Bioprocessing of bacteriophages and bacteriocins: continuous culture and downstream purification

2020-01-13T08:50:08Z (GMT) by Francesco Mancuso
Antimicrobial resistance poses a major problem to health and new alternatives to antibiotics are required. Bacteriophages are viruses able to selectively kill bacteria and they can be exploited to treat infections. Another alternative to antibiotics that can be used to treat bacterial infections are bacteriocins, which are proteins produced by bacteria, meant to kill other strains competing for the same nourishment source. Future industrial demand for large quantities of bacteriophages and bacteriocins imposes the development of a scalable production platform.
In this PhD thesis, research on bioprocessing of bacteriophages K and T3 and one bacteriocin belonging to the subcategory of colicins, E9, has been conducted. The aims were to find the parameters involved in production of phages and colicin E9 using shake flasks and then exploiting them to perform continuous production of both phages and colicin E9 and showing the different outcomes when using complex medium (LB) and a synthetic medium (SM) using glucose as only carbon source.
For the colicin E9 bioprocessing, tests for expression were carried out to measure when and for how long to induce for optimal protein production and the best conditions for production were an induction of 3 h with 1 mM of IPTG in LB medium and >10 h of induction with 1 mM of IPTG in SM. These parameters were used for continuous production of colicin E9 that was carried out using a chemostat. Optimal dilution rates were used to ensure maximum productivity and a production of 1 mg mL-1 of colicin E9 was achieved using either LB and synthetic medium. Then, the influence of flowrates and of different growth media on the first step of purification was assessed using affinity chromatography, showing that the synthetic medium allowed higher recovery of the product in the chromatography step.
Bioprocessing of the phages K and T3 was carried out in shake flasks and 5L fermenters, researching the best parameters for producing the highest titres. The effect on the final titre of a variety of multiplicities of infection (MOI), the ratio of phages out of bacteria at the moment of infection, was evaluated. For both phages the MOI that ensured the highest phage titre, 1011 PFU mL-1, was 0.01. The parameters measured were then used for continuous production of bacteriophage T3.
Continuous phage T3 production was carried out using a novel reactors layout, composed by 3 separated stages. This set up allowed to divide the production of the host cells, carried out in the first reactor, from the infection, carried out in the second reactor, and from the final amplification step, carried out in the third reactor. The synthetic medium with glucose as only carbon source and the dilution rate (D, the volume of media that flows in the reactor per hour) were used to control the growth rate of the host, which strongly influenced the final production of bacteriophages. Different D
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were tested, from 0.1 to 0.6 h-1 and the independent control of the dilution rate of the first and the second reactor allowed to produce phage lysates at the same concentrations as in batch process, reaching a constant production of 2x1011 PFU mL-1 when D = 0.5 h-1 was used in the first reactor.
Moreover, research on the downstream process of phage bioprocessing was conducted, aiming to reduce the host cell protein burden from phage samples using scalable techniques. The purification of bacteriophages from complex medium was performed using ultrafiltration in batch mode in a stirred cell (SC) and in using tangential flow filtration (TFF). The high shear stress produced SC decreased the viability of the tailed phage K of 2 log10 in 2 h and the positive effects of purification were cancelled by the loss of titre. Phage T3, which has a short tail, showed no side effects due to shear stress. TFF was then tested and both phage K and T3 were purified without significant viability loss. Size exclusion chromatography was used to assess the final protein concentration and showed a decrease of host cell protein load in both phages samples purified by ultrafiltration. Samples of both phages were mixed with a positively charged resin used for anion exchange chromatography and it was shown that phages that have been filtered using ultrafiltration can bind 10 times more than phages of the lysate.
The main aim of purification phages produced from Gram-negative bacteria, such as T3, was to reduce the endotoxin levels (or lipopolysaccharide, LPS). Endotoxins are a major contaminant that can cause serious problems if administered and must be removed from drug products. The target was to bring the LPS levels from the initial concentration of 5x106 EU mL-1 (Endotoxin Units per mL) below the limit of 5 EU mL-1. Endotoxin removal was performed using ultrafiltration and liquid-liquid extraction (LLE).
The first method tested was the TFF ultrafiltration, carried out in batch mode or in diafiltration mode. Batch ultrafiltration showed a decrease of LPS of 2 log10, either starting from a phage T3 sample from LB or from SM. Diafiltration TFF reached 3 log10 reduction of LPS in both LB or SM samples, but in LB samples it took 20 h and in SM samples only 4 h, showing how a chemically defined medium could improve the purification of phages produced from Gram-negative hosts.
The other method tested to further reduce LPS concentration was liquid-liquid extraction, using 1- octanol as organic phase. Many different conditions of LLE were tested, such as length of contact time between aqueous and organic phases, different mixing methods and number of extractions. Liquid- liquid extraction using octanol ensured a reduction of endotoxins of 4 log10 from the initial concentration. This technique was then followed by ion exchange chromatography, which allowed to reach a final concentration of 102 EU mL-1.
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Finally, analysis on how endotoxins concentrations affected the aggregation of phage T3 were performed, showing how phage T3 aggregates in presence of endotoxins whilst it has a low polidspersivity at low endotoxins concentrations.
This thesis showed how is it possible to produce bacteriophages and an antimicrobial protein at high concentrations using a continuous production mode and a chemically defined medium. The positive effects of the synthetic medium on the downstream steps were also illustrated, such as a lower burden for ion exchange columns and a faster reduction of endotoxin concentration from the samples.