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Purification of the Staphylococcus aureus bacteriophages VDX-10 on methacrylate monoliths
Journal of Virological Methods 166 (2010) 60–64
Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Purification of the Staphylococcus aureus bacteriophages VDX-10 on methacrylate monoliths Petra Kramberger a,∗ , Richard C. Honour b , Richard E. Herman b , Franci Smrekar a , Matjaˇz Peterka a a b
BIA Separations, d.o.o., Teslova 30, SI-1000 Ljubljana, Slovenia Viridax Corporation, 270 NW 3rd Court, Boca Raton, FL 33432-3720, USA
a b s t r a c t Article history: Received 16 October 2009 Received in revised form 11 February 2010 Accepted 18 February 2010 Available online 25 February 2010 Keywords: CIM monolithic support Phage purification Down-stream processing Ion-exchange chromatography
Bacteriophages (phages) are known to be useful in many fields from medicine to agriculture, and for a broad range of applications, including phage therapy and phage display. For some applications, especially in medicine, high purity and viability of phages are required. Methacrylate monoliths (Convective Interaction Media [CIM] monolithic columns), designed for purification of bionanoparticles, were applied for the purification of Staphylococcus aureus phages VDX-10 from bacterial lysate. With a single step purification method, more than 99% of host cell DNA and more than 90% of proteins were removed, with 60% recovery of viable phages. Comparable results were obtained when the purification method was scaledup from a CIM monolithic disk to a larger CIM monolithic column. Additionally, the dynamic binding capacity of a methacrylate monolith column for S. aureus phages VDX-10 was determined. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Bacteriophages (phages) are viruses that only infect bacteria. Following their discovery at the beginning of the previous century (Duckworth, 1976), phages were recognized as agents with antimicrobial activity. The role of phages in medicine today would probably be different if their discovery had not been coincident with the discovery of penicillin and other antibiotics. As a direct result of the introduction of antibiotics, the use of phage therapy for treating bacterial infections during the 20th century was limited to Eastern Europe. However, during the past few decades, phages have been recognized as potential tools in the fight against antibiotic resistant bacteria, as well as having potential applications in a wide range of other fields. Phages are used today as delivery vehicles for DNA and protein vaccines (Clark and March, 2004; Jepson and March, 2004; Ren et al., 2008; Wan et al., 2001), as phage display libraries for screening protein–protein and protein–DNA interactions in drug discovery (Benhar, 2001; Sergeeva et al., 2006), for delivery of genes (Kassner et al., 1999; Larocca and Baird, 2001) and for the detection and identification of bacterial strains (Balasubramanian et al., 2007; Bhowmick et al., 2007; Kumar et al., 2008). With the growing problem of antibiotic resistance leading to untreatable bacterial infections, there is renewed interest in phages
∗ Corresponding author. Tel.: +386 1 426 5649; fax: +386 1 426 5650. E-mail address: [email protected] (P. Kramberger). 0166-0934/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2010.02.020
as antimicrobial agents, not only in medicine (Bradbury, 2004; Clark and March, 2006; Donlan, 2009; Miedzybrodzki et al., 2005), but also in veterinary medicine (Wagenaar et al., 2005), the food industry (Bigwood et al., 2008; Guenther et al., 2008) and in agriculture for the control of plant pathogens (Goodridge, 2004). Phages intended for use as antimicrobial agents, especially those for human use, need to be purified of contaminants. Although different techniques can be used for the purification of biological materials, such as phages, liquid chromatography is the preferred method, as it reaches the purity required by regulatory agencies. Phages size (usually between 20 and 200 nm) dictates careful selection of an appropriate chromatographic support for their purification. For the efficient and economic purification of viruses in general, chromatographic supports should have large pore diameters to enable access to a large binding surface area, resulting in high binding capacity of the support. As a result of their structure, methacrylate monoliths have large flow-through channels, enabling convective mass transport of molecules, which leads to flow-independent dynamic binding capacity and separation (Jungbauer and Hahn, ˇ 2008; Podgornik and Strancar, 2005), making purification of virussized particles highly efficient. Methacrylate monoliths, referred to as Convective Interaction Media (CIM), have already proven to be an efficient tool for the purification, concentration and in-process control of different viruses (Boben et al., 2007; Gutiérrez-Aguirre et al., 2009; Kramberger et al., 2004, 2007; Whitfield et al., 2009), including T4 phages (Smrekar et al., 2008). The goal of this work was to develop a single step purification method for a S. aureus phages VDX-10 (VDX-10) using methacrylate monolith.
P. Kramberger et al. / Journal of Virological Methods 166 (2010) 60–64
2. Materials and methods 2.1. Bacteriophages preparation VDX-10 phages were produced in the host strain Staphylococcus aureus ATCC 19685. A 1% inoculum of the bacteria was incubated with shaking at 37 ◦ C to mid-log phase, infected with the phages at low multiplicity of infection (0.1 phage/colony forming unit), and the incubation continued with shaking at 30 ◦ C until lysis was observed. The lysate was centrifuged (10,500 × g for 10 min at 4 ◦ C) and the supernatant was passed through a 0.45 m filter and then stored at 4 ◦ C. This cleared and filtered bacterial lysate was the starting material for purification of phages VDX-10 by CIM.
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Table 1 Variability of the plaque assay method: titre of phages in a bacterial lysate determined on 9 different days and intra-day variability. Day of plaque assay
Phages titre (pfu/ml)
1 2 3 4 5 6 7 8 9 Average titre Standard deviation Intra-day variability (%)
3.0 × 108 3.4 × 108 6.0 × 108 6.8 × 108 2.5 × 108 3.5 × 108 3.3 × 108 4.3 × 108 5.6 × 108 4.3 × 108 1.5 × 108 36
2.2. HPLC, stationary and mobile phases Experiments were conducted using a Knauer (Berlin, Germany) gradient HPLC system, consisting of two K-500 pumps, a UV–VIS detector K-2500, which operated at 280 nm, and a data acquisition and control station. Additionally, a conductivity meter from Amersham Biosciences was added. All components were connected with PEEK capillary tubes (i.d. 0.75 mm). CIM monolithic disk columns (12 mm × 3 mm i.d., bed volume 0.34 ml) of two different chemistries (QA [quaternary amine] and DEAE [diethylamine]) were tested during buffer and stationary phase screening. The purification method for VDX-10 phages was optimized on a CIM QA monolithic disk and then scaled-up to a larger 8 ml CIM QA monolithic column (Do: 15 mm, Di: 1.5 mm, L: 45 mm, bed volume: 8 ml). For chromatography experiments, different mobile phases were used. The equilibration buffers tested included 20 mM sodium acetate (pH 5.5), 100 mM phosphate buffer (pH 7) and 50 mM HEPES buffer (pH 8.5). Equilibration buffers with added sodium chloride were used as elution buffers. 2.3. Viable phages particle enumeration Phages were enumerated by the plaque assay method. LB liquid medium (25 mg LB/ml deionised water) was aseptically inoculated with S. aureus at the ratio of 500:1 and incubated overnight at 37 ◦ C with shaking at 130 rpm. The overnight culture of S. aureus was then mixed with a chromatographic fraction (phages sample) in a ratio of 1:1 and added to autoclaved top agar (25 mg LB and 6 mg of agar/ml deionised water, tempered at 50 ◦ C), mixed vigorously and poured onto an LB agar plate. For each sample, several dilutions (using chromatography equilibration buffer) were prepared. Plates were incubated overnight at 37 ◦ C, plaques were counted and the phages titre (in plaque forming units/ml) determined.
and followed by RNase digestion (1 mg/ml). Genomic DNA was extracted from 2 ml of overnight bacterial culture grown in LB medium resulting in 200 l of S. aureus gDNA at a concentration of 500 g/ml. 2.6. Quantitation of S. aureus genomic DNA by qPCR A TaqMan® Staphylococcus aureus Detection Kit (Applied Biosystems) was used to detect host cell gDNA. Samples (chromatographic fractions) and standard (S. aureus gDNA) were diluted in water and stored at 4 ◦ C. Each PCR reaction consisted of 15 l 2× Environmental Master Mix, 3 l of 10× Target Assay Mix and 12 l of diluted sample. S. aureus gDNA isolated following the protocol by Flamm was used to establish a calibration curve. The concentration of gDNA was determined by measuring A260, and was found to be 500 g/ml. A ‘no template’ control (water) was also included in each run. The plate was covered with foil and centrifuged at 1000 × g for 1 min. Real-time PCR reactions were performed in a Roche LC480 real time Light Cycler under universal conditions: 10 min at 95 ◦ C followed by 45 cycles of 15 s at 95 ◦ C and 1 min at 60 ◦ C. 3. Results 3.1. Variability of the plaque assay method The plaque assay method was used for enumeration of viable phages. A cleared and filtered bacterial lysate containing VDX-10 phages was used as a positive control and was always assayed together with unknown samples. Reproducibility of the method was then evaluated by averaging the phages titre in the bacterial lysate determined on different days. The standard deviation of the method was calculated to be 36% (Table 1).
2.4. Total protein determination Total protein was determined by the Bradford Ultra Assay (Expedeon, BFU05L), according to manufacturer’s instructions. Samples (chromatographic fractions) were assayed undiluted and diluted in the chromatography equilibration buffer. Accuracy of the assay was followed by adding a known concentration of BSA to the sample (spiked sample), and the measurement was compared to the non-spiked sample. 2.5. Isolation of S. aureus genomic DNA Isolation of S. aureus genomic DNA (gDNA) was performed by the protocol described by Flamm et al. (1984) with the following changes. After the pellet was washed with SSC buffer, an additional step including lysostaphin digestion (0.1 mg/ml) was added. Proteinase K was used instead of Pronase B. Finally, nucleic acid was precipitated with 80% ethanol, resuspended with sterile water
3.2. Buffer system and monolithic support screening for purification of phages VDX-10 Three equilibration buffers were used for screening: 20 mM sodium acetate, pH 5.5; 100 mM phosphate, pH 7; and 50 mM HEPES buffer, pH 8.5. A cleared and filtered bacterial lysate containing VDX-10 phages was diluted 4 times with equilibration buffer (conductivity less than 20 mS/cm) and separated on CIM DEAE (diethylamine) and CIM QA (quaternary amine) disk monolithic columns using elution buffer in a linear gradient mode (0–1 M NaCl in 100 column volumes). Phages were enumerated in chromatographic fractions and phages recoveries were calculated. Phages recoveries of nearly 100% were achieved in three buffer–monolith combinations: CIM DEAE-phosphate buffer, CIM QA-phosphate buffer and CIM DEAE-HEPES buffer (Fig. 1). Although purification of VDX-10 phages would be possible with either of the above-mentioned conditions, the process was further optimized (as
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P. Kramberger et al. / Journal of Virological Methods 166 (2010) 60–64
Fig. 1. Screening of buffers, pH and monolithic chemistry for optimal purification of VDX-10 phages. Equilibration buffers were 20 mM SA (sodium acetate buffer), 100 mM PB (phosphate buffer), 50 mM HEPES buffer and elution buffers were equilibration buffers with 2 M NaCl. All buffer systems were tested on CIM DEAE and QA monolithic disks.
described in Section 3.3) on a CIM QA disk monolithic column using 100 mM phosphate buffer, pH 7, which resulted in high recovery of phages. 3.3. Optimization of phages VDX-10 purification on CIM QA disk monolithic column The chromatographic profile of VDX-10 phages separated in linear gradient mode, plaque assay results and conductivity profile were the parameters for estimating the elution conditions for the phages (Fig. 2). The concentration of NaCl in equilibration buffer needed for elution of phages from chromatographic support was determined to be 0.6 M. Additional washing steps were then introduced to elute contaminants before elution of the phages. Two wash steps were tested: 0.2 and 0.3 M NaCl in equilibration buffer. With 0.2 M NaCl in equilibration buffer, washing was effective and no phages were eluted from the chromatographic support. Loading conditions were also optimized by increasing the conductivity of bacterial lysate containing phages to 30 mS/cm. Purification of VDX-10 phages from the bacterial lysate using these optimized conditions is shown in Fig. 3. Host cell DNA and protein depletion during VDX-10 phages purification on the CIM QA disk monolithic column were determined by qPCR (TaqMan® Staphylococcus aureus Detection Kit) and Bradford Ultra assay (total protein quantitation). As summarized in Table 2, only 0.02% of the loaded host
Fig. 3. Purification of VDX-10 phages in step gradient mode. Conditions: CIM QA disk monolithic column loaded with 11 ml of bacterial lysate containing VDX-10 phages diluted 2× in buffer A (100 mM phosphate buffer, pH 7); wash buffer 0.3 M NaCl in buffer A; elution buffer: 0.6 M NaCl in buffer A; regeneration buffer (buffer B): 2 M NaCl in buffer A; gradient: as indicated on the figure (conductivity is presented as percentage of buffer B in mobile phase); flow rate: 4 ml/min (212 cm/h); UV detection at 280 nm.
cell DNA was recovered in the eluate, indicating greater than 99% removal, and only 10% of the loaded protein was recovered resulting in depletion greater than 90%. Meanwhile, 54% of the loaded phages were recovered in just a 3 ml volume. 3.4. Dynamic binding capacity of the CIM QA monolith for VDX-10 phages The dynamic binding capacity of a methacrylate monolith for VDX-10 phages was determined by loading 88 ml of bacterial lysate containing known concentration of VDX-10 phages, diluted with 100 mM phosphate buffer, pH 7, on a CIM QA disk monolithic column (chromatogram not shown). Flow-through fractions were collected and the phages titre in each fraction was determined. Plaque assay results showed that 84 ml of the bacterial lysate with VDX-10 phages can be loaded on CIM QA monolithic column with less than 1% of the phages in the flow-through fractions. Since the phages titre of the loaded material was 4.6 × 107 pfu/ml, the dynamic binding capacity for phages VDX-10 was determined to be 1.1 × 1010 pfu/ml of methacrylate monolith. After loading of VDX10 phages on the monolithic support, we eluted and recovered 60% of the phages with 0.6 M NaCl in equilibration buffer. 3.5. Scale-up of the purification method The purification method for VDX-10 phages developed on a CIM QA monolithic disk was transferred to a larger 8 ml CIM QA monolithic column. Since the flow rate for the disk format was 4 ml/min (212 cm/h), the same linear velocity was applied to the 8 ml CIM QA monolithic column (30 ml/min). Purification was performed at 20% of dynamic binding capacity of the methacrylate monolith for VDX-10 phages. Chromatographic fractions were analysed by the plaque assay method, Bradford Ultra and qPCR (Table 3). Phages recovery in single elution fraction was 65%. Only 0.06% of the host cell DNA eluted with phages, indicating that host DNA depletion was 99%. 9% of host cell proteins eluted with phages, indicating that protein depletion was grater than 90%. The results of VDX-10 phages purification on an 8 ml CIM QA monolithic column (Fig. 4 and Table 3) are comparable with purification of phages on a CIM QA monolithic disk (Table 2).
Fig. 2. Separation of VDX-10 phages in linear gradient mode. Conditions: CIM QA disk monolithic column was loaded with 11 ml of bacterial lysate containing VDX10 phages diluted 4× in buffer A (100 mM phosphate buffer, pH 7) and eluted using a gradient of buffer A and buffer B (2 M NaCl in buffer A) as indicated on the figure (conductivity is presented as percentage of buffer B in mobile phase) at a flow rate of 4 ml/min (212 cm/h) with UV detection at 280 nm.
4. Discussion Development of a purification method demands reproducible quantitation method of the target molecule. Accuracy of phages
P. Kramberger et al. / Journal of Virological Methods 166 (2010) 60–64
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Table 2 Purification of VDX-10 phages on CIM QA monolithic disk. Equilibrated lysate represents material loaded on CIM QA monolithic disk: bacterial lysate containing VDX-10 phages, diluted with 100 mM phosphate buffer, pH 7, to the final conductivity of 30 mS/cm.
Equilibrated lysate loaded on CIM Elution from CIM QA disk
Volume
Titre (Plaque assay)
(ml)
(pfu/ml)
11 3
1.3 × 108 2.6 × 108
Proteins (Bradford Ultra)
DNA (qPCR)
(%)
(g/ml)
(%)
(ng/ml)
(%)
54
98.2 35.5
10
23043 21
0.02
Table 3 Purification of VDX-10 phages on 8 ml CIM QA monolithic column. Equilibrated lysate represents material loaded on CIM QA monolithic column: bacterial lysate containing VDX-10 phages, diluted with 100 mM phosphate buffer, pH 7, to the final conductivity of 30 mS/cm.
Equilibrated lysate loaded on CIM Elution from CIM QA 8 ml column
Volume
Titre (Plaque assay)
Proteins (Bradford Ultra)
DNA (qPCR)
(ml)
(pfu/ml)
(%)
(g/ml)
(%)
(ng/ml)
(%)
470 22
3.4 × 10 4.7 × 108
65
25.6 48.1
9
21024 263
0.06
7
quantitation method (plaque assay) was proved to be robust and reproducible enough to be used as analytical method for determination of phages recovery during development of purification method. Purification method development for VDX-10 phages started with screening of two different chemistries (strong and weak anion exchanger) in combination with three buffer systems. Although three chromatographic support–buffer combinations resulted in very high recovery of phages, indicating that purification of VDX10 phages would be possible on either of the above-mentioned chemistries, purification method for VDX-10 was developed and optimized on a strong anion exchanger. It was suggested previously (Kalbfuss et al., 2007) that elution of virus particles from chromatographic support with lower ligand density can be more efficient. Since ligand density of CIM QA is lower then of CIM DEAE monoliths, CIM QA monolith in combination with phosphate buffer was chosen. An optimized purification method for VDX-10 enables efficient removal of host cell proteins and DNA with 60% recovery of viable phages in a single elution fraction. Phages recovery corresponds well with the results described by Smrekar et al. (2008) for purification of T4 phages on the same chromatographic support.An important feature for large scale production of any molecule is dynamic binding capacity of selected chromatographic support for the target molecule. Since dynamic binding capacity describes the amount of target molecule which will bind to a chromatographic
Fig. 4. Scale-up from disk format to 8 ml CIM QA monolithic column. Conditions: loaded material: bacterial lysate containing VDX-10 phages adjusted to 30 mS/cm; buffer A: 100 mM phosphate buffer, pH 7 (12 mS/cm); wash buffer: 0.2 M NaCl in buffer A (29 mS/cm), elution buffer: 0.6 M NaCl in buffer A (60 mS/cm) and buffer B: 2 M NaCl in buffer A (152 mS/cm); gradient: as indicated on the figure (conductivity is presented as percentage of buffer B in mobile phase); flow rate: 30 ml/min (212 cm/h); UV detection at 280 nm.
support under defined conditions dynamic binding capacity consequently determines the number of runs needed on selected chromatographic support to produce a certain amount of the target molecule and thus influences the production costs. The dynamic binding capacity of chromatographic support for a target molecule depends on the level of host cell proteins and host cell DNA in the material; the scale of down-stream process (or the number of purification runs) needed is therefore dependent and can only be determined when the up-stream process is well characterized. Dynamic binding capacity of CIM monolith for VDX-10 phages was determined by loading bacterial lysate containing VDX-10 phages under conditions optimized previously for phages purification. Dynamic binding capacity was determined to be 1.1 × 1010 pfu/ml of methacrylate monolith and even when working at 100% of dynamic binding capacity, VDX-10 phages were still bound efficiently and eluted from the chromatographic support with similar recovery. The method developed for purification of VDX-10 phages on small scale CIM monolithic columns was transferred successfully to a larger chromatographic unit (scale-up). Phages recovery, together with protein and host cell DNA depletion on both scales was comparable. The method developed on a small scale CIM monolithic column was scalable easily to a larger unit with no further optimization. Additionally, the structure of CIM monoliths allowed high flow velocities, resulting in short purification time. In the case of VDX-10 phages purification almost 0.5 l of clarified and equilibrated harvest, containing VDX-10 phages was purified in less than 25 min. All experiments were performed at a linear flow rate of 212 cm/h in order to maintain the same flow velocity at different scales. If the maximal allowed flow velocity for CIM 8 ml monolithic column (up to 290 cm/h) would be used the purification time could be even shorter. Chromatographic purification of phages is relatively new and only few attempts were made to date. In this paper chromatographic method for purification of specific phages based on methacrylate monoliths is presented. The down stream process described for VDX-10 consists of centrifugation, filtration, dilution and column purification and yields phages purified from host cell DNA and proteins. Although substantially high level of purity was achieved, an additional purification step would be needed for phages to be used for clinical application. Acknowledgement The authors would like to thank Tony Brazzale, Vice President of Business Development at BIA Separations, for his respective contribution to the project.
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Contents lists available at ScienceDirect
Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet
Purification of the Staphylococcus aureus bacteriophages VDX-10 on methacrylate monoliths Petra Kramberger a,∗ , Richard C. Honour b , Richard E. Herman b , Franci Smrekar a , Matjaˇz Peterka a a b
BIA Separations, d.o.o., Teslova 30, SI-1000 Ljubljana, Slovenia Viridax Corporation, 270 NW 3rd Court, Boca Raton, FL 33432-3720, USA
a b s t r a c t Article history: Received 16 October 2009 Received in revised form 11 February 2010 Accepted 18 February 2010 Available online 25 February 2010 Keywords: CIM monolithic support Phage purification Down-stream processing Ion-exchange chromatography
Bacteriophages (phages) are known to be useful in many fields from medicine to agriculture, and for a broad range of applications, including phage therapy and phage display. For some applications, especially in medicine, high purity and viability of phages are required. Methacrylate monoliths (Convective Interaction Media [CIM] monolithic columns), designed for purification of bionanoparticles, were applied for the purification of Staphylococcus aureus phages VDX-10 from bacterial lysate. With a single step purification method, more than 99% of host cell DNA and more than 90% of proteins were removed, with 60% recovery of viable phages. Comparable results were obtained when the purification method was scaledup from a CIM monolithic disk to a larger CIM monolithic column. Additionally, the dynamic binding capacity of a methacrylate monolith column for S. aureus phages VDX-10 was determined. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Bacteriophages (phages) are viruses that only infect bacteria. Following their discovery at the beginning of the previous century (Duckworth, 1976), phages were recognized as agents with antimicrobial activity. The role of phages in medicine today would probably be different if their discovery had not been coincident with the discovery of penicillin and other antibiotics. As a direct result of the introduction of antibiotics, the use of phage therapy for treating bacterial infections during the 20th century was limited to Eastern Europe. However, during the past few decades, phages have been recognized as potential tools in the fight against antibiotic resistant bacteria, as well as having potential applications in a wide range of other fields. Phages are used today as delivery vehicles for DNA and protein vaccines (Clark and March, 2004; Jepson and March, 2004; Ren et al., 2008; Wan et al., 2001), as phage display libraries for screening protein–protein and protein–DNA interactions in drug discovery (Benhar, 2001; Sergeeva et al., 2006), for delivery of genes (Kassner et al., 1999; Larocca and Baird, 2001) and for the detection and identification of bacterial strains (Balasubramanian et al., 2007; Bhowmick et al., 2007; Kumar et al., 2008). With the growing problem of antibiotic resistance leading to untreatable bacterial infections, there is renewed interest in phages
∗ Corresponding author. Tel.: +386 1 426 5649; fax: +386 1 426 5650. E-mail address: [email protected] (P. Kramberger). 0166-0934/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2010.02.020
as antimicrobial agents, not only in medicine (Bradbury, 2004; Clark and March, 2006; Donlan, 2009; Miedzybrodzki et al., 2005), but also in veterinary medicine (Wagenaar et al., 2005), the food industry (Bigwood et al., 2008; Guenther et al., 2008) and in agriculture for the control of plant pathogens (Goodridge, 2004). Phages intended for use as antimicrobial agents, especially those for human use, need to be purified of contaminants. Although different techniques can be used for the purification of biological materials, such as phages, liquid chromatography is the preferred method, as it reaches the purity required by regulatory agencies. Phages size (usually between 20 and 200 nm) dictates careful selection of an appropriate chromatographic support for their purification. For the efficient and economic purification of viruses in general, chromatographic supports should have large pore diameters to enable access to a large binding surface area, resulting in high binding capacity of the support. As a result of their structure, methacrylate monoliths have large flow-through channels, enabling convective mass transport of molecules, which leads to flow-independent dynamic binding capacity and separation (Jungbauer and Hahn, ˇ 2008; Podgornik and Strancar, 2005), making purification of virussized particles highly efficient. Methacrylate monoliths, referred to as Convective Interaction Media (CIM), have already proven to be an efficient tool for the purification, concentration and in-process control of different viruses (Boben et al., 2007; Gutiérrez-Aguirre et al., 2009; Kramberger et al., 2004, 2007; Whitfield et al., 2009), including T4 phages (Smrekar et al., 2008). The goal of this work was to develop a single step purification method for a S. aureus phages VDX-10 (VDX-10) using methacrylate monolith.
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2. Materials and methods 2.1. Bacteriophages preparation VDX-10 phages were produced in the host strain Staphylococcus aureus ATCC 19685. A 1% inoculum of the bacteria was incubated with shaking at 37 ◦ C to mid-log phase, infected with the phages at low multiplicity of infection (0.1 phage/colony forming unit), and the incubation continued with shaking at 30 ◦ C until lysis was observed. The lysate was centrifuged (10,500 × g for 10 min at 4 ◦ C) and the supernatant was passed through a 0.45 m filter and then stored at 4 ◦ C. This cleared and filtered bacterial lysate was the starting material for purification of phages VDX-10 by CIM.
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Table 1 Variability of the plaque assay method: titre of phages in a bacterial lysate determined on 9 different days and intra-day variability. Day of plaque assay
Phages titre (pfu/ml)
1 2 3 4 5 6 7 8 9 Average titre Standard deviation Intra-day variability (%)
3.0 × 108 3.4 × 108 6.0 × 108 6.8 × 108 2.5 × 108 3.5 × 108 3.3 × 108 4.3 × 108 5.6 × 108 4.3 × 108 1.5 × 108 36
2.2. HPLC, stationary and mobile phases Experiments were conducted using a Knauer (Berlin, Germany) gradient HPLC system, consisting of two K-500 pumps, a UV–VIS detector K-2500, which operated at 280 nm, and a data acquisition and control station. Additionally, a conductivity meter from Amersham Biosciences was added. All components were connected with PEEK capillary tubes (i.d. 0.75 mm). CIM monolithic disk columns (12 mm × 3 mm i.d., bed volume 0.34 ml) of two different chemistries (QA [quaternary amine] and DEAE [diethylamine]) were tested during buffer and stationary phase screening. The purification method for VDX-10 phages was optimized on a CIM QA monolithic disk and then scaled-up to a larger 8 ml CIM QA monolithic column (Do: 15 mm, Di: 1.5 mm, L: 45 mm, bed volume: 8 ml). For chromatography experiments, different mobile phases were used. The equilibration buffers tested included 20 mM sodium acetate (pH 5.5), 100 mM phosphate buffer (pH 7) and 50 mM HEPES buffer (pH 8.5). Equilibration buffers with added sodium chloride were used as elution buffers. 2.3. Viable phages particle enumeration Phages were enumerated by the plaque assay method. LB liquid medium (25 mg LB/ml deionised water) was aseptically inoculated with S. aureus at the ratio of 500:1 and incubated overnight at 37 ◦ C with shaking at 130 rpm. The overnight culture of S. aureus was then mixed with a chromatographic fraction (phages sample) in a ratio of 1:1 and added to autoclaved top agar (25 mg LB and 6 mg of agar/ml deionised water, tempered at 50 ◦ C), mixed vigorously and poured onto an LB agar plate. For each sample, several dilutions (using chromatography equilibration buffer) were prepared. Plates were incubated overnight at 37 ◦ C, plaques were counted and the phages titre (in plaque forming units/ml) determined.
and followed by RNase digestion (1 mg/ml). Genomic DNA was extracted from 2 ml of overnight bacterial culture grown in LB medium resulting in 200 l of S. aureus gDNA at a concentration of 500 g/ml. 2.6. Quantitation of S. aureus genomic DNA by qPCR A TaqMan® Staphylococcus aureus Detection Kit (Applied Biosystems) was used to detect host cell gDNA. Samples (chromatographic fractions) and standard (S. aureus gDNA) were diluted in water and stored at 4 ◦ C. Each PCR reaction consisted of 15 l 2× Environmental Master Mix, 3 l of 10× Target Assay Mix and 12 l of diluted sample. S. aureus gDNA isolated following the protocol by Flamm was used to establish a calibration curve. The concentration of gDNA was determined by measuring A260, and was found to be 500 g/ml. A ‘no template’ control (water) was also included in each run. The plate was covered with foil and centrifuged at 1000 × g for 1 min. Real-time PCR reactions were performed in a Roche LC480 real time Light Cycler under universal conditions: 10 min at 95 ◦ C followed by 45 cycles of 15 s at 95 ◦ C and 1 min at 60 ◦ C. 3. Results 3.1. Variability of the plaque assay method The plaque assay method was used for enumeration of viable phages. A cleared and filtered bacterial lysate containing VDX-10 phages was used as a positive control and was always assayed together with unknown samples. Reproducibility of the method was then evaluated by averaging the phages titre in the bacterial lysate determined on different days. The standard deviation of the method was calculated to be 36% (Table 1).
2.4. Total protein determination Total protein was determined by the Bradford Ultra Assay (Expedeon, BFU05L), according to manufacturer’s instructions. Samples (chromatographic fractions) were assayed undiluted and diluted in the chromatography equilibration buffer. Accuracy of the assay was followed by adding a known concentration of BSA to the sample (spiked sample), and the measurement was compared to the non-spiked sample. 2.5. Isolation of S. aureus genomic DNA Isolation of S. aureus genomic DNA (gDNA) was performed by the protocol described by Flamm et al. (1984) with the following changes. After the pellet was washed with SSC buffer, an additional step including lysostaphin digestion (0.1 mg/ml) was added. Proteinase K was used instead of Pronase B. Finally, nucleic acid was precipitated with 80% ethanol, resuspended with sterile water
3.2. Buffer system and monolithic support screening for purification of phages VDX-10 Three equilibration buffers were used for screening: 20 mM sodium acetate, pH 5.5; 100 mM phosphate, pH 7; and 50 mM HEPES buffer, pH 8.5. A cleared and filtered bacterial lysate containing VDX-10 phages was diluted 4 times with equilibration buffer (conductivity less than 20 mS/cm) and separated on CIM DEAE (diethylamine) and CIM QA (quaternary amine) disk monolithic columns using elution buffer in a linear gradient mode (0–1 M NaCl in 100 column volumes). Phages were enumerated in chromatographic fractions and phages recoveries were calculated. Phages recoveries of nearly 100% were achieved in three buffer–monolith combinations: CIM DEAE-phosphate buffer, CIM QA-phosphate buffer and CIM DEAE-HEPES buffer (Fig. 1). Although purification of VDX-10 phages would be possible with either of the above-mentioned conditions, the process was further optimized (as
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Fig. 1. Screening of buffers, pH and monolithic chemistry for optimal purification of VDX-10 phages. Equilibration buffers were 20 mM SA (sodium acetate buffer), 100 mM PB (phosphate buffer), 50 mM HEPES buffer and elution buffers were equilibration buffers with 2 M NaCl. All buffer systems were tested on CIM DEAE and QA monolithic disks.
described in Section 3.3) on a CIM QA disk monolithic column using 100 mM phosphate buffer, pH 7, which resulted in high recovery of phages. 3.3. Optimization of phages VDX-10 purification on CIM QA disk monolithic column The chromatographic profile of VDX-10 phages separated in linear gradient mode, plaque assay results and conductivity profile were the parameters for estimating the elution conditions for the phages (Fig. 2). The concentration of NaCl in equilibration buffer needed for elution of phages from chromatographic support was determined to be 0.6 M. Additional washing steps were then introduced to elute contaminants before elution of the phages. Two wash steps were tested: 0.2 and 0.3 M NaCl in equilibration buffer. With 0.2 M NaCl in equilibration buffer, washing was effective and no phages were eluted from the chromatographic support. Loading conditions were also optimized by increasing the conductivity of bacterial lysate containing phages to 30 mS/cm. Purification of VDX-10 phages from the bacterial lysate using these optimized conditions is shown in Fig. 3. Host cell DNA and protein depletion during VDX-10 phages purification on the CIM QA disk monolithic column were determined by qPCR (TaqMan® Staphylococcus aureus Detection Kit) and Bradford Ultra assay (total protein quantitation). As summarized in Table 2, only 0.02% of the loaded host
Fig. 3. Purification of VDX-10 phages in step gradient mode. Conditions: CIM QA disk monolithic column loaded with 11 ml of bacterial lysate containing VDX-10 phages diluted 2× in buffer A (100 mM phosphate buffer, pH 7); wash buffer 0.3 M NaCl in buffer A; elution buffer: 0.6 M NaCl in buffer A; regeneration buffer (buffer B): 2 M NaCl in buffer A; gradient: as indicated on the figure (conductivity is presented as percentage of buffer B in mobile phase); flow rate: 4 ml/min (212 cm/h); UV detection at 280 nm.
cell DNA was recovered in the eluate, indicating greater than 99% removal, and only 10% of the loaded protein was recovered resulting in depletion greater than 90%. Meanwhile, 54% of the loaded phages were recovered in just a 3 ml volume. 3.4. Dynamic binding capacity of the CIM QA monolith for VDX-10 phages The dynamic binding capacity of a methacrylate monolith for VDX-10 phages was determined by loading 88 ml of bacterial lysate containing known concentration of VDX-10 phages, diluted with 100 mM phosphate buffer, pH 7, on a CIM QA disk monolithic column (chromatogram not shown). Flow-through fractions were collected and the phages titre in each fraction was determined. Plaque assay results showed that 84 ml of the bacterial lysate with VDX-10 phages can be loaded on CIM QA monolithic column with less than 1% of the phages in the flow-through fractions. Since the phages titre of the loaded material was 4.6 × 107 pfu/ml, the dynamic binding capacity for phages VDX-10 was determined to be 1.1 × 1010 pfu/ml of methacrylate monolith. After loading of VDX10 phages on the monolithic support, we eluted and recovered 60% of the phages with 0.6 M NaCl in equilibration buffer. 3.5. Scale-up of the purification method The purification method for VDX-10 phages developed on a CIM QA monolithic disk was transferred to a larger 8 ml CIM QA monolithic column. Since the flow rate for the disk format was 4 ml/min (212 cm/h), the same linear velocity was applied to the 8 ml CIM QA monolithic column (30 ml/min). Purification was performed at 20% of dynamic binding capacity of the methacrylate monolith for VDX-10 phages. Chromatographic fractions were analysed by the plaque assay method, Bradford Ultra and qPCR (Table 3). Phages recovery in single elution fraction was 65%. Only 0.06% of the host cell DNA eluted with phages, indicating that host DNA depletion was 99%. 9% of host cell proteins eluted with phages, indicating that protein depletion was grater than 90%. The results of VDX-10 phages purification on an 8 ml CIM QA monolithic column (Fig. 4 and Table 3) are comparable with purification of phages on a CIM QA monolithic disk (Table 2).
Fig. 2. Separation of VDX-10 phages in linear gradient mode. Conditions: CIM QA disk monolithic column was loaded with 11 ml of bacterial lysate containing VDX10 phages diluted 4× in buffer A (100 mM phosphate buffer, pH 7) and eluted using a gradient of buffer A and buffer B (2 M NaCl in buffer A) as indicated on the figure (conductivity is presented as percentage of buffer B in mobile phase) at a flow rate of 4 ml/min (212 cm/h) with UV detection at 280 nm.
4. Discussion Development of a purification method demands reproducible quantitation method of the target molecule. Accuracy of phages
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Table 2 Purification of VDX-10 phages on CIM QA monolithic disk. Equilibrated lysate represents material loaded on CIM QA monolithic disk: bacterial lysate containing VDX-10 phages, diluted with 100 mM phosphate buffer, pH 7, to the final conductivity of 30 mS/cm.
Equilibrated lysate loaded on CIM Elution from CIM QA disk
Volume
Titre (Plaque assay)
(ml)
(pfu/ml)
11 3
1.3 × 108 2.6 × 108
Proteins (Bradford Ultra)
DNA (qPCR)
(%)
(g/ml)
(%)
(ng/ml)
(%)
54
98.2 35.5
10
23043 21
0.02
Table 3 Purification of VDX-10 phages on 8 ml CIM QA monolithic column. Equilibrated lysate represents material loaded on CIM QA monolithic column: bacterial lysate containing VDX-10 phages, diluted with 100 mM phosphate buffer, pH 7, to the final conductivity of 30 mS/cm.
Equilibrated lysate loaded on CIM Elution from CIM QA 8 ml column
Volume
Titre (Plaque assay)
Proteins (Bradford Ultra)
DNA (qPCR)
(ml)
(pfu/ml)
(%)
(g/ml)
(%)
(ng/ml)
(%)
470 22
3.4 × 10 4.7 × 108
65
25.6 48.1
9
21024 263
0.06
7
quantitation method (plaque assay) was proved to be robust and reproducible enough to be used as analytical method for determination of phages recovery during development of purification method. Purification method development for VDX-10 phages started with screening of two different chemistries (strong and weak anion exchanger) in combination with three buffer systems. Although three chromatographic support–buffer combinations resulted in very high recovery of phages, indicating that purification of VDX10 phages would be possible on either of the above-mentioned chemistries, purification method for VDX-10 was developed and optimized on a strong anion exchanger. It was suggested previously (Kalbfuss et al., 2007) that elution of virus particles from chromatographic support with lower ligand density can be more efficient. Since ligand density of CIM QA is lower then of CIM DEAE monoliths, CIM QA monolith in combination with phosphate buffer was chosen. An optimized purification method for VDX-10 enables efficient removal of host cell proteins and DNA with 60% recovery of viable phages in a single elution fraction. Phages recovery corresponds well with the results described by Smrekar et al. (2008) for purification of T4 phages on the same chromatographic support.An important feature for large scale production of any molecule is dynamic binding capacity of selected chromatographic support for the target molecule. Since dynamic binding capacity describes the amount of target molecule which will bind to a chromatographic
Fig. 4. Scale-up from disk format to 8 ml CIM QA monolithic column. Conditions: loaded material: bacterial lysate containing VDX-10 phages adjusted to 30 mS/cm; buffer A: 100 mM phosphate buffer, pH 7 (12 mS/cm); wash buffer: 0.2 M NaCl in buffer A (29 mS/cm), elution buffer: 0.6 M NaCl in buffer A (60 mS/cm) and buffer B: 2 M NaCl in buffer A (152 mS/cm); gradient: as indicated on the figure (conductivity is presented as percentage of buffer B in mobile phase); flow rate: 30 ml/min (212 cm/h); UV detection at 280 nm.
support under defined conditions dynamic binding capacity consequently determines the number of runs needed on selected chromatographic support to produce a certain amount of the target molecule and thus influences the production costs. The dynamic binding capacity of chromatographic support for a target molecule depends on the level of host cell proteins and host cell DNA in the material; the scale of down-stream process (or the number of purification runs) needed is therefore dependent and can only be determined when the up-stream process is well characterized. Dynamic binding capacity of CIM monolith for VDX-10 phages was determined by loading bacterial lysate containing VDX-10 phages under conditions optimized previously for phages purification. Dynamic binding capacity was determined to be 1.1 × 1010 pfu/ml of methacrylate monolith and even when working at 100% of dynamic binding capacity, VDX-10 phages were still bound efficiently and eluted from the chromatographic support with similar recovery. The method developed for purification of VDX-10 phages on small scale CIM monolithic columns was transferred successfully to a larger chromatographic unit (scale-up). Phages recovery, together with protein and host cell DNA depletion on both scales was comparable. The method developed on a small scale CIM monolithic column was scalable easily to a larger unit with no further optimization. Additionally, the structure of CIM monoliths allowed high flow velocities, resulting in short purification time. In the case of VDX-10 phages purification almost 0.5 l of clarified and equilibrated harvest, containing VDX-10 phages was purified in less than 25 min. All experiments were performed at a linear flow rate of 212 cm/h in order to maintain the same flow velocity at different scales. If the maximal allowed flow velocity for CIM 8 ml monolithic column (up to 290 cm/h) would be used the purification time could be even shorter. Chromatographic purification of phages is relatively new and only few attempts were made to date. In this paper chromatographic method for purification of specific phages based on methacrylate monoliths is presented. The down stream process described for VDX-10 consists of centrifugation, filtration, dilution and column purification and yields phages purified from host cell DNA and proteins. Although substantially high level of purity was achieved, an additional purification step would be needed for phages to be used for clinical application. Acknowledgement The authors would like to thank Tony Brazzale, Vice President of Business Development at BIA Separations, for his respective contribution to the project.
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