Detailed project description

Project objective & research approach

Project objective

 

The aim is to be able to demonstrate within the duration of the project that after bacterial typing, effective bacteriophages can be attracted and multiplied within a clinically acceptable period of up to two weeks and that a therapy can be started successfully. The aim is to demonstrate the practicability of phage therapy under the infrastructural conditions already available in Germany and in compliance with legal requirements. It could be shown that an individual phage therapy adapted to the patient situation with adequate indication (multi-resistance) could actually be implemented already today, also in Germany, in the inpatient care everyday life (personalized infection treatment).

 

 

 

 

 

 

Research approach

 

The PhagoFlow project addresses the problem by testing the practicability of patient-specific cocktails of phages that are designed to act against the bacteria in question. The pharmacy of the Bundeswehrkrankenhaus Berlin will mix the phage “cocktails” on medical order for the individual case. Indispensable prerequisites for therapy with lytic phage are: they must be biologically well characterized and efficient, i.e. they must cover (lyse) a broad spectrum of bacterial strains within one bacterial species and they must meet safety and purity criteria according to the German Drug Law. On this basis, collections of complementary phages for particularly frequently multi-resistant bacterial species are assembled in the host spectrum at the Leibniz Institute DSMZ so that they can be passed on to the Fraunhofer Institute ITEM for pharmaceutical purification, where they are manufactured as active pharmaceutical ingredients under GMP conditions, Good Manufacturing Practice, and made available to the pharmacy of the Bundeswehr Hospital in Berlin. After a phagogram has been carried out, patients will receive several suitable phages, which will work together as synergistically as possible. In individual cases, the phagogram provides information on the spectrum of effective phages. This will enable PhagoFlow to implement an individualised therapy against multi-resistant bacteria in Germany.

Magistral Application

Why PhagoFlow is not a clinical trial

 

Health care research is a branch of health system research that lies at the interface of clinical research, public health research and health economics. The object of health care research is to answer general questions of health care.

 

From a scientific point of view, health care research is defined as basic and problem-oriented, interdisciplinary research that describes, causally explains and develops health care concepts, their implementation and evaluation under everyday conditions.

 

Our PhagoFlow project serves to evaluate the benefits of a new form of therapy with bacteriophages for the future introduction into the field of patients with health insurance.

 

In contrast, clinical studies in evidence-based medicine and clinical research are concerned with data collection. It is carried out with patients or healthy volunteers and is a prerequisite for official drug approval or approval extension. The aim of clinical studies is to test the efficacy and safety of drugs, certain forms of treatment, medical interventions or medical devices. Clinical trials are conducted to answer scientific questions and improve medical treatment.

Leibniz Institute DSMZ GmbH

Biological characterization and selection of phages suitable for therapy

 

In order for a patient who is colonized/infected with a multi-resistant bacterium (e.g. Staphylococcus aureus in the form of MRSA or 3/4MRGN Pseudomonas aeruginosa) to be treated with bacteriophages (phages for short), suitable lytic phages must first be identified and provided. A well-suited phage is characterized by the fact that it can destroy as many bacterial variants (“strains”, “isolates”) of a species as possible and is classified as harmless. For this purpose, phages from the collection of the Leibniz Institute DSMZ GmbH are tested on a diverse collection of patient isolates. In addition, these phages must be genetically tested in order to exclude the possibility that they carry virulence factors or antibiotic resistance markers. Promising candidates are subsequently investigated in vitro (in the laboratory) for their efficiency in destroying bacteria. At the same time, the search for further new phages in different environmental samples is carried out in order to enlarge the repertoire. This is followed by clonal isolation/clonal purification of the phages, reproduction on a small scale and also examination of their suitability for application purposes. Since each phage within a bacterial species can only destroy certain patient isolates, it is necessary to find different phages which together cover as many bacterial isolates as possible. For each bacterial species prioritized in the PhagoFlow project, a set of phages with high genetic and morphological diversity is to be compiled, thus increasing the probability of sufficient coverage of patient isolates.

 

In the course of the PhagoFlow project, the procedure of a phagogram for the clinical laboratory routine to test the sensitivity of patient isolates to phages is to be developed in good time. For this purpose, it is necessary to compare different methods with each other in terms of feasibility, evaluability and reproducibility. The goal is a simple implementation and harmonization of the incubation conditions, so that this procedure can be well integrated into the everyday routine of diagnostics.

 

First, efficient new phages are sought for three bacterial species from the ESKAPE group: 1.) Staphylococcus aureus (very frequently detected as an infectious agent in Bundeswehr hospital) is a spherical gram-positive bacterium, a normal inhabitant of the skin and the nasopharynx. However, strains of this type of bacteria are often capable of producing various toxins, and the bacterium is also known as a pyogenic agent in inflammatory processes. If the bacterium enters the bloodstream, serious consequences can occur; it is one of the most frequently known pathogens of bacteremia and sepsis, but also settles on organs such as heart valves and foreign surfaces such as artificial heart valves or joint replacement. In S. aureus, the MRSA variants, methicillin-resistant strains, are particularly feared, the proverbial “hospital germ” whose antibiotic treatment is difficult. 2.) Pseudomonas aeruginosa is also a very common infectious agent found in Bundeswehr hospital, a gram-negative rod bacterium that occurs virtually everywhere in the environment and in and on us, has an extremely flexible genome and is very adaptable to genetic make-up due to this flexibility. P. aeruginosa occurs particularly typically in biofilms, microbial communities in which the bacteria “rest”, but maintain intensive exchange of genes and information (see also “Quorum sensing”), ready to exchange virulence factors and antibiotic resistances for mutual selection advantage. The mentioned undesirable properties of S. aureus are almost entirely true for P. aeruginosa, although P. aeruginosa is more common in various infections. In this bacterial species, not only multi- but even pan-resistant strains are now known in which not a single antibiotic suitable for therapy is effective. 3.) In Bundeswehr hospital, the gram-negative rod Escherichia coli, a typical intestinal inhabitant, is diagnosed with moderate frequency. Within its species, Escherichia coli has an enormous range of variants, from the status of an important microbial inhabitant to the life-threatening pathotype EHEC, with strains that neither form toxins nor carry virulence factors up to extremely dangerous toxin formers with sophisticated infection mechanisms and antibiotic multiresistances. The importance of fine diagnostics in the case of E. coli in patients is therefore particularly clear. These three bacterial species also have in common that they multiply very rapidly. They also occur in mixed infections. In the further course of the project, other bacterial species may come to the fore, depending on requirements and possibilities.

Fraunhofer ITEM

Production of phage active pharmaceutical ingredients in compliance with GMP

 

In the PhagoFlow project, Fraunhofer ITEM is producing a large number of phages in compliance with GMP. Since these phages can differ e.g. in their lysekinetics and biochemical properties, a standardized production platform for phage active pharmaceuticals ingredients is being developed, which can be adapted to the specific phages.

 

This platform comprises standardized cultivation and purification steps and is intended to make it possible to transfer an established manufacturing process for phage active pharmaceutical ingredients from one phage to another by fine-tuning of parameters. After receiving the phages and their respective production strain from the DSMZ, the Fraunhofer ITEM will initially establish manufacturing processes on a laboratory scale. After the production strains have been cultivated, they are infected with phages, which leads to their propagation up to the complete lysis of the production strain. The purification of the phages is optimized by up to 2 chromatography steps and ultrafiltration to such an extent that media components and residues of the production strain, such as host cell proteins and endotoxins, are separated as far as possible.

 

The process development is followed by the production of the phages as active pharmaceutical ingredients in compliance with the requirements of the pharmaceutical quality assurance system GMP. For this purpose, a master phage stock or master cell bank is first created for each individual phage and its production strain, which is then used for initial production as well as later post-production of the phage active pharmaceutical ingredient. The production sequence previously developed in the laboratory is scaled up and carried out depending on the concentration of the phage in the culture supernatant, its yield after purification, its stability and clinical consumption. After production, each phage active pharmceutical ingredient is filled into aliquots, tested for content, identity, biological activity (potency) and purity and finally released for further processing into magistral preparations by hospital pharmacies via an analysis certificate (CoA) and a quality certificate (CoC).