-finding, medical record review and abstraction, and laboratory investigation, including sterility testing of products and whole-genome sequencing of patient and product isolates. Participants included patients who developed bacterial infections following administration of umbilical cord blood-derived products marketed as stem cell treatment during August 2017 to September 2018. Data analysis was performed from March 2019 to September 2021. Umbilical cord blood-derived products marketed as stem cell treatment. Data were collected on patient infections and exposures. The Centers for Disease Control and Prevention performed sterility testing on undistributed and distributed vials of product marketed as stem cell treatment and performed whole-genome sequencing to compare patient and product bacterial isolates. Culture-confirmed bacterial infections were identified in 20 patients (median [range] age, 63 [2-89] years; 13 male patients [65%]) from 8 US states who sought stem cell treatment for conditions including pain, osteoarthritis, rheumatoid arthritis, and injury; all but 1 required hospitalization. The most frequently isolated bacteria from patients with infections were common enteric species, including Escherichia coli (14 patients) and Enterobacter cloacae (7 patients). Of unopened, undistributed products sampled for testing, 65% (22 of 34 vials) were contaminated with at least 1 of 16 bacterial species, mostly enteric. A patient isolate from Arizona matched isolates obtained from products administered to patients in Florida, and patient isolates from Texas matched undistributed product sent from the company in California. Unapproved stem cell products can expose patients to serious risks without proven benefit. Sequencing results suggest a common source of extensive contamination, likely occurring during the processing of cord blood into product. Patients and health care practitioners who are considering the use of unapproved products marketed as stem cell treatment should be aware of their unproven benefits and potential risks, including serious infections.

Performance and safety of femoral central venous catheters in pediatric autologous peripheral blood stem cell collection.

Autologous peripheral blood hematopoietic progenitor cell collection (A-HPCC) in children typically requires placement of a central venous catheter (CVC) for venous access. There is scant published data regarding the performance and safety of femoral CVCs in pediatric A-HPCC. Seven-year, retrospective study of A-HPCC in pediatric patients collected between 2009 and January 2017. Inclusion criteria were an age ≤ 21 years and A-HPCC using a femoral CVC for venous access. Femoral CVC performance was examined by CD34 collection rate, inlet rate, collection efficiency (MNC-FE, CD34-FE), bleeding, flow-related adverse events (AE), CVC removal, and product sterility testing. Statistical analysis and graphing were performed with commercial software. A total of 75/119 (63%) pediatric patients (median age 3 years) met study criteria. Only 16% of children required a CVC for ≥ 3 days. The CD34 collect rate and CD34-FE was stable over time whereas MNC-FE decreased after day 4 in 80% of patients. CD34-FE and MNC-FE showed inter- and intra-patient variability over time and appeared sensitive to plerixafor administration. Femoral CVC showed fewer flow-related AE compared to thoracic CVC, especially in pediatric patients (6.7% vs. 37%, P = 0.0005; OR = 0.12 (95%CI: 0.03-0.45). CVC removal was uneventful in 73/75 (97%) patients with hemostasis achieved after 20-30 min of pressure. In a 10-year period, there were no instances of product contamination associated with femoral CVC colonization. Femoral CVC are safe and effective for A-HPCC in young pediatric patients. Femoral CVC performance was maintained over several days with few flow-related alarms when compared to thoracic CVCs.

Sterility Testing of Injectable Products: Evaluation of the Growth-based BacT/ALERT® 3D™ Dual T Culture System.

Sterility is defined as the freedom from the presence of viable microorganisms. Injectable pharmaceutical products that are sometimes used for treatment of patients need to be free of microorganisms and therefore tested in the laboratory to confirm sterility. The testing procedures are described in detail in the European Pharmacopoeia. To test for sterility, the products need to be incubated for 14 days at two different temperatures and using two different growth media to ensure growth of various microorganisms. These tests, however, are cost- and labor-intensive. Therefore, we sought to use an automated system to reduce costs and hands-on time, as well as to improve quality assurance. The study objective was to evaluate the applicability of the automated BacT/ALERT® 3D™ Dual T system (Biomérieux, Nürtingen, Germany) for detection of microorganisms according to current pharmacopeia standards. We compared the BacT/ALERT® 3D™ Dual T system to standard sterility testing using the so-called direct inoculation method. The results showed no major disadvantages of sterility testing by the automated system compared to the standard method with regard to the detection level of microorganisms. Furthermore, product testing using the BacT/ALERT® 3D™ Dual T system met the compendia requirements for method qualification. Additionally, the new automated method provided reliable results and promises a higher robustness to human errors than do standard manual methods, which might reduce the potential for errors and improves quality assurance. Altogether, our data provide evidence that the BacT/ALERT® 3D™ Dual T system is a promising alternative for sterility testing of injectable products.

Product Sterility Testing . . . To Test or Not to Test? That Is the Question.

The applications for sterility testing in the validation and routine control of sterilization of medical devices have changed dramatically over the years. As the definition of sterility assurance has evolved, so has the state of the science associated with product sterility testing. Historically, product sterility testing has been applied to such things as sterilization validation, sterilization lot release, packaging qualification, aseptic processing qualification, and determination of shelf life for the packaged medical device. In most of these cases, however, the results obtained from performing sterility testing on products do not provide the desired confirmation and assurance. Utilizing sterility testing on fully processed finished product is not appropriate for determination of sterilization process effectiveness, sterility assurance level, package integrity, or shelf life. The industry has developed more robust methods for validation of these applications to assure sterility and package performance. This article outlines the appropriate applications for sterility testing and highlight the applications currently in use that have significant limitations within the results and introduce undesirable risk to the validity of the data.

Validation of an alternative microbiological method for tissue products.

According to the European Pharmacopoeia sterility testing of products includes an incubation time of 14 days in thioglycollate medium and soya-bean casein medium. In this case a large period of time is needed for product testing. So we designed a study to evaluate an alternative method for sterility testing. The aim of this study was to reduce the incubation time for the routinely produced products in our tissue bank (cornea and amnion grafts) by obtaining the same detection limit, accurateness and recovery rates as the reference method described in the European Pharmacopoeia. The study included two steps of validation. Primary validation compared the reference method with the alternative method. Therefore eight bacterial and two fungi test strains were tested at their preferred milieu. A geometric dilution series from 10 to 0.625 colony forming unit per 10 ml culture media was used. Subsequent to the evaluation the second part of the study started including the validation of the fertility of the culture media and the parallel testing of the two methods by investigating products. For this purpose two product batches were tested in three independent runs. Concerning the validation we could not find any aberration between the alternative and the reference method. In addition, the recovery rate of each microorganism was between 83.33 and 100 %. The alternative method showed non-inferiority regarding accuracy to the reference method. Due to this study we reduced the sterility testing for cornea and amniotic grafts to 9 days.

Elimination of the antimicrobial action of the organoarsenical cancer therapeutic, 4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid, before finished product sterility testing

Arsenical compounds have been used therapeutically for over 2000 years finding particular relevance as antimicrobials. After being replaced by more selective and consequently less toxic antibiotics in the last century, arsenicals have recently made a resurgence as anticancer drugs (specifically arsenic trioxide and its derivatives). Arsenical parenteral formulations require post-manufacture sterility testing; however, their intrinsic antimicrobial activity must be neutralised before testing to eliminate the possibility of false (no-growth) test results. A range of thiol-containing compounds was screened to establish a suitable deactivation agent for the novel organoarsenical compound, 4-(N-(S-glutathionylacetyl)amino) phenylarsonous acid (GSAO). Dimercatopropanol (DMP) was found to successful deactivate GSAO and was validated according to pharmacopoeial sterility test guidelines (specifically the method suitability test/sterility validation test). DMP is an effective way of deactivating GSAO before sterility testing and can be used for pharmacopoeial sterility tests. Our results affirm previous research highlighting the sensitivity of Staphylococcus aureus to arsenical compounds A method of deactivating the arsenical drug GSAO before the post-manufacture sterility test was established and validated. DMP is a commonly used chelator/deactivation agent so this work may have implications for other inorganic therapeutic agents.

Sterility testing of minimally manipulated cord blood products: validation of growth‐based automated culture systems

The St Louis Cord Blood Bank submitted a biologics license application for cord blood (CB) products processed by PrepaCyte-CB (BioE), supported with a validation study of a microbial detection system for product sterility testing (BACTEC-FX, Becton Dickinson). This article provides the validation approach followed to fulfill Food and Drug Administration requirements pertinent to sterility testing method. System qualification, culture media quality verification, and validation of CB processing by-product (CB-BP) sample as surrogate to final product for sterility testing were followed by studies evaluating method sensitivity, specificity, reproducibility, ruggedness or robustness, and bacteriostatic or fungistatic effect of CB-BP sample. CB-BP cultures and control samples were formulated using BACTEC Plus Aerobic/F, Plus Anaerobic/F, and Myco F/Lytic media. Samples were seeded with selected test organisms (n = 13 at 10-100 colony-forming units [CFUs] per vial) and cultured for 14 days (bacterial) and 30 days (fungal). Under testing conditions, no stasis effect of test sample on microbial growth and no false-positive or false-negative results were reported. Although a 7-day culture was sufficient to detect all validation test organisms seeded at ≤ 26 CFUs/vial, growth in actual product sterility testing practice may require a 10- to 14-day culture. Assay reproducibility was uncertain at very low bioburden (< 10 CFUs/vial). Growth time to detection neither varied between different media lots nor prolonged in culture vials with loading delay (6-8 hr at room temperature). BACTEC-FX culture and detection system and BACTEC media formulae have high detection capability and can be effectively validated for sterility testing of CB products.