Study of robustness of filamentous bacteriophages for industrial applications

Authors

  • Steven Branston,

    1. Department of Biochemical Engineering, The Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK; telephone: +44-20-7679-2961; fax: +44-20-7209-0703
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  • Emma Stanley,

    1. Research Department of Structural and Molecular Biology, University College London, The Darwin Building, Gower Street, London, UK
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  • John Ward,

    1. Research Department of Structural and Molecular Biology, University College London, The Darwin Building, Gower Street, London, UK
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  • Eli Keshavarz-Moore

    Corresponding author
    1. Department of Biochemical Engineering, The Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK; telephone: +44-20-7679-2961; fax: +44-20-7209-0703
    • Department of Biochemical Engineering, The Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London WC1E 7JE, UK; telephone: +44-20-7679-2961; fax: +44-20-7209-0703.
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Abstract

The development of a whole new class of industrial agents, such as biologically based nanomaterials and viral vectors, has raised many challenges for their large-scale manufacture, principally due to the lack of essential physical data and bioprocessing knowledge. A new example is the promise of filamentous bacteriophages and their derivatives. As a result, there is now an increasing need for the establishment of strong biochemical engineering foundations to serve as a guide for future manufacture. This article investigates the effect of high-energy fluid flow on filamentous bacteriophage M13 to determine its robustness for large-scale processing. By the application of well-understood ultra scale-down predictive techniques, the viability of bacteriophage M13 was studied as a measure of its robustness and as a function of energy dissipation rate and fluid conditions. These experiments suggested that despite being perceived as a relatively fragile molecule in the literature, bacteriophage M13 should tolerate processing conditions in existing large-scale equipment designs. No loss of viability was noted up to a maximum energy dissipation rate of 2.9 × 106 W kg−1. Furthermore, significant losses above this threshold only occurred over periods well in excess of the exposure times expected in a bioprocess environment. Filamentous bacteriophages may therefore be regarded as a viable process material for industrial applications. Biotechnol. Bioeng. 2011; 108:1468–1472. © 2011 Wiley Periodicals, Inc.

Introduction

In nature, the Ff group of filamentous bacteriophages are viruses that reproduce by the non-lethal infection of Escherichia coli. Instead of release by cell lysis, progeny bacteriophages are continuously extruded through the cell wall to the surroundings. For many years, they have found utility in molecular biology as tools for expression and sequencing, following the discovery of their ability to display foreign gene products on the viral surface (Smith, 1985). This technique has become known as phage display, and also underpins most of the commercial uses of filamentous bacteriophages that have been derived so far. Today, filamentous bacteriophages feature as potential vectors for gene therapy (Liang et al., 2006) and as immunization tools against diseases such as Alzheimer's disease (Solomon, 2008). Furthermore, in the nanotechnology arena the use of phage display in conjunction with the inherent properties of the filamental viral structure have led to the construction of useful materials such as functioning Li-ion battery cathodes (Lee et al., 2009).

However, the filamentous bacteriophage industry is very much in its infancy. On the road from research to market, a key challenge will be the application of biochemical engineering principles to translate production from the laboratory to the large-scale process. The task of scale-up is hindered by a relative lack of published data in the area. For filamentous bacteriophages, a recent study on the 1 L scale fermentation of a phage display derivative virtually stands alone in the field (Grieco et al., 2009). Similarly, studies focusing on purification processes are sparse. One example is the bench-scale purification by expanded bed anion exchange chromatography (Ling et al., 2004).

Shear-associated degradation by large-scale process equipment has been identified as a problem for several industrially relevant macromolecules, including plasmid DNA (Zhang et al., 2007), whole antibodies (Biddlecombe et al., 2007) and protein precipitates (Boychyn et al., 2004). However, little equivalent information exists concerning the general response of filamentous bacteriophages to large-scale processing. The response of filamentous bacteriophage to turbulent fluid flow is of key importance. High-energy turbulent conditions can occur throughout a bioprocess, such as at the fermentation, filtration, pumping, and continuous centrifugation stages (Boychyn et al., 2004; Levy et al., 1999; Zhang et al., 2007). From existing literature, the physical disruption of filamentous bacteriophages has been reported as a result of exposure to the extreme conditions of ultrasonication (Fareed and Valentine, 1968) and pressure homogenization (Grant et al., 1981).

Ff Filamentous bacteriophages are semi-flexible tubes in nature, 900 nm in length but only 6.5 nm in diameter. They consist almost entirely of 2,700 copies of a small 5.5 kDa protein (named pVIII) arranged in a helical array about a tightly bound 6.6 kb single-stranded DNA core (Fig. 1). Therefore, each virus is in effect a large protein rod 100-fold greater in mass than a full length IgG antibody and of contour length approaching the characteristic dimensions of a considerably shear-sensitive supercoiled 20 kb plasmid (Levy et al., 1999; Zhang et al., 2007). If M13 proves to be an extensively shear sensitive molecule then the choice of large-scale operations could be constrained.

Figure 1.

Schematic diagram of bacteriophage M13 particle with enlarged cross-section (A) and TEM diagram of several M13 bacteriophages (B). The bacteriophage M13 particle appears as a semi-flexible tube 900 nm in length, 6.5 nm in diameter. Bacteriophages were negatively stained with 0.25% (w/v) uranyl acetate for 10–20 s.

Instead of committing liters of material for trials in large-scale equipment, milliliter-scale tools have been developed at University College London to simulate the large-scale fluid flow environment. The benefit of this ultra scale-down (USD) approach is that predictions of the performance or effect of large-scale operations can be made more rapidly using considerably less material. In particular, a USD rotating disk shear device has been developed to predict the effects of turbulent fluid flow from large-scale pumps (Zhang et al., 2007) and continuous centrifuges (Boychyn et al., 2004; Hutchinson et al., 2006; Zhang et al., 2007). A solid base of small and large-scale verification experimentation exists, validating its use to determine patterns of shear-sensitive behavior for materials such as plasmid DNA, mammalian cell and antibody feeds (Biddlecombe et al., 2007; Hutchinson et al., 2006; Levy et al., 1999).

In this study we have utilized a USD rotating disk shear device to study the resistance of the Ff filamentous bacteriophage M13 to turbulent fluid flow of increasing intensity. We then examined the effect of the suspension fluid and predicted the likelihood of shear damage from conditions up to a maximum energy dissipation rate of 5.9 × 106 W kg−1, expressed as the rate of energy (W, Watts) dissipated per kilogram of fluid. This energy dissipation rate deliberately exceeded that predicted for the potentially damaging pumping and continuous centrifugation stages, and was chosen to elicit the strongest response from the bacteriophage. If no damage was found to occur at this rate, then it would be possible to predict that no damage was likely in most large-scale equipment designs.

Results and Discussion

Bacteriophage M13 suspensions of concentration of 3 × 1011 plaque forming units (pfu) mL−1 in 10 mM Tris were subject to maximum energy dissipation rates in the range 1.3–5.9 × 106 W kg−1. Bacteriophage concentrations were chosen to represent those typically achieved by fermentation (Grieco et al., 2009). The resulting effect on bacteriophage viability is shown in Figure 2, where a significant decrease in viability occurred only at maximum energy dissipation rates in excess of 4.2 × 106 W kg−1 (P = 0.001, two tailed t-test). Increases in the maximum energy dissipation rate beyond 4.2 × 106 W kg−1 resulted in significantly greater reductions in bacteriophage viability, suggesting that a threshold resistance had been exceeded. A maximum 62% loss of viability occurred at the highest energy dissipation rate (5.9 × 106 W kg−1) after 25 min.

Figure 2.

Relationship between the maximum energy dissipation rate and the fraction of viable bacteriophage M13 remaining after a 25-min exposure time. The initial bacteriophage concentration (C0) was 3 × 1011 pfu mL−1. Viability was determined by the surface-droplet technique (see Materials and Methods Section), where viability is defined as the continuing ability to infect E. coli. No significant decrease in bacteriophage viability was observed to occur over 25 min exposure to maximum energy dissipation rates of 1.3 × 106 or 2.9 × 106 W kg−1. Error bars represent the standard error of the mean fractions, N = 3.

At the three highest energy dissipation rates the loss of viability at 5-min intervals was subsequently recorded (Fig. 3). To estimate the rate of decay of bacteriophage M13 against maximum energy dissipation, each set of data in Figure 3 was fitted to a first order decay process of the form:

equation image(1)

where Ct is the concentration of viable bacteriophage at time t, C0 the initial concentration of viable bacteriophages prior to shearing, and K the decay rate constant, representing the proportional loss in bacteriophage viability per minute. Such shear degradation profiles are consistent with those reported for other shear sensitive macromolecules (Biddlecombe et al., 2007; Zhang et al., 2007). Finally, the decay rate constants were found to increase in exponential proportion to the corresponding maximum energy dissipation rate, ε (data not shown), according to the equation:

equation image(2)
Figure 3.

The fraction of viable bacteriophage M13 remaining over time at maximum energy dissipation rates 4.2 × 106 W kg−1 (Δ), 5.0 × 106 W kg−1 (□), and 5.9 × 106 W kg−1 (●). The initial bacteriophage concentration (C0) was 3 × 1011 pfu mL−1. Viability was determined by the surface-droplet technique. Error bars represent the standard error of the mean fractions, N = 3. Solid lines represent the lines of best fit for each data set.

Although the cumulative exposure time to turbulent shear conditions in large-scale processing may be of the order of minutes (Zhang et al., 2007), the highest maximum energy dissipation rates within mechanically agitated fermenters are typically only up to 103 W kg−1 and within pumps up to 105 W kg−1 (Yim and Shamlou, 2000). Since a maximum energy dissipation rate of 2.9 × 106 W kg−1 was found to result in no significant bacteriophage viability loss over 25 min (Fig. 2), bacteriophage damage from these operations is unlikely.

Over the course of a large-scale purification scheme the composition of the process fluid would alter considerably as contaminants are progressively removed, buffers changed, and chemicals added. Since the action of turbulent shear was shown to decrease the viability of bacteriophage M13 in buffer, it was considered important to investigate the effect of the process fluid composition on shear sensitivity. Four bacteriophage-containing process fluids were compared at a maximum energy dissipation rate of 5.9 × 106 W kg−1. The four process fluids were (1) post-fermentation M13-infected E. coli culture (OD600 = 2.9); (2) culture supernatant (cells removed by bench-scale centrifugation, 14,000g, 15 min); (3) precipitated M13 (culture supernatant with 3.3% (w/v) PEG 6 000 and 330 mM NaCl); (4) purified M13 in 10 mM Tris.

Similarly to Figure 3, losses in bacteriophage viability were recorded at 5-min intervals over 25 min and each data set fitted to a first order decay process. However, the composition of the process fluid was found to exhibit little effect on the sensitivity of bacteriophage M13 to turbulent shear conditions (data not shown). Furthermore, since in a large-scale process the exposure time to the critical high-energy areas in the most damaging equipment (such as continuous centrifuges, where energy dissipation rates are up to 106 W kg−1 in magnitude) is of the order of a fraction of a second (Byrne et al., 2002), the absolute decreases in bacteriophage viability are unlikely to be significant either.

This study described the effect of quantified turbulent shear conditions on the filamentous bacteriophage M13. The USD methodology allowed for the rapid assessment of bacteriophage fragility under a range of maximum energy dissipation rates and fluid types. Conditions imposing a maximum energy dissipation rate of 2.9 × 106 W kg−1 did not cause a significant drop in bacteriophage viability. Furthermore, the viability decay rate at higher energy dissipation rates was small considering the short exposure time to such conditions within large-scale equipment. Since verification studies have demonstrated the link between the USD rotating disk device and pumping (Zhang et al., 2007) and continuous centrifugation performance (Boychyn et al., 2004), these results imply that the shear imposed by these large-scale stages will not have an adverse effect. In practical terms, the large-scale manufacture of filamentous bacteriophage-based products should be feasible using existing process equipment.

Materials and Methods

Bacteriophage Propagation and Purification

Wild-type M13 (ATCC 15669B1) was propagated in E. coli Top10F′ (Invitrogen, Paisley, UK). Cultures (400 mL) were grown in Nutrient Broth Number 2 (Oxoid, Hants, UK) with 0.2 g L−1 polypropylene glycol (PPG) in 2 L shakeflasks overnight at 37°C and 200 rpm. Cultures were harvested by centrifugation at 14,000g at 4°C for 15 min and the supernatants decanted. M13 was precipitated from the supernatant fraction by the addition, to a final concentration, of 3.3% (w/v) polyethylene glycol (PEG) 6000 and 330 mM sodium chloride (NaCl), and incubated for 1 h at 4°C. Precipitated M13 particles were pelleted by centrifugation at 14,000g at 4°C for 10 min and resuspended in 8 mL 10 mM Tris–HCl (pH 7.6 at 25°C). Resuspended bacteriophage pellets were filtered through a 0.22 µm filter (Millipore, Watford, UK) and subjected to cesium chloride density centrifugation. Banding occurred at a cesium chloride concentration of 0.4 g mL−1 at 110,000g (Beckman, Buckinghamshire, UK) for 23 h, 15°C. Bands of concentrated M13 were extracted and dialyzed against 3 L of 10 mM Tris–HCl (pH 7.6 at 25°C) using 10,000 MWCO dialysis tubing (Pierce, Rockford, IL) for 24 h at 4°C. Dialysis solution was changed after 12 h. For all subsequent experimentation a portion of bacteriophage stock was diluted in 10 mM Tris–HCl (pH 7.6 at 25°C) to a concentration of 3 × 1011 pfu mL−1.

Rotating Disk Shear Device Operation

The use of a rotating disk shear device to mimic the shear conditions experienced during bioprocessing has been previously described (Hutchinson et al., 2006; Levy et al., 1999; Zhang et al., 2007). Here, shearing was performed in a device consisting of a single rotating stainless steel disk (diameter 40 mm, thickness 0.14 mm) mounted centrally in a 20-mL capacity stainless steel cylindrical chamber (diameter 50 mm). The disk rotation speed within the device has been previously characterized using computational fluid dynamics (CFD) in terms of the resulting maximum energy dissipation rate (Boychyn et al., 2004), a general parameter for which estimates exist for many large-scale equipment designs (Yim and Shamlou, 2000). In particular, verification studies using shear sensitive material have shown a good correlation between the shear effects of the feed-zone of large-scale centrifuge designs and the rotating disk mimic (Boychyn et al., 2004; Hutchinson et al., 2006). In this study, the disk device was operated at rotation speeds of 300, 400, 467, 500, and 533 revolutions per second (rps). The top speed of 533 rps corresponded with a maximum energy dissipation rate of 5.9 × 106 W kg−1.

Analyses

Viability of Bacteriophage M13

To be viable, a bacteriophage must remain intact since infection of E. coli occurs from the interaction of one tip of the virus with an F-pilus. The surface droplet technique (Miles and Misra, 1938) was employed to calculate the quantity of viable bacteriophage in a sample in terms of pfu mL−1. Samples containing bacteria and bacteriophages were centrifuged to pellet the cells (10 min, 17,000g) and the supernatants used for bacteriophage enumeration. Serial dilutions of each sample were spotted onto a soft agar overlay (0.7% Difco agar, 1× Nutrient Broth No. 2) containing 2 × 109 colony forming units (cfu) of stationary phase Top10F′. The agar base comprised 1.4% Difco agar, 1× Nutrient Broth No. 2. Plates were incubated at 37°C overnight to allow bacterial growth and bacteriophage infection to occur. Dilutions giving rise to 3–30 plaques per 10 µL droplet were used for enumeration.

Transmission Electron Microscopy (TEM)

Bacteriophage M13 was visualized by TEM as follows. A carbon grid (carbon film supported on 3 mm, 400 mesh copper grid, Agar Scientific, Stansted, UK) was placed mesh-side down onto a 50 µL droplet of 109 pfu mL−1 caesium chloride purified bacteriophage M13 for twenty seconds and removed. The excess was removed with absorbent paper. Each grid was placed onto a 50 µL droplet of uranyl acetate (0.25% w/v) for 10–20 s to negatively stain the bacteriophages followed by sterile RO water for 1 min. The excess was removed with absorbent paper and the grid allowed to air dry. Samples were imaged by JEOL 100CX electron microscope.

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