Rapid Sterility Test Systems in the Pharmaceutical Industry: Applying a Structured Approach to Their Evaluation, Validation and Global Implementation.

The current compendial sterility test has a 14-day incubation time and is often the time-limiting step in the Assess and Release Process of pharmaceutical products. There is an ever-increasing number of technologies available on the market that have benefits in addition to faster Time to Result, such as standardization and automation of readout (eliminating analyst subjectivity) and improved data integrity (including eliminating the need for contemporaneous verification of the result by another analyst). Regulators have been encouraging the pharmaceutical industry to adopt these innovative systems; however, it has taken a considerable time before receiving the first approvals from various health authorities (including both the European Medicines Agency and Food and Drug Administration) for the use of an alternative and rapid sterility test for the release of sterile drug product lots. This article describes a systematic 9-step approach to the evaluation, equipment qualification, validation, and deployment of alternative sterility tests that can be applied by pharmaceutical companies wanting to take advantage of the numerous benefits of alternative sterility tests. Two case studies are presented to illustrate the validation and implementation approach, including statistical methods. Although most of the steps toward implementation are aligned, the validation and transfer have been approached differently for each of the case studies because of differences in the chosen technology as well as independent company internal decisions to comply with validation guidelines. However, both case studies show successful implementation of an alternative sterility test for sterile drug products with an ∼50% reduced incubation time.

Comparison of pyrogen assays by testing products exhibiting low endotoxin recovery.

The use of pyrogen tests to assess the risk of endotoxin in biological products has increased recently due to concerns of some regulatory authorities about products exhibiting low endotoxin recovery (LER). Manufacturers increasingly seek to reduce the use of animals unless essential to assure patient safety. The current study compares the ability of the monocyte activation test (MAT) and the bacterial endotoxin test (BET) to the rabbit pyrogen test (RPT) to detect endotoxin spikes in samples of products shown to exhibit LER. Product samples or water were spiked with endotoxin and held for three days or tested immediately in the BET, the RPT, and two variations of the MAT at the same time. Results show high sensitivity to endotoxin of both the BET and MAT, and much lower sensitivity of the RPT, indicating that much higher levels of reference standard endotoxin are required to induce pyrogenicity in the RPT than the 5 endotoxin units (EU) per kg common threshold. The results of the BET and MAT correlated well for the detection of endotoxin spike in water. We also show that LER (masking of endotoxin) found in the BET is also seen in the MAT and RPT, suggesting that the products themselves elicit a biological inactivation of spiked endotoxin over time, thereby rendering it less or non-pyrogenic. We conclude that the non-animal MAT option is a suitable replacement for the RPT to measure spiked endotoxin in biopharmaceuticals.

A Systematic Approach for the Evaluation, Validation, and Implementation of Automated Colony Counting Systems.

For several years, automated colony counting systems have been available with varying degrees of automation. Ever more sophisticated instruments are now increasingly used in microbiological laboratories of pharmaceutical quality control. In addition to the colony counting device, the instruments are now also equipped with robotic systems performing the entire handling of the petri dishes, e.g., automated internal transportation of petri dishes from the incubator chamber to the instrument's enumeration device and back. Moreover, the subjective evaluation of microbial enumeration tests by analysts is replaced with a more accurate and precise process. This leads to significant improvements to data integrity compliance. Automated colony counting systems also often enable cost reduction in the microbiological laboratory, e.g., by not requiring a contemporaneous verification by a second analyst. They also enable direct integration of count data into an existing laboratory information management system, reducing the hands-on time, costs per test and also preventing human errors caused by manual transcription. Altogether, these instruments will lead to improved monitoring and assurance of control of biopharmaceutical processes and manufacturing environments, as well as shortened cycle times in the supply chain. Regulators are encouraging the biopharmaceutical industry to adopt these innovative systems. For example, this year a BioPhorum member company received the first health authority approvals from EU, US, CH, Canada, Australia, and China for the use of automated colony counting systems for in-process bioburden testing and the release of drug substance lots, with an incubation time reduced by about 50%. Although these approvals are for release testing of drug substance lots, the instruments can also be used for environmental monitoring, testing of water samples, etc. This article describes a systematic 9-step approach to the evaluation, equipment qualification, and deployment of automated colony counting systems, which can be applied by biopharmaceutical companies wanting to take advantage of their numerous benefits.

Validation of a NAT-based Mycoplasma assay according European Pharmacopoiea.

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 bioreactors 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 determined 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 consuming indicator cell culture and the culture method for the detection of Mollicutes (requested sensitivity of at least 10 colony-forming-units/mL).