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Within this first part of the two-part series on phage manufacturing, we will give an overview of the process leading to bacteriophages as a drug substance, before covering the formulation into a drug product in the second part. The principal goal is to provide the reader with a comprehensive framework of the challenges and opportunities that present themselves when developing manufacturing processes for bacteriophage-based products. We will examine cell line development for manufacture, upstream and downstream processes, while also covering the additional opportunities that engineered bacteriophages present.

The AOAC Research Institute Performance Tested MethodsSM Program certified Sample6 DETECT/L™ in April 2014 (Certification No. 041401) for the detection of Listeria species (L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. marthii, L. welshimeri) on stainless steel environmental surfaces. A modification was approved in January 2016, increasing the concentration of sanitizer-neutralizing reagents in detection reagents, increasing the number of phage in the detection solution, and increasing the sample test volume. Moreover, changes to reduce the number of negative controls and add compatibility with polyurethane sponges were also approved. In this modification, to ensure that DETECT/L continues to meet performance expectations, Sample6 evaluated workflow changes to enhance sensitivity and the ease-of-use of the assay. Changes to the phage concentration and detection threshold, plus the inclusion of a confirmation step (DETECT Check), were validated to obtain better accuracy and optimize assay performance. Inclusivity, exclusivity, and robustness testing were conducted by Sample6 to evaluate the changes. A third-party laboratory compared the DETECT/L assay and the U.S. Department of Agriculture reference method in a stainless steel environmental surface matrix study. The data presented in this report demonstrate that the changes proposed to the DETECT/L assay meet or exceed the performance in the current configuration.

Background: Listeria spp. are an important foodborne human pathogen because of their ability to cause disease and high mortality in individuals, particularly pregnant women, neonates, the elderly, immunocompromised individuals, and children. The Sample6 DETECTTM HT/L Kit is a semi-automated qualitative pathogen detection system designed to detect Listeria spp. (L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri, and L. marthii) in environmental samples using the Sample6 BioIlluminationTM technology. Objective: The study was done to evaluate the Sample6 DETECT HT/L Kit. The assay was evaluated for inclusivity, exclusivity, robustness, product consistency, and stability, and a matrix study of one environmental surface. Methods: The performance of the Sample6 DETECT HT/L was compared with U.S. Food and Drug Administration reference culture method for Listeria using an unpaired study design. Results: The Sample6 DETECT HT/L assay correctly identified all 50 inclusivity isolates and correctly excluded all 30 nontarget strains evaluated. The assay was not affected by minor variations in incubation temperature and time, or sample volume. Results across three production lots spanning the shelf life of the assay were consistent. In the matrix study, the Sample6 DETECT HT/L for Listeria correctly identified each test portion for the presence or absence of Listeria, and there were no statistically significant differences between candidate and reference method results. Conclusions: The data collected in this study demonstrate that the Sample6 DETECT HT/L assay is a reliable method for the detection of Listeria spp. on stainless-steel environmental surfaces after 22 h of enrichment.

Bacteriophages have been used for diagnostic purposes in the past, but a lack of parallelizable engineering methods had limited their applicability to a narrow subset of diagnostic settings. More recently, however, advances in DNA sequencing and the introduction of more sensitive reporter systems have enabled novel engineering methods, which in turn have broadened the scope of modern phage diagnostics. Here we describe advanced methods to engineer the genomes of bacteriophages in a modular and rapid fashion.

Bacteriophage therapy for bacterial infections is a concept with an extensive but controversial history. There has been a recent resurgence of interest into bacteriophages owing to the increasing incidence of antibiotic resistance and virulent bacterial pathogens. Despite these efforts, bacteriophage therapy remains an underutilized option in Western medicine due to challenges such as regulation, limited host range, bacterial resistance to phages, manufacturing, side effects of bacterial lysis, and delivery. Recent advances in biotechnology, bacterial diagnostics, macromolecule delivery, and synthetic biology may help to overcome these technical hurdles. These research efforts must be coupled with practical and rigorous approaches at academic, commercial, and regulatory levels in order to successfully advance bacteriophage therapy into clinical settings.

Synthetic biology is an emerging engineering field focused on designing artificial biological systems with novel functionalities for use in therapeutics, basic science, biotechnology, and diagnostics [1,2]. Continuous advancements in DNA synthesis and sequencing technologies coupled with new techniques for genomic modification and assembly have opened the door for harnessing the power and diversity of biology for applications. For example, natural bacteriophage products, such as ListShield (Intralytix) and Agriphage (Omnilytics), are commercially available for reducing unwanted bacterial contamination.

RNA editing by A-to-I modification has been recognized as an important molecular mechanism for generating RNA and protein diversity. In mammals, it is mediated by a family of adenosine deaminases that act on RNAs (ADARs). The large version of the editing enzyme ADAR1 (ADAR1-L), expressed from an interferon-responsible promoter, has a Z-DNA/Z-RNA binding domain at its N-terminus. We have tested the in vitro ability of the enzyme to act on a 50 bp segment of dsRNA with or without a Z-RNA forming nucleotide sequence. A-to-I editing efficiency is markedly enhanced in presence of the sequence favoring Z-RNA. In addition, an alteration in the pattern of modification along the RNA duplex becomes evident as reaction times decrease. These results suggest that the local conformation of dsRNA molecules might be an important feature for target selectivity by ADAR1 and other proteins with Z-RNA binding domains.