Purification and concentration of mycobacteriophage D29 using monolithic chromatographic columns

Journal of Virological Methods 186 (2012) 7–13

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Purification and concentration of mycobacteriophage D29 using monolithic chromatographic columns Keyang Liu a,b , Zhanbo Wen a,b , Na Li a,b , Wenhui Yang a,b , Lingfei Hu a,b , Jie Wang a,b , Zhe Yin a,b , Xiaokai Dong a,b , Jinsong Li a,b,∗ a State Research Center for Bio protective Equipment & Engineering Technology, State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, China b Department of Biosafety, Beijing Institute of Microbiology and Epidemiology, 20# Dongda Street, 100071 Fengtai District, Beijing, China

a b s t r a c t Article history: Received 13 February 2012 Received in revised form 10 July 2012 Accepted 11 July 2012 Available online 20 July 2012 Keywords: CIM monolithic support Mycobacteriophage D29 Phage purification Polyethylene glycol 8000 Ammonium sulphate

Bacteriophages are used widely in many fields, and phages with high purity and infectivity are required. Convective interaction media (CIM) methacrylate monoliths were used for the purification of mycobacteriophage D29. The lytic phages D29 from bacterial lysate were purified primarily by polyethylene glycol 8000 or ammonium sulphate, and then the resulting phages were passed through the CIM monolithic columns for further purification. After the whole purification process, more than 99% of the total proteins were removed irrespective which primary purification method was used. The total recovery rates of viable phages were around 10–30%. Comparable results were obtained when the purification method was scaled-up from a 0.34 mL CIM DEAE (diethylamine) monolithic disk to an 8 mL CIM DEAE monolithic column. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Phages are viruses that infect bacteria. D’Herelle (1926) first used the phage to control outbreak of avian typhosis among farmed chickens. But the research was abandoned after antibiotics were discovered (Ho, 2001; Summers, 2001). There has been renewed interest in phage therapy because of the emergence of multidrug resistant bacterial infections (Chanishvili et al., 2001; Sharp, 2001). Phages are also used for other purposes, such as phage display (Benhar, 2001; Kassner et al., 1999; Larocca and Baird, 2001; Sergeeva et al., 2006), for DNA and protein vaccines delivery (Clark and March, 2004; Jepson and March, 2004; Ren et al., 2008; Wan et al., 2001), and phage bacteria typing (Balasubramanian et al., 2007; Bhowmick et al., 2007; Kumar et al., 2008). Phages intended for use need to be purified to a high level preserving high infectivity. D29, a lytic phage, can form visible plaques after overnight incubation on a lawn of fast-growing Mycobacterium smegmatis. In the present work, D29 phages were purified by column chromatography. Currently available chromatography supports are mainly designed for protein purification with pore

∗ Corresponding author at: Department of Biosafety, Beijing Institute of Microbiology and Epidemiology, 20# Dongda Street, 100071 Fengtai District, Beijing, China. Tel.: +86 10 66948559; fax: +86 10 63865693. E-mail address: [email protected] (J. Li). 0166-0934/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jviromet.2012.07.016

diameters adjusted to the protein size (Tyn and Gusek, 1990). However, chromatography columns for virus purification should have large pore diameters to enable access to a large binding surface area, which results in a high binding capacity (Kramberger et al., 2010). An appropriate chromatography support should be selected for phage purification. Recently, convective interaction media (CIM) methacrylate monoliths have been proven to be an efficient tool for purification and concentration of different viruses including phages (Boben et al., 2007; Gutiérrez-Aguirre et al., 2009; Kramberger et al., 2004, 2007, 2010; Smrekar et al., 2008; Whitfield et al., 2009). The aim of the study was to develop a purification method for mycobacteriophage D29 using CIM monolithic columns. 2. Material and methods 2.1. Bacteriophage preparation The bacterial host M.smegmatis mc2 155 cells was propagated in 7H9 broth (Difco Middlebrook 7H9 broth; BD, USA) for 60 h. Approximately 2 × 104 plaque forming units (pfu) D29 phages were mixed with 1 mL bacteria stock and about 13 mL 7H9 top agar (containing 0.7% agar) (Michael et al., 1998). The mixture was plated on a 150 mm petri dish containing 7H10 agar (Difco Middlebrook 7H10 broth; BD, USA). Twenty such plates were prepared and incubated overnight at 37 ◦ C. Both 7H9 and 7H10 broths contained 10% (vol/vol) oleic acid–albumin–dextrose–catalase (OADC; BBL

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Middlebrook OADC Enrichment; BD, USA) which was added when temperature of the medium was below 60 ◦ C. After cultivation, the plates showed confluent lysis. Phage particles were collected by the addition of 10 mL phage buffer (containing 100 mM NaCl, 8.5 mM MgSO4 ·7H2 O, 50 mM Tris·Cl (pH 7.5), and 0.01% gelatin) to the surface of each plate. After six hours incubation at 4 ◦ C, the phagecontaining buffer was pipetted off the plates, and then filtered through a 0.45 ␮m pore size filter. The resulting phage solution was kept at 4 ◦ C.

2.2. Viable phages particle enumeration The titer of phage was determined by plaque assay (Adams, 1959). The result is expressed in plaque forming units. The phage suspension was diluted serially 10-fold with phage buffer, and each 100 ␮L phage solution was mixed with 300 ␮L M. smegmatis cells and 3 mL 7H9 top agar. The mixtures were plated on 90 mm petri dishes and incubated at 37 ◦ C. After 16–20 h cultivation, PFU counting was done on plates containing between 30 and 300 plaques.

2.5. HPLC, stationary and mobile phases All experiments were conducted using an AKTATM prime plus system (GE, USA), consisting of two pumps, a UV detector, which operated at 280 nm, and an injection valve with 5 mL sample loop. All components were connected with polyether ether keton (PEEK) capillary tubes. The anion exchange methacrylate-based CIM DEAE (diethylamine) disk monolithic column (BIA Separations) was used for the experiments. The purification method for D29 phages was optimized on a 0.34 mL CIM DEAE monolithic disk (12 mm × 3 mm i.d., bed volume 0.34 mL) and then scaled-up to a larger 8 mL CIM DEAE monolithic column (Do: 15 mm, Di: 1.5 mm, L: 45 mm, bed volume: 8 mL). The columns were sanitized periodically by circulating 1 M NaOH through it. For chromatography experiments, the phage buffer (pH 7.5) mentioned in Section 2.1 was used as an equilibration buffer. The buffer has the function of phage preservation and dilution (Sambrook et al., 1989). Elution buffers were prepared by adding sodium chloride to equilibration buffers to final molarity of 2 M, followed by adjusting pH of the buffers to 7.5 using NaOH. 2.6. Total protein determination

2.3. Determination of phage infectivity at different NaCl conditions Different NaCl molarity of phage solution (0.1 M, 0.5 M and 1 M) was obtained by dissolving appropriate salt to phage buffer. Phage solutions were kept at 4 ◦ C for 4 h, 12 h, 24 h and 7 d, respectively. Phage infectivity at each time point was determined with plaque assay technique.

2.4. Preliminary purification of phage D29 2.4.1. Preliminary purification of the phage by polyethylene glycol (PEG) precipitation The 100 mL of phage suspension was precipitated by PEG 8000 as described by Sambrook (1989). First, sodium chloride was added to the suspension to 1 mol/L. After 1 h incubation at 0 ◦ C, phage particles were precipitated by the addition of PEG 8000 to 10%, followed by incubation at 0 ◦ C for at least one hour. After the centrifugation at 11,000 × g for 15 min at 4 ◦ C, the phage-containing pellet was resuspended by 10 mL phage buffer. The phage particles were separated out by rinsing the precipitate repeatedly. Precipitate was broken down by blowing and sucking the mixture gently using wide-bore pipette tips and was then stored at 4 ◦ C for 24 h. Afterwards, the mixture was centrifuged at 6000 × g for 15 min at 4 ◦ C. The phage-remaining in the supernatant was stored at 4 ◦ C, and the precipitate was resuspended again by 10 mL phage buffer. The whole process was repeated for four times. Finally, all the incubated phage-containing suspensions were placed together, followed by filteration through a 0.45 ␮m filter, and then stored at 4 ◦ C. The resulting phage solution was the starting material for purification by CIM column.

2.4.2. Preliminary purification of the phage by ammonium sulphate precipitation The 100 mL of phage suspension was precipitated by the addition of ammonium sulphate to 60% saturation at 0 ◦ C. After 2 h incubation at 0 ◦ C, the suspension was centrifuged at 20,000 × g for 30 min at 4 ◦ C. The precipitate was resuspended in 20 mL phage buffer. The suspension was shaken by hand, and then stored at 4 ◦ C for 12 h to dissolve the precipitate completely. The phagecontaining suspension was then filtered through 0.45 ␮m filter and stored at 4 ◦ C. The resulting phage solution was the starting material for purification by the CIM column.

Total protein was determined by the BCA (bicinchoninic acid) assay (Cwbio, Beijing, China), according to the manufacturer’s instructions using bovine serum albumin (BSA) as a standard. 3. Results 3.1. Effect of NaCl concentration on phage D29 infectivity An important limitation during purification of biological materials is stability under different conditions. When ion exchange chromatography is used, phage infectivity under different NaCl conditions has to be determined. In this experiment, phages D29 have been exposed to different NaCl concentrations for 4, 12, 24, and 7 d, respectively. The results showed that the phage was essentially stable in the NaCl concentration between 0.1 and 1 M when stored at 4 ◦ C (data not shown). The phage D29 had infectivity retention of almost 100% up to 7 days in all NaCl concentrations. 3.2. Variability of plaque assay The plaque assay was used for enumeration of viable phages. In order to determine variability of the plaque assay for phage D29, ten plates with the same sample having appropriate dilution were inoculated. This procedure was repeated twice and one result is shown in Fig. 1. Standard deviations were found to be 33% and 27%, respectively. The average value is 30%. The method was proved to be robust and reproducible, and can be used as an analytical method for evaluation during the development of purification methods. 3.3. Comparisons of methods for primary purification of the phage The recovery rates of PEG 8000 and ammonium sulphate precipitation for phage D29 were 68.9% and 81.2%, respectively. Taking into account that variability of the plaque assay method is around 30%, it can be concluded that the recovery rates of the two methods are comparable. The protein removal capacity of the two methods is shown in Table 1. 3.4. Optimization of purification method using a CIM DEAE disk monolithic column After primary purification, a portion of the resulting phage suspension (1 mL) was diluted four times with equilibration buffer

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Table 1 Primary purification of D29 phages in bacterial lysate. Volume (mL)

Titer (Plaque assay) (pfu/mL)

Phage incubation PEG precipitation Ammonium sulphate precipitation

100 35 20

Fig. 1. Determination of the plaque assay repeatability. 10 plates were inoculated with the same sample, and the phage titer was determined for each plate according to the plaque assay method.

(conductivity of diluted sample close to that of equilibration buffer) and separated on the 0.34 mL CIM DEAE disk monolithic column using elution buffer in a linear gradient mode (0–2 M NaCl in 10 column volumes). The phage was enumerated in chromatographic fractions and the recovery rates were calculated. The result is shown in Fig. 2. Most phages were eluted in the second peak. The

3.3 × 109 6.5 × 109 1.34 × 1010

Proteins (BCA assay) (%)

(␮g/mL)

depletion (%)

68.94 81.21

3559.44 139.56 477.89

98.63 97.31

volume of phage suspension eluted from the 0.34 mL monolithic column is around 1.5 mL. The chromatographic profile of D29 phages separated in linear gradient mode, plaque assay results and the conductivity profile were parameters used to optimize the elution conditions. The stepwise gradient elutions, where narrower peaks are achieved and fraction collection is easier, were performed. Four stepwise gradient modes which consist of three steps were introduced to determine the best elution condition, 0.4 M–0.7 M–2 M NaCl, 0.5 M–0.8 M–2 M NaCl, 0.6 M–0.9 M–2 M NaCl, and 0.6 M–1 M–2 M NaCl. Collected fractions were analyzed via plaque assays to determine the presence of infective phage particles. The results are shown in Fig. 3. With 1 M NaCl in elution buffer, most phages were eluted from the chromatographic support. With 0.6 M NaCl in elution buffer, a sharp peak came up with few phages eluted, and can be performed as the washing step. Therefore, the optimal step gradient elution is determined to be 0.6 M–1 M–2 M NaCl. The data are summarized in Table 2. Host cell protein depletion during D29 phages purification on the CIM DEAE monolithic columns was determined by the BCA assay (total protein quantitation). As the data summarized in Table 3, the total protein depletion after two cycles of purification is approximately more than 99%. 3.5. Scale-up of the purification method The purification method for D29 phages developed on a 0.34 CIM DEAE disk monolithic column was transferred successfully to a larger 8 mL CIM DEAE monolithic column without further optimization. The stepwise gradient mode (0.6 M–1 M–2 M) was

Fig. 2. Linear gradient elution of phages D29 on the 0.34 mL monolithic column. Conditions: mobile phase: buffer A (phage buffer, pH 7.5); buffer B (phage buffer with 2 M NaCl, pH 7.5); stationary phase: CIM DEAE disk monolithic column. Flow rate: 2 mL/min; gradient, 0–100% B (10 column volumes). Sample: phage suspension diluted 4× in buffer A; injection volume: 5 mL; detection: UV at 280 nm.

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Fig. 3. Purification of D29 phages in the step gradient mode. Conditions: mobile phase: buffer A (phage buffer, pH 7.5); buffer B (phage buffer with 2 M NaCl, pH 7.5); stationary phase: 0.34 mL CIM DEAE disk monolithic columns. Flow rate: 3 mL/min; injection volume: 5 mL; detection: UV at 280 nm. (a) Sample: phage suspensions (purified by ammonium sulphate); gradient: stepwise consisting four steps: 0% B (loading), 30% B (wash), 50% B (elution), 100% B (regeneration); (b) Sample: phage suspensions (purified by polyethylene glycol 8000); gradient: stepwise consisting four steps: 0% B (loading), 30% B (wash), 50% B (elution), 100% B (regeneration).

applied to the 8 mL CIM DEAE column with the flow rate of 30 mL/min corresponding to the linear velocity of 212 cm/h. The result is shown in Fig. 4. The washing step with 0.6 M NaCl in equilibration buffer was introduced to elute contaminants and phage elution took place with 1 M NaCl in equilibration

buffer. The volume of eluted phage suspension is around 10 mL no matter how much sample is loaded. Chromatographic fractions were analyzed by the plaque assay method, BCA assay. Phage recovery and protein depletion rates are summarized in Table 4.

Table 2 Fine purification of D29 phages on 0.34 mL CIM DEAE disk monolithic column. Primary purified by

Volume (mL)

Titer (Plaque assay)

Equilibrated sample loaded on CIM

PEG Ammonium sulphate

5 5

5 × 108 6.67 × 108

Elution from CIM DEAE disk

PEG Ammonium sulphate

1.5 1.5

4.9 × 108 3.3 × 108

(pfu/mL)

Proteins (BCA assay) (%)

(␮g/mL)

Depletion (%)

10.74 13.54 29.4 14.84

<20 <20

>44.11 >55.68

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Fig. 4. Purification of D29 phages in the step gradient mode. Conditions: mobile phase: buffer A (phage buffer, pH 7.5); buffer B (phage buffer with 2 M NaCl, pH 7.5); stationary phase: 8 mL CIM DEAE monolithic columns. Flow rate: 30 mL/min; injection volume: 50 mL; detection: UV at 280 nm. (a) Sample: phage suspensions (purified by ammonium sulphate); gradient: stepwise consisting four steps: 0% B (loading), 30% B (wash), 50% B (elution), 100% B (regeneration); (b) Sample: phage suspensions (purified by polyethylene glycol 8000); gradient: stepwise consisting four steps: 0% B (loading), 30% B (wash), 50% B (elution), 100% B (regeneration).

4. Discussion In order to obtain infective phages during and after purification, the effect of different salt concentrations on the infectivity of D29 phages was investigated. This information is necessary to

Table 3 Protein removing rates after two cycles of purification for D29 phages. Chromatographic support

Primary purified by

Total protein depletion (%)

0.34 mL CIM DEAE disk

PEG Ammonium sulphate

>99.23 >98.81

8 mL CIM DEAE monolith

PEG Ammonium sulphate

>99.16 >98.99

adjust the chromatographic method accordingly to avoid undesired phage inactivation during and after processing. In parallel, we also investigated reproducibility of the phage assay method to be able to evaluate determined amount of infective phages properly. Considerable impurities are contained in phage suspensions pipetted from plates after cultivation, i.e. mycolic acid. Some impurities may have influence on the purification process by the CIM monolithic column. When a large volume of phage suspension passed through the 8 mL CIM DEAE monolithic column, impurities may block up channels of the chromatographic support, and increase the back-pressure of the HPLC system a lot, and few phages are eluted. To avoid this, phages were purified and concentrated in the present study by two methods before column chromatography, PEG 8000 and ammonium sulphate precipitation. The recovery rates of the two methods are comparable. The difference in

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Table 4 Fine purification of D29 phages on 8 mL CIM DEAE monolithic column. Primary purified by

Volume (mL)

Titer (Plaque assay) (pfu/mL)

Equilibrated sample loaded on CIM

PEG Ammonium sulphate

Elution from CIM DEAE monolith

PEG Ammonium sulphate

50 49 8.5 9

protein removal capacity is only 1%, but the corresponding protein quantity is nearly 4.6 mg. This result is consistent with Trépanier et al. (1981), who reported that the protein content of PEG concentrates was much lower than that of ammonium sulphate. PEG precipitation was the normal preparation method for CsCl density gradient centrifugation (Sambrook et al., 1989). Usually, PEG was extracted by chloroform after precipitation. However, most D29 phages lost infectivity during the extraction process if chloroform which is detrimental to the phage is used. In the present work, phages precipitated by PEG were separated by rinsing the precipitate repeatedly with the phage buffer. The whole process took four days, but the resulting phage suspensions still contain dissociative and associative PEG molecules. The ammonium sulphate precipitation method, which is commonly used for protein purification and virus concentration, was also employed in this study for the phage D29 purification (Asenjo and Andrews, 2011; Caul et al., 1978; Raweeritha and Ratanabanangkoon, 2003; Takemori et al., 2012; Trépanier et al., 1981; Yang et al., 1993; Ziai et al., 1988). As previous work described (Yang et al., 1993; Ziai et al., 1988), most phages were precipitated with 60% ammonium sulphate. Although PEG precipitation performed better in protein depletion, ammonium sulphate precipitation is more rapid and easier to perform. Further purification was developed on the 0.34 mL CIM DEAE disk monolithic column. Most phages were eluted with 1 M NaCl in elution buffer. The elution condition for phages D29 is completely different from other phages, Escherichia coli bacteriophage T4 and Staphylococcus aureus bacteriophage VDX-10. The phage elutions take place in 0.5 M and 0.6 M NaCl concentration steps for phages T4 and VDX-10 on CIM QA disk monolithic columns, respectively (Kramberger et al., 2010; Smrekar et al., 2008). The host cell protein depletion was determined with the commercial kit. The working range of the BCA protein assay kit is 20–2000 ␮g/mL, but the measured protein content in purified sample is below the lower limit. Therefore, the protein depletion was expressed as the form of greater than the rate calculated by the concentration of 20 ␮g/mL. Although the protein depletion rate after primary purification is as high as 97% or 98% (Table 1), further purification is necessary because the remaining protein contents are still hundreds of ␮g per mL. The method developed on small scale CIM monolithic columns for purification of D29 phages was transferred successfully to a larger chromatographic unit. Taking into account the variability of plaque assay method, the phage recovery and protein depletion are comparable with purification of D29 phages on a 0.34 mL CIM DEAE disk monolithic column. For the 8 mL monolithic column, the phage recovery could be improved if the sample loadings on the monolith could be higher.

5. Conclusions In the present study, D29 phages were purified and concentrated in two steps. First, phages were precipitated by PEG 8000 and ammonium sulphate. Compared with PEG precipitation, ammonium sulphate precipitation is rapid and easy to perform. After primary purification, the resulting phages were passed through

Proteins (BCA assay) (%)

2.6 × 108 2.68 × 108 5.4 × 108 6.25 × 108

(␮g/mL)

Depletion (%)

5.58 9.75 35.31 42.83

<20 <20

>39.1 >62.33

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