European Journal of Pharmaceutical Sciences 67 (2015) 119–125
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps
Chromatographic purification of adenoviral vectors on anion-exchange resins Huaben Bo a, Jun Chen b, Ting Liang a, Senhai Li a, Hongwei Shao a, Shulin Huang a,⇑ a School of Bioscience and Biopharmaceutics, Guangdong Province Key Laboratory for Biotechnology Drug Candidates, Guangdong Pharmaceutical University, 510006 Guangzhou, Guangdong, PR China b College of Pharmacy, Guangdong Pharmaceutical University, 510006 Guangzhou, Guangdong, PR China
a r t i c l e
i n f o
Article history: Received 21 May 2014 Received in revised form 14 November 2014 Accepted 14 November 2014 Available online 26 November 2014 Keywords: Anion-exchange chromatography Adenoviral vector Gene therapy Vaccines
a b s t r a c t Anion-exchange chromatography is a useful and effective tool for adenoviral vectors purification. However, due to the different functional groups and matrices, both binding capacity and resolution of most AEC resins are different. In this study, four different AEC resins are compared by the binding capacity, resolution and recovery. Using Fractogel TMAE as an adsorbent to purify adenoviral vectors has obvious advantages over the other resins, namely (1) dynamic binding capacity is higher than other resins; (2) unprecedented sharpness (1,570,000 ± 250,000) and symmetry of adenoviral vectors peak (1.67 ± 0.06); (3) higher resolution with other contaminants (2.16 ± 0.08); (4) no enzymatic treatment; (5) the recovery can reach 75%; (6) the purity is higher and the total virion to infectious particle ratios can reach 18.9. In the present work, we confirmed the possibility of purifying pharmaceutical-grade adenoviral vectors by AEC. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Adenoviral vectors (adenovectors) are currently the most attractive vehicle for gene therapy, oncolytic virotherapy, and vaccination because of their wide cell tropism in quiescent and nonquiescent cells, the poor ability to integrate the host genome and the high production titers obtained in culture (Dormond et al., 2009). As of 2013, 23.4% of all gene therapy clinical trials utilized adenovirus as a vector (Anonymous, 2014), with the majority of trials being associated with cancer gene therapy. To meet the needs for clinical trials, scalable and robust production processes that allow maintenance of viral infectivity, high recovery of infectious particles, and removal of host cell DNA, removal of host cell proteins are required. CsCl-density gradient ultracentrifugation can be used to rapidly purify adenovectors in sufficient quantities for research purposes (Green and Pina, 1963). However, difficulties of scaling up production, costs and toxicity of CsCl limit its large-scale application. The use of chromatographic steps, instead of the traditional CsCl gradient purification, facilitates process scale-up (Kamen and Henry,
⇑ Corresponding author. Tel.: +86 20 39352199; fax: +86 20 39352201. E-mail address: [email protected] (S. Huang). http://dx.doi.org/10.1016/j.ejps.2014.11.004 0928-0987/Ó 2014 Elsevier B.V. All rights reserved.
2004; Lusky, 2005). Techniques include: anion exchange (Green et al., 2002; Blanche et al., 2000; Goerke et al., 2005; Konz et al., 2005; Vellekamp et al., 2001; Huyghe et al., 1995; Eglon et al., 2009; Peixoto et al., 2006), hydrophobic interaction (Vellekamp et al., 2001; Huyghe et al., 1995), size exclusion (Vellekamp et al., 2001; Huyghe et al., 1995; Peixoto et al., 2006), immobilized metal chelate (Vellekamp et al., 2001; Huyghe et al., 1995), etc. In most processes described in the literature, AEC is the ‘‘heart’’ of the process as it offers the advantages of rapid separation, no solvent requirement, sanitization with sodium hydroxide and a wide selection of industrial media. However the currently available chromatographic matrices have pore dimensions (typically 30–80 nm). Adenovirus is a non-enveloped 70–90 nm double stranded DNA virus (Albinana-Gimenez et al., 2009). So the adenovectors are too big to diffuse into the pores. As a result, both the dynamic binding capacities (DBC) and purification efficiencies using conventional resins are compromised. This drawback can be circumvented by using tentacle supports, membrane adsorbers, and monoliths (Wolf and Reichl, 2011; Segura et al., 2011; Nestola et al., 2014). In this paper, we have an attempt to better understand the column performances, dynamic binding capacity, resolution and recovery of adenovectors on various AEC resins including conventional resin and tentacle supports. So that we can find an AEC resin that meets the needs of large-scale purification of adenovectors.
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H. Bo et al. / European Journal of Pharmaceutical Sciences 67 (2015) 119–125
2. Materials and methods 2.1. Materials The adenovector (Ad5-GFP) used in this study which is a replication competent recombinant human adenovirus type 5 (Ad5) expressing green fluorescent protein (GFP) was constructed in our laboratory. HEK-293 cells were purchased from ATCC. AEC resins were purchased as follows: HiTrap Q XL 7/25 and Mono Q™ 5/50 GL from Amersham Bioscience, Fractogel TMAE, Fractogel DMAE and Fractogel DEAE scout column (1 ml) from Merck. AKTA Basic system and Frac-920 fraction collector were all purchased from Amersham Bioscience. Beckman Avanti J-25 centrifuge was provided by Beckman Co. Tianli gel scanner was purchased from Tianli Biotech. Dulbecco’s modified Eagle medium (DMEM) and Fetal bovine serum (FBS) were from Invitrogen. Other reagents used were of analytical-reagent grade or with equivalent purity. 2.2. Cell culture and virus propagation HEK 293 cells were cultivated in DMEM supplemented with 10% FBS at 37 °C and a 5% CO2 atmosphere. Cells were seeded in 15-cm plate. When the cell monolayers reached approximately 70–80% confluence by microscopic observation, adenovirus was thoroughly mixed with fresh media and added to the cells at a multiplicity of infection (MOI) of 10 pfu/cell and medium exchange at time of infection. 2.3. Adenovirus harvest and clarification Infected cells were collected with the aid of a scraper when substantial cytopathic effect was observed 2–3 days after infection. The cells were centrifuged at 3000 g for 10 min at 4 °C. The supernatants were discarded and the pellets were resuspended in 5 ml of buffer A [40 mM Tris, 2 mM MgCl2, 5% glycerol (v/v), pH 8.0] per 15-cm plate. The concentrated cells were vortexed and lysed by three consecutive freeze–thaw cycles. Cell lysates were clarified to remove cellular debris by centrifugation at 3000 g for 10 min at 4 °C. Adenovirus contained in the supernatant was filtered through 0.45 lm filter and pooled with the buffer A to obtain a homogeneous adenovirus stock for purification experiments. 2.4. Preparative anion-exchange chromatography All chromatography purifications were performed on an AKTA Basic low-pressure liquid chromatography system. The system is equipped with an UV, conductivity and pH meters and controlled by UNICORN software. Absorbance at 280 nm and 260 nm was measured on-line. The running flow rate was 0.5 ml/min (78 cm/ h) for all runs. For resin screening experiments, conventional media (Q Sepharose XL) and tentacle support (Fractogel TMAE, Fractogel DMAE and Fractogel DEAE) were packed into a 7/25 column to a final volume of 1 ml. All columns were equilibrated with five column volumes (CV) of buffer A. Up to 0.5 ml of the filtered crude lysate was loaded on each column. After washing columns with 5 CV of buffer A, a 20 CV gradient of 0 to 1 M NaCl in buffer A was applied to purify adenovirus. Fractions containing the adenovirus peak and the flow-through were collected using Frac 920 fraction collector and pooled for purity and activity analysis. To preliminarily assess the ability of the best resin (Fractogel TMAE) to resolve the impurities, we adjusted the buffer A pH value to 6.0, 7.0, 8.0 and 9.0. Other conditions remained the same. The dynamic binding capacities of Q Sepharose XL were estimated by increasing flow rate (0.1 and 0.5 ml/min) in independent
runs. Flow-through fractions were collected, pooled, and analyzed by biochemical analysis. All columns were then regenerated with 4 CV of 1 M NaCl in buffer A, sanitized with 5 CV of 0.5 M NaOH followed by 5 CV reequilibration in buffer A. Height Equivalent to a Theoretical Plate (HETP) values at 50% peak height, asymmetry (As) values at 10% peak height and resolution were calculated automatically with UNICORN software. Column efficiencies were expressed as N/m, the number of theoretical plates per linear meter of column. 2.5. Analytical methods 2.5.1. Biochemical analysis GFP expression in HEK 293 cells was determined by fluorescence microscope. HEK 293 cells were seeded in a 96-well plate at a density of 1 104 cells/well. After 24 h, 10 ll of collected fractions were added to the wells and incubated at 37 °C and a 5% CO2 atmosphere for 24 h. GFP expression was revealed under a fluorescence microscope. 2.5.2. Quantification of infective adenovector by TCID50 assay HEK 293 cells were seeded at a density of 1 104 cells/well in a 96-well plate and cultivated in DMEM supplemented with 5% FBS. Test samples were prepared by serial log-fold dilution in the same medium. At 70–80% confluence, cells were infected across rows of a 96-well plate. Each well was checked for cytopathic effect (CPE) 10 days after infection and scored as positive or negative. The number of infectious units (IU) per ml was calculated using the following formula: Titer (IU/ml) = 10(s+1.5), where ‘‘s’’ is the sum of the fractions of CPE-positive wells in each row. 2.5.3. Analytical chromatography Adenovector purity was determined by anion-exchange HPLC on a Mono Q™ 5/50 GL column. Chromatography was performed on an AKTA Basic low-pressure liquid chromatography system. The column was equilibrated with 2 CV of loading buffer [40 mM Tris, 2 mM MgCl2, 0.3 M NaCl, 5% glycerol (v/v), pH 8.0] at a flow rate of 0.5 ml/min. Injection of 100 ll of sample was followed by a linear gradient of 0.3 M to 1 M NaCl for 10 CV. The column was cleaned with 2 CV of 0.5 M NaOH. 2.5.4. Spectrophotometry analysis The concentration of adenovector was determined by optical density at 260 nm (OD260). Samples was denatured with an equal volume of 0.1% of SDS and incubated at room temperature for 1 h. The optical density of the samples was detected with spectrophotometer (Thermo). The total physical viral particle was calculated using the conversion factor of 1.1 1012 (Maizel et al., 1968). 2.5.5. SDS–PAGE analysis Samples were mixed with 2 SDS–PAGE loading buffer to approximately 30 ll, boiled for 5 min, spun for 10 min at 30,000 rpm in an Eppendoff centrifuge and separated by 10% SDS– PAGE for 2 h at 25 mA. Gels were stained using the silver method. 3. Results 3.1. Column performances In the course of this study, four AEC resins with different matrices and functional groups were evaluated for their performances with adenovectors particles. The identification of the peak as adenovectors relied on Biochemical analysis. Namely the elution fractions were used to infect HEK 293 cells later analyzed by fluorescence
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Fig. 1. Elution profile of adenovectors on a 1 ml Q Sepharose XL (A), Fractogel TMAE (B), Fractogel DEAE (C) and Fractogel DMAE (D) column equilibrated with 5 CV of buffer A. Up to 0.5 ml of the filtered crude lysate was loaded on each column. After washing columns with 5 CV of buffer A, a 20 CV gradient of 0–1 M NaCl in buffer A was applied to purify adenovirus. Fractions were collected using Frac 920 fraction collector and pooled for biochemical analysis. Inset, GFP expression was revealed under a fluorescence microscope.
Table 1 Comparative column performances of several anion-exchange resin for adenovirus (data are expressed as means ± standard deviations). Chromatography adsorbent
Retention time (min)
Efficiency (N/m)
Asymmetry (As)
Resolution (R)
Q Sepharose XL Fractogel TMAE Fractogel DMAE Fractogel DEAE
36.2 34.2 32.1 35.5
377,000 ± 26,000 1,570,000 ± 250,000 90,000 ± 14,000 264,000 ± 24,000
1.24 ± 0.11 1.67 ± 0.06 3.7 ± 0.43 1.62 ± 0.15
0.95 ± 0.11 2.16 ± 0.08 0.96 ± 0.04 1.21 ± 0.02
microscopy (Fig. 1, inset). Results showed that the adenovectors retention time at different resins were different (Fig. 1, Table 1), and the adenovectors peak purified by Fractogel TMAE was more sharp than by other resins (Fig. 1).
The chromatographic performance of four AEC resins were evaluated by column efficiency (N/m), peak asymmetry (As) and resolution. Both column efficiency and resolution of Fractogel TMAE were significantly better (1,570,000 ± 250,000 and 2.16 ± 0.08,
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1.15 1.25 1.14 1.21 75 91 76 74 53 75 33 68 20.3 18.9 17.5 19.8
Ratio (OD260/ OD280) Purity by HPLC Recovery (%) Infectious particle ratio (vp/IU)
1.3 1011 1.7 1011 0.7 1011 1.6 1011 0.64 1010 0.9 1010 0.4 1010 0.81 1010 0.8 1010 1.8 1010 0.4 1010 0.9 1010 1.2 1010 1.2 1010 1.2 1010 1.2 1010 0.5 0.5 0.5 0.5 Q Sepharose XL Fractogel TMAE Fractogel DMAE Fractogel DEAE
2.4 1010 2.4 1010 2.4 1010 2.4 1010
0.8 0.5 1 0.9
Total virus output (vp) Total infectious output (pfu) Output infectious titer (pfu/ml) Total infectious input (pfu) Input volume (ml)
Input infectious titer (pfu/ml)
Output volume (ml)
Chromatography adsorbent
Dynamic capacity (pfu adenovectors/ml gel)
Q Sepharose XL Fractogel TMAE Fractogel DMAE Fractogel DEAE
1.2 1010
Contents lists available at ScienceDirect
European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps
Chromatographic purification of adenoviral vectors on anion-exchange resins Huaben Bo a, Jun Chen b, Ting Liang a, Senhai Li a, Hongwei Shao a, Shulin Huang a,⇑ a School of Bioscience and Biopharmaceutics, Guangdong Province Key Laboratory for Biotechnology Drug Candidates, Guangdong Pharmaceutical University, 510006 Guangzhou, Guangdong, PR China b College of Pharmacy, Guangdong Pharmaceutical University, 510006 Guangzhou, Guangdong, PR China
a r t i c l e
i n f o
Article history: Received 21 May 2014 Received in revised form 14 November 2014 Accepted 14 November 2014 Available online 26 November 2014 Keywords: Anion-exchange chromatography Adenoviral vector Gene therapy Vaccines
a b s t r a c t Anion-exchange chromatography is a useful and effective tool for adenoviral vectors purification. However, due to the different functional groups and matrices, both binding capacity and resolution of most AEC resins are different. In this study, four different AEC resins are compared by the binding capacity, resolution and recovery. Using Fractogel TMAE as an adsorbent to purify adenoviral vectors has obvious advantages over the other resins, namely (1) dynamic binding capacity is higher than other resins; (2) unprecedented sharpness (1,570,000 ± 250,000) and symmetry of adenoviral vectors peak (1.67 ± 0.06); (3) higher resolution with other contaminants (2.16 ± 0.08); (4) no enzymatic treatment; (5) the recovery can reach 75%; (6) the purity is higher and the total virion to infectious particle ratios can reach 18.9. In the present work, we confirmed the possibility of purifying pharmaceutical-grade adenoviral vectors by AEC. Ó 2014 Elsevier B.V. All rights reserved.
1. Introduction Adenoviral vectors (adenovectors) are currently the most attractive vehicle for gene therapy, oncolytic virotherapy, and vaccination because of their wide cell tropism in quiescent and nonquiescent cells, the poor ability to integrate the host genome and the high production titers obtained in culture (Dormond et al., 2009). As of 2013, 23.4% of all gene therapy clinical trials utilized adenovirus as a vector (Anonymous, 2014), with the majority of trials being associated with cancer gene therapy. To meet the needs for clinical trials, scalable and robust production processes that allow maintenance of viral infectivity, high recovery of infectious particles, and removal of host cell DNA, removal of host cell proteins are required. CsCl-density gradient ultracentrifugation can be used to rapidly purify adenovectors in sufficient quantities for research purposes (Green and Pina, 1963). However, difficulties of scaling up production, costs and toxicity of CsCl limit its large-scale application. The use of chromatographic steps, instead of the traditional CsCl gradient purification, facilitates process scale-up (Kamen and Henry,
⇑ Corresponding author. Tel.: +86 20 39352199; fax: +86 20 39352201. E-mail address: [email protected] (S. Huang). http://dx.doi.org/10.1016/j.ejps.2014.11.004 0928-0987/Ó 2014 Elsevier B.V. All rights reserved.
2004; Lusky, 2005). Techniques include: anion exchange (Green et al., 2002; Blanche et al., 2000; Goerke et al., 2005; Konz et al., 2005; Vellekamp et al., 2001; Huyghe et al., 1995; Eglon et al., 2009; Peixoto et al., 2006), hydrophobic interaction (Vellekamp et al., 2001; Huyghe et al., 1995), size exclusion (Vellekamp et al., 2001; Huyghe et al., 1995; Peixoto et al., 2006), immobilized metal chelate (Vellekamp et al., 2001; Huyghe et al., 1995), etc. In most processes described in the literature, AEC is the ‘‘heart’’ of the process as it offers the advantages of rapid separation, no solvent requirement, sanitization with sodium hydroxide and a wide selection of industrial media. However the currently available chromatographic matrices have pore dimensions (typically 30–80 nm). Adenovirus is a non-enveloped 70–90 nm double stranded DNA virus (Albinana-Gimenez et al., 2009). So the adenovectors are too big to diffuse into the pores. As a result, both the dynamic binding capacities (DBC) and purification efficiencies using conventional resins are compromised. This drawback can be circumvented by using tentacle supports, membrane adsorbers, and monoliths (Wolf and Reichl, 2011; Segura et al., 2011; Nestola et al., 2014). In this paper, we have an attempt to better understand the column performances, dynamic binding capacity, resolution and recovery of adenovectors on various AEC resins including conventional resin and tentacle supports. So that we can find an AEC resin that meets the needs of large-scale purification of adenovectors.
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2. Materials and methods 2.1. Materials The adenovector (Ad5-GFP) used in this study which is a replication competent recombinant human adenovirus type 5 (Ad5) expressing green fluorescent protein (GFP) was constructed in our laboratory. HEK-293 cells were purchased from ATCC. AEC resins were purchased as follows: HiTrap Q XL 7/25 and Mono Q™ 5/50 GL from Amersham Bioscience, Fractogel TMAE, Fractogel DMAE and Fractogel DEAE scout column (1 ml) from Merck. AKTA Basic system and Frac-920 fraction collector were all purchased from Amersham Bioscience. Beckman Avanti J-25 centrifuge was provided by Beckman Co. Tianli gel scanner was purchased from Tianli Biotech. Dulbecco’s modified Eagle medium (DMEM) and Fetal bovine serum (FBS) were from Invitrogen. Other reagents used were of analytical-reagent grade or with equivalent purity. 2.2. Cell culture and virus propagation HEK 293 cells were cultivated in DMEM supplemented with 10% FBS at 37 °C and a 5% CO2 atmosphere. Cells were seeded in 15-cm plate. When the cell monolayers reached approximately 70–80% confluence by microscopic observation, adenovirus was thoroughly mixed with fresh media and added to the cells at a multiplicity of infection (MOI) of 10 pfu/cell and medium exchange at time of infection. 2.3. Adenovirus harvest and clarification Infected cells were collected with the aid of a scraper when substantial cytopathic effect was observed 2–3 days after infection. The cells were centrifuged at 3000 g for 10 min at 4 °C. The supernatants were discarded and the pellets were resuspended in 5 ml of buffer A [40 mM Tris, 2 mM MgCl2, 5% glycerol (v/v), pH 8.0] per 15-cm plate. The concentrated cells were vortexed and lysed by three consecutive freeze–thaw cycles. Cell lysates were clarified to remove cellular debris by centrifugation at 3000 g for 10 min at 4 °C. Adenovirus contained in the supernatant was filtered through 0.45 lm filter and pooled with the buffer A to obtain a homogeneous adenovirus stock for purification experiments. 2.4. Preparative anion-exchange chromatography All chromatography purifications were performed on an AKTA Basic low-pressure liquid chromatography system. The system is equipped with an UV, conductivity and pH meters and controlled by UNICORN software. Absorbance at 280 nm and 260 nm was measured on-line. The running flow rate was 0.5 ml/min (78 cm/ h) for all runs. For resin screening experiments, conventional media (Q Sepharose XL) and tentacle support (Fractogel TMAE, Fractogel DMAE and Fractogel DEAE) were packed into a 7/25 column to a final volume of 1 ml. All columns were equilibrated with five column volumes (CV) of buffer A. Up to 0.5 ml of the filtered crude lysate was loaded on each column. After washing columns with 5 CV of buffer A, a 20 CV gradient of 0 to 1 M NaCl in buffer A was applied to purify adenovirus. Fractions containing the adenovirus peak and the flow-through were collected using Frac 920 fraction collector and pooled for purity and activity analysis. To preliminarily assess the ability of the best resin (Fractogel TMAE) to resolve the impurities, we adjusted the buffer A pH value to 6.0, 7.0, 8.0 and 9.0. Other conditions remained the same. The dynamic binding capacities of Q Sepharose XL were estimated by increasing flow rate (0.1 and 0.5 ml/min) in independent
runs. Flow-through fractions were collected, pooled, and analyzed by biochemical analysis. All columns were then regenerated with 4 CV of 1 M NaCl in buffer A, sanitized with 5 CV of 0.5 M NaOH followed by 5 CV reequilibration in buffer A. Height Equivalent to a Theoretical Plate (HETP) values at 50% peak height, asymmetry (As) values at 10% peak height and resolution were calculated automatically with UNICORN software. Column efficiencies were expressed as N/m, the number of theoretical plates per linear meter of column. 2.5. Analytical methods 2.5.1. Biochemical analysis GFP expression in HEK 293 cells was determined by fluorescence microscope. HEK 293 cells were seeded in a 96-well plate at a density of 1 104 cells/well. After 24 h, 10 ll of collected fractions were added to the wells and incubated at 37 °C and a 5% CO2 atmosphere for 24 h. GFP expression was revealed under a fluorescence microscope. 2.5.2. Quantification of infective adenovector by TCID50 assay HEK 293 cells were seeded at a density of 1 104 cells/well in a 96-well plate and cultivated in DMEM supplemented with 5% FBS. Test samples were prepared by serial log-fold dilution in the same medium. At 70–80% confluence, cells were infected across rows of a 96-well plate. Each well was checked for cytopathic effect (CPE) 10 days after infection and scored as positive or negative. The number of infectious units (IU) per ml was calculated using the following formula: Titer (IU/ml) = 10(s+1.5), where ‘‘s’’ is the sum of the fractions of CPE-positive wells in each row. 2.5.3. Analytical chromatography Adenovector purity was determined by anion-exchange HPLC on a Mono Q™ 5/50 GL column. Chromatography was performed on an AKTA Basic low-pressure liquid chromatography system. The column was equilibrated with 2 CV of loading buffer [40 mM Tris, 2 mM MgCl2, 0.3 M NaCl, 5% glycerol (v/v), pH 8.0] at a flow rate of 0.5 ml/min. Injection of 100 ll of sample was followed by a linear gradient of 0.3 M to 1 M NaCl for 10 CV. The column was cleaned with 2 CV of 0.5 M NaOH. 2.5.4. Spectrophotometry analysis The concentration of adenovector was determined by optical density at 260 nm (OD260). Samples was denatured with an equal volume of 0.1% of SDS and incubated at room temperature for 1 h. The optical density of the samples was detected with spectrophotometer (Thermo). The total physical viral particle was calculated using the conversion factor of 1.1 1012 (Maizel et al., 1968). 2.5.5. SDS–PAGE analysis Samples were mixed with 2 SDS–PAGE loading buffer to approximately 30 ll, boiled for 5 min, spun for 10 min at 30,000 rpm in an Eppendoff centrifuge and separated by 10% SDS– PAGE for 2 h at 25 mA. Gels were stained using the silver method. 3. Results 3.1. Column performances In the course of this study, four AEC resins with different matrices and functional groups were evaluated for their performances with adenovectors particles. The identification of the peak as adenovectors relied on Biochemical analysis. Namely the elution fractions were used to infect HEK 293 cells later analyzed by fluorescence
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Fig. 1. Elution profile of adenovectors on a 1 ml Q Sepharose XL (A), Fractogel TMAE (B), Fractogel DEAE (C) and Fractogel DMAE (D) column equilibrated with 5 CV of buffer A. Up to 0.5 ml of the filtered crude lysate was loaded on each column. After washing columns with 5 CV of buffer A, a 20 CV gradient of 0–1 M NaCl in buffer A was applied to purify adenovirus. Fractions were collected using Frac 920 fraction collector and pooled for biochemical analysis. Inset, GFP expression was revealed under a fluorescence microscope.
Table 1 Comparative column performances of several anion-exchange resin for adenovirus (data are expressed as means ± standard deviations). Chromatography adsorbent
Retention time (min)
Efficiency (N/m)
Asymmetry (As)
Resolution (R)
Q Sepharose XL Fractogel TMAE Fractogel DMAE Fractogel DEAE
36.2 34.2 32.1 35.5
377,000 ± 26,000 1,570,000 ± 250,000 90,000 ± 14,000 264,000 ± 24,000
1.24 ± 0.11 1.67 ± 0.06 3.7 ± 0.43 1.62 ± 0.15
0.95 ± 0.11 2.16 ± 0.08 0.96 ± 0.04 1.21 ± 0.02
microscopy (Fig. 1, inset). Results showed that the adenovectors retention time at different resins were different (Fig. 1, Table 1), and the adenovectors peak purified by Fractogel TMAE was more sharp than by other resins (Fig. 1).
The chromatographic performance of four AEC resins were evaluated by column efficiency (N/m), peak asymmetry (As) and resolution. Both column efficiency and resolution of Fractogel TMAE were significantly better (1,570,000 ± 250,000 and 2.16 ± 0.08,
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1.15 1.25 1.14 1.21 75 91 76 74 53 75 33 68 20.3 18.9 17.5 19.8
Ratio (OD260/ OD280) Purity by HPLC Recovery (%) Infectious particle ratio (vp/IU)
1.3 1011 1.7 1011 0.7 1011 1.6 1011 0.64 1010 0.9 1010 0.4 1010 0.81 1010 0.8 1010 1.8 1010 0.4 1010 0.9 1010 1.2 1010 1.2 1010 1.2 1010 1.2 1010 0.5 0.5 0.5 0.5 Q Sepharose XL Fractogel TMAE Fractogel DMAE Fractogel DEAE
2.4 1010 2.4 1010 2.4 1010 2.4 1010
0.8 0.5 1 0.9
Total virus output (vp) Total infectious output (pfu) Output infectious titer (pfu/ml) Total infectious input (pfu) Input volume (ml)
Input infectious titer (pfu/ml)
Output volume (ml)
Chromatography adsorbent
Dynamic capacity (pfu adenovectors/ml gel)
Q Sepharose XL Fractogel TMAE Fractogel DMAE Fractogel DEAE
1.2 1010
