Differential interactions of plasmid DNA, RNA and endotoxin with immobilised and free metal ions

Journal of Chromatography A, 1141 (2007) 226–234

Differential interactions of plasmid DNA, RNA and endotoxin with immobilised and free metal ions Lihan Tan a , Wen-Bin Lai b , Chew Tin Lee c , Duck Sang Kim d , Woo-Seok Choe a,e,∗ a

Department of Chemical and Biomolecular Engineering, National University of Singapore, 10 Kent Ridge Crescent, 117576 Singapore b SingVax Pte Ltd., 11 Biopolis Way, Helios #04-03/04, 138667 Singapore c University of Malaya, Malaysia d Sartorius Korea Biotech, Republic of Korea e Department of Chemical Engineering, Sungkyunkwan University, Suwon 440-746, Republic of Korea Received 30 August 2006; received in revised form 2 December 2006; accepted 5 December 2006 Available online 20 December 2006

Abstract Separation of negatively charged molecules, such as plasmid DNA (pDNA), RNA and endotoxin forms a bottleneck for the development of pDNA vaccine production process. The use of affinity interactions of transition metal ions with these molecules may provide an ideal separation methodology. In this study, the binding behaviour of pDNA, RNA and endotoxin to transition metal ions, either in immobilised or free form, was investigated. Transition metal ions: Cu2+ , Ni2+ , Zn2+ , Co2+ and Fe3+ , typically employed in the immobilised metal affinity chromatography (IMAC), showed very different binding behaviour depending on the type of metal ions and their existing state, i.e. immobilised or free. In the alkaline cell lysate, pDNA showed no binding to any of the IMAC chemistries tested whereas RNA interacted significantly with Cu2+ -iminodiacetic acid (IDA) and Ni2+ -IDA but showed no substantial binding to the rest of the IMAC chemistries. pDNA and RNA, however, interacted to varying degrees with free metal ions in the solution. The greatest selectivity in terms of pDNA and RNA separation was achieved with Zn2+ which enabled almost full precipitation of RNA while keeping pDNA soluble. For both immobilised and free metal ions, ionic strength of solution affected the metal ion-nucleic acid interaction significantly. Endotoxin, being more flexible, was able to interact better with the immobilised metal ions than the nucleic acids and showed binding to all the IMAC chemistries. The specific interactions of immobilised and/or free metal ions with pDNA, RNA and endotoxin showed a good potential, by selectively removing RNA and endotoxin at high efficiency, to develop a simplified pDNA purification process with improved process economics. © 2006 Elsevier B.V. All rights reserved. Keywords: Plasmid DNA vaccine; RNA; Endotoxin; IMAC; Metal ion precipitation

1. Introduction Plasmid DNA (pDNA) vaccine comprises gene of an antigen inserted into a bacterial plasmid [1]. Following pDNA vaccination, the bacterial plasmid enables in situ expression of protein antigen which primes humoral (antibody) and cellular (T cell) immune responses against the antigenic organism [2]. Some benefits of the pDNA vaccine over conventional vaccine include stability and potential life-long immunity against multiple diseases in a single inoculation [3]. The specifications of pDNA for therapeutic applications are as follows: appearance (clear, colourless

∗

Corresponding author. Tel.: +82 31 290 7344; fax: +82 31 290 7272. E-mail address: [email protected] (W.-S. Choe).

0021-9673/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2006.12.023

solution), plasmid homogeneity (>90% supercoiled), proteins (not detectable, BCA (<5 ␮g/ml)), RNA (not detectable, 0.8% agarose gel), genomic DNA (gDNA) (<0.05 ␮g/␮g plasmid, PCR) and endotoxin (<0.1 EU/␮g plasmid, LAL assay) [4,5]. Current purification processes for pDNA typically comprise several unit operations including cell harvest, lysis, cell debris/solid separation, affinity precipitation, adsorption, buffer exchange and polishing steps prior to attaining pDNA suitable for therapeutic applications [6], thus rendering the processes time and cost ineffective. With gene therapies and pDNA vaccines moving towards the stage of market approval [7], largescale, intensified plasmid purification process must be in place. One of the major impurities in pDNA production using E. coli host cell is endotoxin. Endotoxin, also named lipopolysaccharide (LPS), forms part of the outer cell membrane of

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gram-negative bacteria and generally comprises three subunits: O-specific chain (i.e. bacteria strain-specific repeating oligosaccharide units), core oligosaccharide and a lipid moiety (lipid A) [8], with exceptions of certain gram-negative bacteria strains (e.g. E. coli K12) which lack the O-specific chain [9]. Lipid A, the endotoxic carrier of endotoxin (e.g. induction of fever and shock) [8], consists of both anionic phosphoric acid and hydrophobic lipophilic regions [10]. Endotoxin removal by harnessing the hydrophobic interaction between Polymyxin B and lipid A was reported [11]. However, Polymyxin B-endotoxin interaction is not selective since positively charged amino groups of Polymyxin B interacted indiscriminately with phosphate backbone of pDNA, hence resulting in low pDNA recovery (∼50%) despite the 200–104 fold reduction in endotoxin level from pDNA preparation [9,12,13]. Besides, excessively long processing time (∼16 h) probably due to the slow binding kinetics between Polymyxin B and endotoxin makes the polymyxin-based process less attractive to implement [9,14]. Other methods utilising the interaction of histamine or histidine with endotoxin exhibited low endotoxin clearance in the presence of negatively charged proteins (e.g. BSA) [15], thus not so effective for the direct purification of pDNA from the alkaline cell lysate. Various types of chromatography, based on differences in size, charge, hydrophobicity and affinity of different molecules in a mixture, have been employed to purify pDNA [16]. Sizeexclusion chromatography (SEC) has limited capacity and selectivity for pDNA [17] and hence is not preferred as an initial pDNA purification step. SEC is more ideally used as a finishing step for separation of supercoiled pDNA from open circular pDNA and residual contaminants (e.g. gDNA and RNA) [5]. For anion-exchange chromatography (AEC), selectivity towards pDNA or impurities (e.g. RNA, gDNA and endotoxin) is poor due to their non-specific binding to the anion-exchange resin [16], thus AEC is often used in series with other purification processes, such as SEC [18]. Hydrophobic interaction chromatography (HIC), on the other hand, will only capture pDNA in high salt condition (e.g. 3 M ammonium sulphate) as hydrophobicity of pDNA is low [19] while reverse-phase chromatography (RPC), which also exploits the hydrophobic interaction between target molecules and resin, requires organic solvents for elution of pDNA [17]. The use of organic solvents which are often toxic is a major disadvantage for RPC. Largescale purification of pDNA using affinity chromatography, such as immobilised metal affinity chromatography (IMAC), on the contrary, is largely unexplored [16]. IMAC harnesses the affinity interactions between the metal ions (immobilised on the support through the chelating compound) and the target molecules, thus enabling high-efficiency separation of the target molecules from other components present in a mixture [20]. It was reported that IMAC exhibited potential for removal of RNA and damaged pDNA from the alkaline cell lysate [21]. The use of IMAC for endotoxin removal was also reported elsewhere. For instance, Ni2+ -nitrilotriacetic acid (NTA) in His-tagged protein purification [22] and Cu2+ -iminodiacetic acid (IDA) in Fv antibody purification [23] showed removal of endotoxin from crude preparations.

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Besides the chromatographic procedures, selective precipitation of pDNA or impurities (e.g. RNA) has often been used for the purification of pDNA. For example, specific pDNA precipitation using poly(N,N’-dimethyldiallylammonium) chloride (∼80% plasmid recovery and ∼95% RNA removal) [24] or spermine [25] was employed to purify pDNA from the alkaline cell lysate. Selective precipitation of RNA in high salt condition (e.g. 1.5 M sodium sulphate, 2 M ammonium acetate, 1.5 M tri-potassium citrate, 1.5 M ammonium sulphate and 1.5 M calcium chloride) was also reported [26]. Among which, calcium chloride showed the highest efficiency in RNA precipitation (i.e. 100% plasmid recovery, 84.6% RNA removal and 91% endotoxin removal) [26] but it was not efficient in precipitating low molecular weight RNA (<0.5 kb) and endotoxin, thus requiring additional purification step, such as chromatography for the removal of residual RNA and endotoxin. In this study, the feasibility of using IMAC for purification of pDNA from alkaline cell lysate with simultaneous removal of RNA and endotoxin was explored. For this purpose, the binding behaviour of pDNA, RNA and endotoxin in solutions of different chemistry including the alkaline cell lysate was investigated with various transition metal ions (i.e. Cu2+ , Ni2+ , Zn2+ , Co2+ and Fe3+ ) immobilised on commercially available IMAC resins (i.e. IDA-Chelating Sepharose Fast Flow, Ni-NTA Agarose and TALON Metal Affinity Resin). The interactions of pDNA and RNA to the same metal ions, as used in the IMAC but in the free form, were also studied to assess the possibility of employing free metal ions for selective precipitation of nucleic acids or impurities. 2. Experimental All experiments were conducted at room temperature unless otherwise stated. 2.1. Materials IDA-Chelating Sepharose Fast Flow (Amersham, 17-057501), Ni-NTA Agarose (Qiagen, 30210) and TALON Metal Affinity Resin (BD Biosciences Clontech, 635502) were the IMAC resins employed. Reagents used were 2-amino2-hydroxymethyl-1,3-propanediol, also named tris (Numi, Singapore), sodium acetate trihydrate (Riedel-de Haen, 32318), sodium chloride (HCS, S6001-1-1000), cobalt (II) chloride hexahydrate (Avocado, 7791-13-1), EDTA disodium dihydrate (Duchefa Biochemie, E0511.0500), Seakem LE Agarose (Cambrex, 50004), ethidium bromide solution (Bio-rad, 1610433), 1 kb DNA ladder (Promega, G5711) and Blue/Orange Loading Dye 6x (Promega, G1881). Baker’s yeast RNA (R6750), purified endotoxin (L4524), cupric chloride dihydrate (C6641), nickel chloride hexahydrate (N5756), zinc chloride (Z0152) and ferric chloride hexahydrate (F1513) were from Sigma. SnakeSkin Pleated Dialysis Tubing (68035) was supplied by PIERCE and QIAprep Spin Miniprep Kit (27104) (miniprep) by Qiagen.

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2.2. Fermentation DH5-␣ mutant (originating from E. coli strain K12) harbouring plasmid pcDNA3.1D, a 7.3 kb high copy plasmid (100–200/cell) consisting of 1.8 kb dengue fever antigenic gene (NS3), was kindly provided by Bioprocessing Technology Institute, Singapore. Cells were grown on 12 g/l yeast extract (BD BBL 211929), 6 g/l tryptone (BD Bacto), 5 g/l glucose (Sigma, G8270), 6 g/l K2 HPO4 (US Biological, P5100) and 0.48 g/l MgSO4 (Sigma, M7506) using a 30 l fermenter (B. Braun Biotech International). Filter-sterilised ampicillin was added to a final concentration of 100 ␮g/ml immediately before inoculation (400 ml of flask culture grown for 16 h at 37 ◦ C, 250 rpm using a shaking incubator (New Brunswick Scientific Co. Inc.)). A 20 l working volume was used and the fermenter was operated at 37 ◦ C, 350 rpm, pH 7 and airflow of 20 standard litre per min (slpm). The fermentation was terminated at a final OD600 of approximately 11 (10 h after inoculation). 2.3. Alkaline cell lysis The harvested cell culture was centrifuged at 11000 × g and 4 ◦ C for 45 min (Beckman J2-21M, Rotor JA-10). Cell pellets obtained from 250 ml of cell culture were resuspended in 100 ml Buffer 1 (50 mM tris-Cl, 10 mM EDTA, pH 8), followed by cell pellet disruptions using 100 ml Buffer 2 (200 mM NaOH, 1% (w/v) SDS) and the mixture gently inverted (6x) before 5 min incubation. Subsequently, 100 ml neutralising Buffer 3 (3 M potassium acetate (P1147, Sigma), adjusted to pH 5.5 using glacial acetic acid (Merck)) was added and the mixture gently inverted (6x) before 30 min incubation on ice. The mixture was then centrifuged as above and the supernatant (alkaline cell lysate) collected. 2.4. Interactions of pDNA, RNA and endotoxin with 15 IMAC chemistries IDA-Chelating Sepharose Fast Flow, Ni-NTA Agarose and TALON Metal Affinity Resin were each washed with water (2 × 5 settled resin volumes (SV)) and stripped of any chelated metal ions with 50 mM EDTA (5 SV). The resins were then: washed with water (5 × 10 SV); charged with 100 mM metal chloride (2 SV); washed with water (5 × 5 SV); and finally, equilibrated with Acetate-NaCl Buffer (20 mM sodium acetate, 0.75 M NaCl, pH 5) (3 × 5 SV). Chelating compounds, IDA, NTA and carboxymethylated aspartate (CM-Asp), on the above resins were charged with Cu2+ , Ni2+ , Zn2+ , Co2+ and Fe3+ , giving a total of 15 IMAC chemistries. Centrifugation was performed at 700 × g for 1 min (Denville, 260D) and the supernatant removed after each mixing of solution with the resin. 0.5 SV of alkaline cell lysate was then mixed with the resin and incubated on a mixer at 10 rpm for 10 min (intelli-mixer MyLab, SLRM-2M). The adsorbent was then removed by centrifugation as above and the collected supernatant assayed for nucleic acids and endotoxin by gel electrophoresis and LAL test, respectively. The above procedures were repeated with miniprep purified pDNA, Baker’s yeast RNA, pDNA-RNA mixture and puri-

fied endotoxin in Tris-NaCl Buffer (20 mM tris, 0.75 M NaCl, pH 7) or Tris Buffer (20 mM tris, pH 7). Concentrations of pDNA, RNA and endotoxin used were 0.017, 1.00 mg/ml and 700 EU/ml, respectively. After equilibrating the resins with TrisNaCl or Tris Buffer, 0.5 SV of the pDNA, RNA, pDNA-RNA mixture or endotoxin was mixed with the resins. The supernatants were assayed for nucleic acids or endotoxin by gel electrophoresis or LAL test, respectively. 2.5. Interactions of pDNA and RNA with free metal ions: Solubility profiles Miniprep purified pDNA and Baker’s yeast RNA were mixed separately with various metal chloride solutions (Cu-, Ni-, Zn-, CoCl2 , FeCl3 ) to obtain final concentrations of 0.05, 0.10, 0.25, 0.50, 0.75, 1.00, 1.25 M for metal chlorides, 1.85 mg/ml for RNA and 0.017 mg/ml for pDNA in Tris-NaCl Buffer, Tris Buffer or Cell Lysis Buffer (mixture of Buffers 1, 2 and 3). Each mixture was incubated on the mixer at 10 rpm for 15 min, centrifuged (14000 × g, for 1 min) and the collected supernatant assayed for nucleic acid by gel electrophoresis. 2.6. Analytical methods 2.6.1. pDNA and RNA measurements For assay of pDNA or RNA content, 1% agarose gel electrophoresis was conducted (100 V, 40 min) in TAE Buffer (40 mM tris-acetate, 1 mM EDTA, pH 8) using Sub-Cell GT Agarose Gel Electrophoresis System (Bio-rad). The gel was then imaged by Gene Genius Bio-imaging System using GeneSnap (SynGene) and pDNA/RNA quantified with pDNA/RNA standard using GeneTools (SynGene). Concentration of miniprep purified pDNA or Baker’s yeast RNA was measured at 260 nm by UV spectrometer (UV-mini 1240, Shimadzu) and used as the pDNA/RNA standard. 2.6.2. Endotoxin measurement Endotoxin reagents used were Limulus Amebocyte Lysate (LAL) Endosafe KTA2 (R19000), Control Standard Endotoxin (E110), Endosafe LAL Reagent Water (W110) and 100 mM Tris Buffer (BT103) from Charles River Laboratories. The samples for endotoxin analysis were prepared in sterile polystyrene culture tubes (Falcon 2057). Endotoxin measurements were performed at 37 ◦ C and absorbance wavelength of 340 nm for 1 h using a microplate reader (Tecan Sunrise). Time taken to reach 0.05 OD (optical density unit) for each sample was recorded and endotoxin concentration calculated according to manufacturer’s instructions. 3. Results and discussion 3.1. Interactions of pDNA, RNA and endotoxin with immobilised metal ions 3.1.1. Affinity of pDNA, RNA and endotoxin in alkaline cell lysate for immobilised metal ions IMAC chemistries consisting of different metal ions and chelating compounds were tested with the alkaline cell lysate

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Fig. 1. Batch binding test of alkaline cell lysate using IDA charged with various metal ions. Lanes: 1, DNA ladder; 2, alkaline cell lysate; 3, supernatant from uncharged resin; 4, supernatant from Cu2+ -IDA; 5, supernatant from Ni2+ -IDA; 6, supernatant from Zn2+ -IDA; 7, supernatant from Co2+ -IDA; 8, supernatant from Fe3+ -IDA.

(0.091 mg/ml pDNA, 2.47 mg/ml RNA, 105 EU/ml endotoxin). Cu2+ , Ni2+ , Zn2+ , Co2+ and Fe3+ were the immobilised metal ions studied herein; IDA (Amersham, 17-0575-01), NTA (Qiagen, 30210) and CM-Asp (BD Biosciences Clontech, 635502) [27], which were bound to 6% agarose support, were the chelating compounds investigated. Among the 15 IMAC chemistries, all combinations displayed no affinity to pDNA (Fig. 1, lanes 4–8, Table 1) in the alkaline cell lysate whereas only Cu2+ IDA and Ni2+ -IDA exhibited almost complete removal of RNA (Fig. 1, lanes 4 and 5). However, all IMAC chemistries showed high endotoxin removal (>99%) from the alkaline cell lysate. Differential interactions of pDNA and RNA with the immobilised metal ions could be attributed to the properties of nucleic acids, metal ions and chelating compounds. First, the inaccessibility of unexposed aromatic nitrogen bases in pDNA to the

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immobilised metal ions [21] might hinder interaction between pDNA and the metal ions, thus pDNA did not show affinity for any IMAC combination in the alkaline cell lysate. In contrast, the exposure of aromatic nitrogen bases in RNA was reported to facilitate interaction of RNA with the immobilised metal ions [21]. Second, the affinity of metal ions towards RNA however varies with metal ion type. Metal ions are divided based on their reactivities towards nucleophiles into three categories: hard, intermediate and soft [28]. Hard metal ions (Fe3+ , Ca2+ , Al3+ ) show a preference for oxygen; intermediate metal ions (Cu2+ , Ni2+ , Zn2+ , Co2+ ), often employed in IMAC, prefer aromatic nitrogen, oxygen and sulphur; soft metal ions (Cu+ , Hg2+ , Ag+ ) prefer sulphur. For intermediate metal ions, the affinity towards electron rich groups follows the order: Cu2+ > Ni2+ > Zn2+ ∼Co2+ [29]. Hence, intermediate metal ions, in particular Cu2+ and Ni2+ , possibly show higher affinity to aromatic nitrogen bases in RNA as compared to other metal ions. Third, affinity capture of a given target by IMAC is also known to be affected by the metal ion-chelating compound combination [20]. Among the chelating compounds used in the present study, IDA is tridentate while NTA and CM-Asp are tetradentates. This means that for six coordination number metal ion, 3 and 2 coordination sites, respectively, remain available to interact with the surrounding molecules. Stronger interactions with other molecules are expected for metal ion chelated by tridentate rather than tetradentate due to the availability of an extra metal ion coordination site. The binding affinity of RNA to Cu2+ -IDA and Ni2+ -IDA is thus stronger among all metal ion-chelating compound combinations. All 15 IMAC chemistries showed affinity to endotoxin probably due to the small molecular weight and flexibility of endotoxin molecule. Endotoxin has an average molecular weight of 10 kDa [30] while pDNA used in the present study is 4818 kDa (or 7.3 kb). Hence, endotoxin is likely to be more accessible to the pore area of the resin than larger molecules, such as pDNA, thereby readily interacting with the immobilised metal ions.

Table 1 Binding of nucleic acids in alkaline cell lysate, Tris-NaCl Buffer or Tris Buffer to various IMAC chemistries Metal ion

Chelating compound

Alkaline cell lysate

Tris-NaCl Buffer

Tris Buffer

pDNA

RNA

pDNA

RNA

pDNA

RNA

IDA

(−) (−) (−) (−) (−)

(+) (+) (−) (−) (−)

−(−) −(−) −(−) −(−) +(−)

+(+) +(+) −(−) −(−) +(+)

−(−) −(−) −(−) −(−) +(−)

+(+) +(+) −(−) −(−) −(−)

Cu2+ Ni2+ Zn2+ Co2+ Fe3+

NTA

(−) (−) (−) (−) (−)

(−) (−) (−) (−) (−)

−(−) −(−) −(−) −(−) +(+)

+(+) −(−) −(−) −(−) +(+)

−(−) −(−) −(−) −(−) +(−)

+(+) −(−) −(−) −(−) +(+)

Cu2+ Ni2+ Zn2+ Co2+ Fe3+

CMAsp

(−) (−) (−) (−) (−)

(−) (−) (−) (−) (−)

−(−) −(−) −(−) −(−) +(+)

+(+) −(−) −(−) −(−) +(+)

−(−) −(−) −(−) −(−) +(−)

−(−) −(−) −(−) −(−) −(−)

Cu2+ Ni2+ Zn2+ Co2+ Fe3+

+: Binding of either pDNA/RNA; −: non-binding of either pDNA/RNA; (): binding behaviour of pDNA/RNA in pDNA-RNA mixture.

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Moreover, owing to the flexibility of endotoxin molecule [31], the molecule can possibly change its conformation to maximise interaction with the charged resin, thus enhancing adsorption. Electrostatic attraction between anionic phosphoric acid group of endotoxin and positively charged immobilised metal ions could have effected adsorption of endotoxin to the charged resin. 3.1.2. Interference of endotoxin with pDNA and RNA binding to immobilised metal ions Preferential interaction of endotoxin, followed by RNA and pDNA with immobilised metal ions in the alkaline cell lysate suggested that endotoxin might have interfered with the interaction of RNA and/or pDNA which would otherwise bind to IMAC. To study the binding characteristics of pDNA and RNA to immobilised metal ions, purified pDNA or Baker’s yeast RNA was tested with the 15 IMAC chemistries in Tris-NaCl Buffer lacking endotoxin (Table 1). Surprisingly, pDNA showed significant binding to Fe3+ -charged chelating compounds in Tris-NaCl Buffer (note that pDNA did not interact with any IMAC chemistry in the alkaline cell lysate). pDNA however showed no affinity to the other immobilised metal ions (i.e. Cu2+ , Ni2+ , Zn2+ and Co2+ ) in Tris-NaCl Buffer, indicating that only immobilised Fe3+ could interact with the exposed phosphate backbone in pDNA. In comparison, calf thymus DNA (10000–15000 kDa) showed no binding towards Fe3+ -IDA [32] while interactions of phosphate group on nucleotides, phosphoamino acids, phosphopeptides and phosphoproteins with Fe3+ -IDA were reported [32,33]. This implies that binding of DNA towards Fe3+ -IMAC chemistries might be dependent on DNA molecular weight and/or conformation, with preference of binding given to nucleotides, followed by pDNA (4818 kDa) and calf thymus DNA. The presence of endotoxin was found to exert significant impact on RNA binding to immobilised metal ions since RNA was bound to seven IMAC combinations (Cu2+ -, Ni2+ -, Fe3+ IDA; Cu2+ -, Fe3+ -NTA; Cu2+ -, Fe3+ -CM-Asp) in Tris-NaCl Buffer devoid of endotoxin whereas only to Cu2+ -IDA and Ni2+ IDA in the alkaline cell lysate. This suggests that endotoxin either interferes with binding of RNA to the immobilised metal ions or competes with RNA for the available binding sites. Apart from the presence of endotoxin, other differences between the alkaline cell lysate and Tris-NaCl Buffer (e.g. buffer composition and presence of impurities including host cell proteins) may have affected the binding of pDNA and RNA to IMAC. To address these issues, alkaline cell lysate was dialysed against Tris-NaCl Buffer using SnakeSkin Pleated Dialysis Tubing with a nominal MWCO of 3.5 kDa and applied to Fe3+ -IDA. Both pDNA and RNA showed negligible binding to Fe3+ -IDA (Fig. 2, lanes 4 and 7). However, when a mixture comprising purified pDNA and Baker’s yeast RNA in Tris-NaCl Buffer was tested against Fe3+ IDA, RNA (though not pDNA) exhibited affinity to Fe3+ -IDA (Table 1). This indicates that pDNA binding to Fe3+ -IDA is interfered by RNA whose binding to IMAC chemistries must be interfered by high molecular weight impurities, such as endotoxin still present following the dialysis of alkaline cell lysate.

Fig. 2. Batch binding test of non-dialysed and dialysed alkaline cell lysate using Fe3+ -IDA. Lanes: 1, DNA ladder; 2, alkaline cell lysate; 3, alkaline cell lysate after incubation with uncharged IDA resin; 4, alkaline cell lysate after incubation with Fe3+ -IDA; 5, alkaline cell lysate after dialysis against Tris-NaCl Buffer; 6, dialysed alkaline cell lysate after incubation with uncharged IDA resin; 7, dialysed alkaline cell lysate after incubation with Fe3+ -IDA.

To further investigate the possible interference of endotoxin with binding of RNA (and hence pDNA) to Fe3+ -charged chelating compounds, alkaline cell lysate lacking endotoxin was prepared by applying the cell lysate to Fe3+ -IDA. Almost complete removal of endotoxin was achieved with no significant binding of RNA (Fig. 3, lane 6). The endotoxin-free supernatant was then applied to fresh Fe3+ -IDA, leading to almost complete

Fig. 3. Sequential removal of endotoxin, RNA and pDNA from alkaline cell lysate using Fe3+ -IDA. Lanes: 1, DNA ladder; 2, alkaline cell lysate; 3, alkaline cell lysate after incubation with uncharged IDA resin; 4, supernatant from lane 3 after incubation with fresh uncharged IDA resin; 5, supernatant from lane 4 after incubation with fresh uncharged IDA resin; 6, alkaline cell lysate after incubation with Fe3+ -IDA; 7, supernatant from lane 6 after incubation with fresh Fe3+ -IDA; 8, supernatant from lane 7 after incubation with fresh Fe3+ -IDA.

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removal of RNA (Fig. 3, lane 7). Subsequent addition of the supernatant containing only pDNA to Fe3+ -IDA showed removal of pDNA (Fig. 3, lane 8). This provided clear and direct evidence that pDNA binding to Fe3+ -IDA is inhibited by the presence of RNA whose binding in turn is interfered with the presence of endotoxin. The possible interferences of proteinaceous contaminants in the alkaline cell lysate with the binding of RNA to Fe3+ -IDA were excluded since no protein was detected from the alkaline cell lysate by SDS-PAGE analysis. 3.1.3. Effect of ionic strength on pDNA, RNA and endotoxin binding to immobilised metal ions Besides the hierarchical binding affinity in the order of endotoxin, RNA and pDNA to IMAC, the presence of salts may affect the interaction of pDNA, RNA or endotoxin with the metal ions. This is possible, if the nature of their interaction is electrostatic, by screening the attraction between pDNA, RNA or endotoxin and the immobilised metal ions through non-selective interaction with the buffer ions. The effect of ionic strength on pDNA, RNA and endotoxin binding to IMAC chemistries was therefore investigated to determine whether the decrease in ionic strength (and hence reduction of screening effect by buffer ions) would enhance capture of pDNA, RNA and endotoxin by the immobilised metal ions. The binding affinity of purified pDNA, Baker’s yeast RNA and purified endotoxin to the 15 IMAC chemistries was investigated at a moderately high ionic strength in Tris-NaCl Buffer (∼70 ms/cm, with 0.75 M NaCl to mimic the ionic strength of alkaline cell lysate) and at a lower ionic strength in Tris Buffer (∼1.8 ms/cm, without NaCl) (Table 1). pDNA showed binding to Fe3+ -charged chelating compounds but not the other immobilised metal ions (i.e. Cu2+ , Ni2+ , Zn2+ and Co2+ ) in both Tris-NaCl and Tris Buffers, confirming the previous result that pDNA does not interact with the immobilised metal ions except Fe3+ despite the absence of endotoxin. Since the effect of ionic strength on pDNA binding to IMAC chemistries was not significant, specific affinity interaction between phosphate backbone of pDNA and Fe3+ -charged chelating compounds, rather than the electrostatic interaction, might be a dominant factor to account for the pDNA binding to Fe3+ -charged IMAC chemistries. For Baker’s yeast RNA, Cu2+ -IDA, Ni2+ -IDA, Cu2+ -NTA and Fe3+ -NTA showed a good capture of RNA in Tris Buffer while Fe3+ -IDA, Cu2+ -CM-Asp and Fe3+ -CM-Asp, in addition to the above-listed chemistries, exhibited significant interaction with RNA in Tris-NaCl Buffer. This implies that increased ionic strength is conducive to the binding of RNA to IMAC, thereby resulting in enhanced RNA binding to more IMAC chemistries which otherwise showed inefficient capture of RNA. The effect of high ionic strength on enhancing RNA binding to the IMAC was however dependent on the IMAC chemistry used, for example, Fe3+ -IDA showed capture of RNA in Tris-NaCl Buffer but not in Tris Buffer while Zn2+ -IDA did not show any capture of RNA in both Tris-NaCl and Tris Buffers. The enhanced binding of RNA to IMAC chemistries in the presence of salt (e.g. NaCl), if any, could be attributed to the increase in two factors: RNA accessibility to pore area of resins and availability of immobilised metal ions to interact with RNA. First, compaction of

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RNA structure might occur due to (1) electrostatic interaction of Na+ with negatively charged RNA and/or (2) increase in RNA hydrophobicity with salt addition, hence increasing accessibility of RNA to the pore area of resins. Second, screening effect of Na+ will reduce repulsion between RNA and the negative charges (induced by hydrolytic complex formations [34]) on the resin surface [35], thus rendering the immobilised metal ions more accessible to the RNA. Na+ could also interact with electron rich buffer constituent, Tris, to mitigate interaction between Tris and the positively charged chelated metal ions [20]. The effect of Na+ on RNA compaction, together with the increased availability of immobilised metal ions to interact with RNA, prevails over the screening effect of Na+ on RNA-immobilised metal ion interaction, thus explaining the increased affinity of RNA to the metal ions. Efficient endotoxin removal (>90%) by all 15 IMAC chemistries was achieved in both Tris-NaCl and Tris Buffers (Table 2). Among which, Fe3+ -charged IMAC chemistries showed the greatest efficiency in endotoxin removal (∼100% in both Tris-NaCl and Tris Buffers), probably due to preference of hard metal ions (i.e. Fe3+ ) for anionic phosphoric acid group of endotoxin (vs. intermediate metal ions, i.e. Cu2+ , Ni2+ , Zn2+ and Co2+ , which prefer aromatic nitrogen base). Comparison of endotoxin removal efficiency by the immobilised metal ions in Tris-NaCl and Tris Buffers revealed that endotoxin removal was facilitated at high ionic strength. This is probably due to the interaction of salt cations (e.g. Na+ ) with negatively charged endotoxin and/or increase in hydrophobicity of endotoxin with salt addition, thus compacting endotoxin molecule and increasing accessibility of endotoxin molecule to the immobilised metal ions. In addition, screening effect of Na+ on endotoxin-negative surface charge repulsion and Tris-immobilised metal ion attraction (as discussed above) increases availability of metal ions to interact with endotoxin. These factors predominate over the screening effect of Na+ on endotoxin-immobilised metal ion Table 2 Removal of endotoxin in Tris-NaCl and Tris Buffers by various IMAC chemistries Metal ion

Chelating compound

Endotoxin removal (%) Tris-NaCl Buffer

Tris Buffer

IDA

100 99 98 99 100

96 96 91 94 100

Cu2+ Ni2+ Zn2+ Co2+ Fe3+

NTA

98 97 98 98 100

90 95 94 98 100

Cu2+ Ni2+ Zn2+ Co2+ Fe3+

CMAsp

99 99 99 99 100

95 91 94 96 100

Cu2+ Ni2+ Zn2+ Co2+ Fe3+

Endotoxin concentration used: 700 EU/ml.

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interaction, thus giving rise to enhanced endotoxin binding to metal ions.

3.2. Interactions of pDNA and RNA with free metal ions: solubility profiles

3.1.4. Preference of pDNA versus RNA binding to immobilised metal ions To further investigate the binding priority of pDNA and RNA towards IMAC chemistries, a mixture of purified pDNA and Baker’s yeast RNA in Tris-NaCl or Tris Buffer was incubated with the 15 IMAC chemistries (Table 1). Generally, with the presence of RNA, no significant pDNA binding was observed for all IMAC chemistries in both buffer conditions. However, pDNA in Tris-NaCl Buffer (despite the presence of RNA) showed binding to Fe3+ -NTA and Fe3+ -CM-Asp which were demonstrated not to interact with pDNA in the alkaline cell lysate containing RNA and endotoxin. This implies that binding of pDNA to Fe3+ -NTA and Fe3+ -CM-Asp at high ionic strength is mainly repressed by endotoxin rather than RNA. Interestingly, in the absence of RNA, pDNA in Tris-NaCl Buffer showed affinity to all Fe3+ -charged chelating compounds. This indicates that the extent of pDNA binding inhibition by RNA would be dependent on the type of chelating compound employed in IMAC chemistry. It is also interesting to note that RNA inhibitions with pDNA binding may occur in two ways. Firstly, pDNA (but not RNA) showed binding to IMAC when each nucleic acid existed separately, however, neither pDNA nor RNA would bind to the same IMAC when both nucleic acids were present (e.g. interactions of pDNA and RNA with Fe3+ -CM-Asp in Tris Buffer). The presence of steric hindrance probably dictates that smaller non-binding molecules (i.e. RNA) gain better access to the immobilised metal ions than the larger molecules (i.e. pDNA), thus interfering with the access of larger molecules to the immobilised metal ions. Secondly, RNA was preferentially bound to IMAC in a pDNA-RNA mixture (e.g. interactions of pDNA and RNA with Fe3+ -NTA in Tris Buffer); pDNA which would otherwise bind to IMAC in the absence of RNA was uncaptured. Regardless of the mode of RNA inhibition on pDNA binding, RNA in the mixture showed almost the same binding behaviour as when it existed alone. Hence, RNA binding to the immobilised metal ions was preferred over pDNA. Taken together, endotoxin was found to exhibit preferential binding to the immobilised metal ions, followed by RNA and pDNA. In the alkaline cell lysate, endotoxin showed binding to all IMAC combinations; while RNA was bound only to Cu2+ IDA and Ni2+ -IDA; and pDNA did not bind to any IMAC. When pure RNA existed, RNA in Tris-NaCl Buffer showed affinity to seven IMAC chemistries (Cu2+ -, Ni2+ -, Fe3+ -IDA; Cu2+ -, Fe3+ -NTA; Cu2+ -, Fe3+ -CM-Asp) whereas RNA in Tris Buffer showed affinity to four IMAC chemistries (Cu2+ -, Ni2+ -IDA; Cu2+ -, Fe3+ -NTA). When pure pDNA existed, in both Tris-NaCl and Tris Buffers, only Fe3+ -chelated IMAC chemistries showed affinity to pDNA. For a pDNA-RNA mixture, in both Tris-NaCl and Tris Buffers, no significant pDNA binding was observed in all IMAC combinations except pDNA in Tris-NaCl Buffer which showed binding to Fe3+ -NTA and Fe3+ -CM-Asp. Binding behaviour of RNA, in the presence of pDNA, towards IMAC chemistries was identical to that of pure RNA.

Binding characteristics of pDNA and RNA with IMAC chemistries varied greatly with the immobilised metal ion type. However, it was postulated that pDNA/RNA-metal ion interaction would be enhanced as the metal ion coordination site increases from 2 or 3 in immobilised metal ion (when chelated by NTA or IDA) to 6 in free metal ion in solution for a six coordination number metal ion. To study the precipitation efficiency of free metal ions towards nucleic acids, different concentrations of metal ions were added to pDNA or RNA in various buffers: Tris-NaCl Buffer, Tris Buffer and Cell Lysis Buffer. Tris-NaCl and Tris Buffers were selected as used in the IMAC chemistry study while Cell Lysis Buffer was chosen to mimic the nucleic acid precipitation from the alkaline cell lysate by the free metal ions. All free metal ions (i.e. Cu2+ , Ni2+ , Zn2+ , Co2+ and Fe3+ ) were found to interact with both pDNA and RNA in all three buffers studied (Figs. 4 and 5). This is in contrast to the situation with immobilised metal ions where pDNA and RNA showed interaction only with the selected metal ions. The precipitation efficiency of free metal ions for nucleic acids generally followed: Fe3+ > Cu2+ > Zn2+ > Ni2+ ∼Co2+ . In addition to the increased coordination sites available, metal ion concentration in bulk solution is readily controllable and the free metal ions are also more mobile, providing easier access to the grooves of pDNA and RNA. Hence, more effective interactions were expected for free metal ions. From Figs. 4 and 5, it was shown that the specific concentration of free metal ions (i.e. metal ion/nucleic acid, mmol/mg) required to effect pDNA or RNA precipitation varied widely depending on the type of nucleic acid, ionic strength and the metal ion used. RNA was precipitated at 100 times lower metal ion/nucleic acid ratio than pDNA, possibly due to the exposed nitrogen bases of RNA which facilitated interaction with the metal ions as compared to pDNA without exposed nitrogen base. With increment in metal ion/nucleic acid ratio, interactions between free metal ions and nucleic acids were enhanced, resulting in the increased precipitation of pDNA and RNA. The presence of salt (i.e. NaCl), however, hampered the precipitation of nucleic acids by possibly interfering free metal ion-nucleic acid interaction. This is in contrast to the observation that high salt condition was conducive to the interactions between immobilised metal ions and nucleic acids. For free metal ion, it is thus likely that the presence of Na+ may compact nucleic acids but this compaction alone is not sufficient to effect precipitation of the nucleic acids. Instead, the screening effect exerted by Na+ is likely to interfere with the free metal ion-nucleic acid interaction. For buffers of the same ionic strength (i.e. Tris-NaCl Buffer and Cell Lysis Buffer), similar solubility profiles for pDNA and RNA were observed for all free metal ions except Zn2+ whose affinity to pDNA could have been affected by other buffer components in Cell Lysis Buffer. Zn2+ showed the greatest selectivity between pDNA and RNA with full precipitation of RNA achieved at a Zn2+ /RNA ratio of 0.4 mmol/mg (Fig. 5C) but negligible precipitation of pDNA even at a significantly higher (∼100 times) Zn2+ /pDNA

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Fig. 4. Solubility profiles of pDNA at various specific metal chloride (䊉 Cu;  Ni;  Zn;  Co;  Fe) concentrations. The initial pDNA concentration was 0.017 mg/ml in (A) Tris-NaCl Buffer; (B) Tris Buffer; (C) Cell Lysis Buffer.

ratio of 40 mmol/mg (Fig. 4C) in Cell Lysis Buffer. This raises a potential to employ ZnCl2 for selective removal of RNA from the alkaline cell lysate with minimal impact on pDNA solubility. Alternatively, simultaneous precipitation of pDNA and RNA by a specific metal ion could be a useful strategy in largescale pDNA purification since early fractionation of nucleic acids from other soluble impurities and resulting reduction of processing volume would be economically advantageous. For this purpose, the use of metal ion with the greatest precipitation efficiency would be desired in order to reduce the metal ion concentration required for the co-precipitation of nucleic

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Fig. 5. Solubility profiles of RNA at various specific metal chloride (䊉 Cu;  Ni;  Zn;  Co;  Fe) concentrations. The initial RNA concentration was 1.85 mg/ml in (A) Tris-NaCl Buffer; (B) Tris Buffer; (C) Cell Lysis Buffer.

acids. Fe3+ and Cu2+ showed the highest precipitation efficacy for the nucleic acids with full precipitation achieved at metal ion/nucleic acid ratios of <45 mmol metal ion/mg pDNA and <0.3 mmol metal ion/mg RNA in Cell Lysis Buffer. Nucleic acids bound to the metal ions could be easily recovered by adding EDTA to the precipitates. EDTA chelates the metal ions and thus resolubilises the nucleic acids. It was observed that Fe3+ , with a higher charge valency of +3 as compared to +2 in Cu2+ , showed a tighter binding to the nucleic acids since the addition of 50 mM EDTA resolubilised and recovered nucleic acids from Cu2+ -nucleic acid but not from Fe3+ -nucleic acid precipitates (results not shown). CuCl2 would thus be preferred, in view of

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developing a process flowsheet for pDNA purification, for the co-precipitation of pDNA and RNA from alkaline cell lysate. Unfortunately, the interaction of endotoxin with free metal ions was difficult to assess quantitatively due to the strong interference of free metal ions with the endotoxin assay. Nevertheless, our results presented in this study clearly demonstrated the differential and/or hierarchical binding behaviour of pDNA, RNA and endotoxin to immobilised or free metal ions, raising a potential to develop simplified process alternatives for the production of pDNA vaccines. 4. Conclusions Differential interactions of plasmid DNA (pDNA), RNA and endotoxin with various transition metal ions, typically employed in immobilised metal affinity chromatography (IMAC), were largely governed by the type, existing state (i.e. immobilised or free) and specific concentration (i.e. metal ion/nucleic acid, mmol/mg) of metal ions as well as the ionic strength of solution. The preference of interaction with immobilised metal ion follows the order: endotoxin > RNA > pDNA. Endotoxin, which showed the strongest binding to all IMAC chemistries tested, was found to inhibit the binding of RNA whose presence in turn interfered with the binding of pDNA. RNA showed selective interactions only to Cu2+ -iminodiacetic acid (IDA) and Ni2+ -IDA in the presence of endotoxin whereas pDNA exhibited negligible affinity to all the immobilised metal ions tested under the co-presence of endotoxin and RNA. In contrast to the immobilised metal ions, the same metal ions in bulk solution showed varying extent of binding to pDNA and RNA. Among which, Zn2+ selectively precipitated RNA without affecting the solubility of pDNA. The differential binding behaviour of immobilised and free metal ions with pDNA, RNA and endotoxin showed potential to intensify pDNA purification processes by achieving selective removal of cumbersome contaminants (i.e. RNA and endotoxin) in fewer process steps, thereby reducing the number of unit operations and processing costs entailed. Cu2+ -IDA, for instance, could be used for simultaneous removal of RNA and endotoxin directly from the alkaline cell lysate. Furthermore, the use of free metal ions for selective precipitation of pDNA or impurities from the alkaline cell lysate can be easily coupled with IMAC process, contributing to the increased IMAC binding capacity through the exclusion of impurities. Taken together, this study showed potential to extend the use of metal ions, typically employed in IMAC for proteins capture, to the intensification of plasmid DNA vaccine purification process with better process economics. Acknowledgment We thank Mr. Baek Seung-Woo and Miss Tan Tanya for their assistance in the endotoxin assay and the solubility curve construction. This work was financially supported by Center for Advanced Bioseparation Technology at Inha University, Korea

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