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.

Bacterial Endotoxin Testing-Fast Endotoxin Masking Kinetics in the Presence of Lauryldimethylamine Oxide.

For release of parenteral drug products, bacterial endotoxin testing is one of a panel of necessary tests. In order to ensure the validity of such tests, various controls are performed, including demonstration of compendial method suitability or method qualification. In addition to compendial suitability testing, quality control (QC) sample hold-time studies are requested by authorities like the Food and Drug Administration (FDA) as described in "Guidance for Industry: Pyrogen and Endotoxins Testing." It is requested to be determine whether the ability to detect endotoxins can be affected by storage and handling of the sample to be tested. To accomplish these studies, endotoxin is introduced or spiked into the undiluted product and held for a certain period of time in process-representative containers. This time period reflects procedural maximum QC sample hold time from sampling until analysis. Inadequate detection of endotoxin can be caused by adsorption of endotoxin to container surfaces or molecular masking effects, in which the binding sites on the endotoxin molecules are prevented from triggering the enzymatic cascade necessary in the assay, are obscured. The endotoxin may form macromolecular structures, such as sheets or blebs, or the binding sites may otherwise be rendered unavailable due to the sample matrix composition. In either case, the endotoxin assay may yield falsely low results if and when masking occurs. In this work, the QC sample hold times of different in-process controls within the production process of a biopharmaceutical product were analyzed. One out of eight different samples showed a strong masking of endotoxin. Analysis of the sample composition revealed that either kifunensine, mycophenolic acid (MPA), or lauryl-N, N-dimethylamine oxide (LDAO) was responsible for masking. Further analysis clearly identified LDAO as the root cause for masking. A novel one-step mechanism for LDAO-induced endotoxin masking is proposed. The principle is similar to an already-proposed two-step mechanism for endotoxin masking, but the LDAO case combines these two steps: the disturbance of the salt bridges and hydrophobic interactions with LPS in one molecule. These molecular interactions occur quickly when both endotoxin and LDAO are present in the same matrix. Thus, depending on the masking agents, low endotoxin recovery (LER) can occur regardless of the QC sample hold duration.

Microbiological Control for Affinity Capture Chromatography Processing: An Industry Perspective.

The purpose of this paper is to provide a summary of a BPOG-led industry survey of the microbiological control aspects of affinity chromatography processing in the biopharmaceutical industry. The document provides a summary of historical microbiological control concerns, coupled with industry-derived best practices, for material, equipment, and storage controls required to mitigate the potential for microbial ingress and contamination of chromatography resin and equipment. These best practice guidelines, which are derived from the members of the BPOG Bioburden Working Group, are intended to assist biopharmaceutical manufacturers to enhance microbial control and monitoring strategies for chromatography systems.