Phactory - A Safe Manufacturing Pipeline
Phactory revolutionizes the production of therapeutic phages independently
from their bacterial hosts. Each step of our bacteriophage production line
has been optimized individually to meet Good Manufacturing Practice and
guarantee that all safety requirements for therapeutic phages are met.
The simplicity of Phactory enables phage assembly by
simply adding high-purity phage genomic DNA to cell extract. This allows for
flexible and individualized medical treatment of patients.
Maximum phage purity and high titers are ensured by filtration and purification steps. Furthermore, Phactory incorporates packaging of our assembled phages in alginate capsules for protection from gastric acid after oral administration and intestinal delivery.
Safety Requirements for Therapeutic Phages
Identification
Contamination
Toxicity
pH
Purity
Preservation
Identification of Bacteriophages
We performed a plague assay to determine the activity of the viable phages (titers) in our assembly batch. By creating serial dilutions we were able to calculate a plague forming unit / milliliter (PFU/ml) value. On average the titer is approximately 108.
Next Generation Sequencing by Nanopore TM made it possible to detect DNA impurities in our isolated bacteriophages. This is necessary to achieve high titers with phage assembly. With Sequ-Into we developed a custom open-source software tool which simplyfies the analysis of sequencing data. Especially stataistical analysis of impurities are faciliated. Based on Sequ-Into we reduced contamination, as the improvement of our purification protocol was clearly detectable.
Transcription-Translation System
First we optimized our cell extract preparation protocols to get highly active
extract which is able to produce high amounts of bacteriophages.
Specifically engineered E. coli strains are the core component for the preparation
of cell extract with low endotoxin levels.
We optimized our strain for therapeutic bacteriophage production by knocking
out myristoyltransferase msbB in the host strain to disrupt the lipid A biosynthesis pathway.
A LAL test was used to measure endotoxin levels. In comparison with commercially available cell extract and cell extract prepared using wild-type strains, the lipid A concentration could be lowered to a fraction of 2%.
We have approved that the assembled phage solution has a physiological pH to ensure the requirement of the correct pH range for therapeutic phages.
Cell extract can be stored and shipped at room temperature if the samples are prepared by lyophilization. To make our cell extract accessible to everyone everywhere, we seeled to ensure long term storage and shipping at room temperature. Therefore we created lyophilized cell extract. The results show, that the tested samples retained 70% and 90% of expression quality respectively after lyophilization.
Furthermore, we tested the functionality of phages assembled in our home made cell extract after lyophilization and found functional phages afterwards.
Phage Assembly
The simplicity of Phactory offers the possibility of producing phages simply by adding the specific phage DNA to the TX-TL machinery. Moreover, Phactory yields excellent titers of assembled phages by far higher as required for therapeutic use.
Phactory makes high yield of phages of up to 1012 PFU/mL in a small reaction volume of 9 µL possible. Because we are using an E.coli based cell extract we avoid biohazardous pathogens traditionally necessary for therapeutic phage production. Bacteriophages can be assembled independent of the living host.
To fulfill the strict requirements for therapeutically used phages further purification is required to produce a high quality product.
The abscence of impurities in the phage solutions produced in Phactory was certified by Transmission Electron Microscopy (TEM) imaging. By ultra-filtration phages got successfully separated from submolecular content.
Phactory yields phages with toxicity levels that allow oral administration to the patient. However, oral delivery requires protection of the phages from rapid degradation in the acidic gastric juice. Therefore, we encapsulated our phages in alginate. This protects our phages successfully until release into the gastric system. We were able to prove functionality of encapsulated and released phages.
Clinically Relevant Bacteriophages
It was of great importance for us to demonstrate the medical relevance of bacteriophages manufactured with Phactory. We received the SEC-3S bacteriophage from the Queen Astrid Hospital as well as the P2 and P3 bacteriophages from the group of Laurent Debarbieux at the Pasteur Institute in Paris. These bacteriophages are specific for the E.Coli strain O104:H4, also known as EHEC, which caused 53 deaths during an outbreak in Germany in 2011. As indicated in the microscopy images, we successfully assembled these medically relevant bacteriophages in our cell-free system. We conducted a LAL-test and compared the results with magistral bacteriophage preparation regulations. According to these results, our bacteriophages are orally applicable. In the future we will work on reaching even higher titers with Phactory, so that we can apply more extensive purification protocols to allow intravenous application of bacteriophage therapy.
Clinical Trial
