high-speed chromatographic purification of plasmid dna with a customized biporous hydrophobic...

Biochemical Engineering Journal 27 (2005) 33–39

High-speed chromatographic purification of plasmid DNA with acustomized biporous hydrophobic adsorbent

Yuan Li, Xiao-Yan Dong, Yan Sun∗

Department of Biochemical Engineering, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China

Received 28 March 2005; received in revised form 2 May 2005; accepted 16 June 2005

Abstract

Interest in producing large quantities of plasmid DNA has recently increased as a result of the rapid evolution of gene therapy and DNAvaccines. Hydrophobic chromatography is a popular technique in the downstream processing of plasmid DNA. However, the low capacityand the high mass transfer resistance of most commercially available packings for bio-macromolecules limit their application in a large-scaleprocess. In this work, a hydrophobic absorbent with wide pores was synthesized by the solid porogenic method. Analyses by scanning electronmicroscopy and mercury intrusion porosimetry revealed that the matrix contained two families of pores, i.e., micropores smaller than 100 nmand superpores of 500–7300 nm. The superpores provided not only convective flow channels for the mobile phase, but also a large surface forb kpressurea ck ando that theh©

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iomolecules binding. So the chromatographic process can be operated at high flow rate with high column efficiency and low bacs identified on a 2-mL column (5 mm i.d., 2 cm length). With a loading up to 2.6 mg of 5.4 kb plasmid (pcDNA3) in 8 mL feedstoperated at a flow rate as high as 20 cm/min, nearly 100% of plasmid was recovered with a purity of 100%. The results indicateydrophobic medium is promising for high-speed purification of plasmid DNA.2005 Elsevier B.V. All rights reserved.

eywords: Plasmid DNA; Hydrophobic interaction chromatography; Biporous medium; Purification; High speed

. Introduction

The interest in plasmid DNA (pDNA) has recentlyncreased due to the rapid evolution of gene therapy andNA vaccines. In gene therapy applications, non-viral vec-

ors would be preferred in clinical applications to minimizehe risk of viral infection[1,2]. This increases the demandor large-scale processes to manufacture pDNA of a highevel of purity for use as therapeutic agent[3,4]. Liquidhromatography is one of the most effective methods forhe analytical and preparative separations of bio-molecules.urrently, pDNA and DNA vaccines have been purifiedy different types of chromatography such as gel filtration

5], ion-exchange[6–10], reversed-phase[11], hydrophobicnteraction[12–15], hydroxyapatite[16], and affinity[17–20]hromatography as well as other adsorbents[21]. Of the chro-atography methods, hydrophobic interaction chromatogra-

∗ Corresponding author. Tel.: +86 22 27404981; fax: +86 22 27406590.E-mail address: [email protected] (Y. Sun).

phy (HIC) takes the advantage of the different hydrophocharacters of pDNA and other impurities, such as RNdenatured genomic DNA, proteins and lipopolysaccharid(LPS) in cell lysate. Plasmids are double-stranded and thydrophobic bases are packed and shielded inside the dohelix. Genomic DNA inEscherichia coli is also double-stranded, but becomes single-strands during alkaline lyRNA molecules are also single-stranded by nature. BecaRNA and denatured gDNA have the structure of singstrand with a higher exposition of the hydrophobic basescompared with double-stranded plasmid, they have stroninteractions with the hydrophobic adsorbents than pDNA.addition, LPS are polyanionic amphiphilic molecules that cform multimolecular aggregates with a complex supramolular structure. The presence of a hydrophobic lipidic moiein these molecules made it possible to clear the residual Lin the lysate with HIC. Hence, hydrophobic matrices cselectively adsorb proteins, LPS and RNA instead of pDNBecause the molecular masses of proteins, LPS and Rare much smaller than pDNA, higher binding capacities

369-703X/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2005.06.011

34 Y. Li et al. / Biochemical Engineering Journal 27 (2005) 33–39

HIC adsorbents for these relatively low molecular-mass sub-stances would be possible. This would therefore lead to thepartial solution to the problem of the low pDNA capacity ofcommon porous media[22–25]. Consequently, high-capacitypurification of pDNA by using HIC is expected because thepDNA could flow through an HIC column without binding.This work is to test the possibility of high-capacity purifica-tion of pDNA using HIC.

In a previous publication, the authors’ laboratory hasreported a novel rigid biporous medium suitable for high-speed protein chromatography[26]. The polymeric micro-sphere was fabricated with calcium carbonate granules andorganic solvents as porogenic agents. Due to the presenceof wide pores created by the solid granules, the medium-based medium has a high dynamic capacity and high columnefficiency at high flow rate. Consequently, it is expected thatonce the medium is substituted into a hydrophobic adsorbent,it would possess a high capacity for the impurities such asproteins and RNA at high flow rate, leading to the high-speedpurification of pDNA. In this article, we report the fabricationof the biporous hydrophobic adsorbent and its application tohigh-throughput purification of plasmid by HIC.

2. Materials and methods

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Tris–HCl, 10 mM EDTA, pH 8.0). Then, solid ammoniumsulfate was dissolved in the plasmid solution up to 2.5 M, fol-lowed by 15 min incubation on ice. The precipitated proteinand RNA were removed by centrifugation at 10,000× g for15 min at 4◦C. The supernatant was used as the feedstock forfurther purification by hydrophobic interaction chromatogra-phy.

2.3. Preparation of pure plasmid

A standard sample of pure pcDNA3 was prepared usinga plasmid purification kit from Promega Corporation (Madi-son, USA) according to the instruction of the manufacturer.

2.4. Preparation of biporous hydrophobic adsorbents

Biporous resin (BiPR) was prepared by the radicalsuspension–polymerization method with a mixture of cyclo-hexanol and dodecanol as the liquid porogenic agent andcalcium carbonate granules as the solid porogenic agent[26].The composition of the polymerization mixture for preparingthe beads was 24/16/45/5/10 (GMA/EDMA/cyclohexanol/dodecanol/solid granule in volumetric ratio). The contentof free radical initiator AIBN was 1% (w/v) with respectto monomers (GMA and EDMA). After degassing and wellmixing in an ultrasonicator for 30 min, 20 mL of the mixturew osedoA tem-p 5 to62 witha ents,c ctionw tiona bon-a Clb Oc shedw

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.1. Materials

Ethylene dimethacrylate (EDMA) was obtained frigma (St. Louis, MO). Glycidyl methacrylate (GM

99%) was purchased from Suzhou Anli Chemical Cany (Jiangsu, China). Yeast extract and tryptone

rom Oxoid Ltd (Hampshire, England). The DNA marL15000 was from TaKaRa Biotech (Dalian, China). Ticro BCA protein assay kit was from Pierce (RockfoSA) Plasmid purification kit was purchased from Promorporation (Madison, USA). Agarose powder, ampici

ris(hydroximethyl) methylamine (Tris), sodium salt of etinediaminetetraacetic acid (EDTA), sodium dodecylsulSDS) and other materials were all received from Dingiotech (Tianjin, China).

.2. Fermentation and crude pcDNA3 preparation

E. coli DH5� strain harboring the plasmid pcDNA3.4 kb was kindly provided by the Hematology Institutehinese Academy of Medical Sciences (Tianjin, China).train was stored in LB medium plus 20% glycerol at−80◦C.

transformed strain was grown in 2× YT medium conaining 150�g/mL ampicillin at 37◦C by shaking culturn 2-L flasks at 200 rpm for 14–16 h till an optical densitypproximately 4 was reached. The cells were harvesteentrifugation at 5000× g for 15 min at 4◦C.

Crude pcDNA3 was prepared by alkaline lysis andropanol precipitation based on the method described inture[27]. The precipitate was dissolved in TE buffer (25 m

as added to 100 mL aqueous solution which was compf 10 g/L poly(vinyl alcohol) and 0.1 g/L gelatin at 45◦C.fter the system was agitated at 500 rpm for 30 min, theerature of the suspension was linearly raised from 45◦C in 1 h, and was kept at 65◦C for 3 h, then 85◦C forh. The resultant white beads were thoroughly washedlarge amount of hot water. The solvent porogenic ag

yclohexanol and dodecanol, were removed by extraith ethanol under reflux for 24 h in a Soxhlet extracpparatus, while the solid porogenic agent, calcium carte, was removed by slowly adding 100 mM glycin–Huffer (pH3.0) to the solid suspension until no bubbles of C2ould be detected. Then, the matrix was thoroughly waith distilled water before modification with phenol.Modification of the matrix with phenol was based

ethod of Kubota et al.[28]. Briefly, 5.0 g of beads was addnto 100 mL of 0.1 M phenol solution whose pH was adjuso 9 with sodium hydroxide. The suspension was heate0◦C and kept at this temperature for 12 h. Thereafter, e

de groups remaining on the polymeric beads were redo hydroxyl groups by suspending the beads in 100 m.1 M NaBH4 solution and shaking the suspension overn

n a shaking incubator of 140 rpm at room temperature.ydrophobic biporous resin (HBiPR) was thoroughly wasith an excess of methanol and distilled water before fuse. The phenyl group coupled to the resin was determy the method of Kubota et al.[28].

Particle size distribution of the matrix was measuredastersizer 2000 particle size analyzer (Malvern Insent Ltd., UK). The pore size distribution analysis was

Y. Li et al. / Biochemical Engineering Journal 27 (2005) 33–39 35

formed by mercury intrusion porosimetry on a QuantachromePoremaster-60 (Quantachrome Corporation, USA). The porestructure of the beads was observed by scanning electronmicroscopy (SEM) on an XL30 ESEM scanning microscope(Phillips, Netherlands).

2.5. Plasmid purification by HIC

An HR5/10 column (5 mm i.d., 10 cm length) obtainedfrom Amersham Biosciences (Uppsala, Sweden) was packedwith the HBiPR by means of the ethanol-slurry packing tech-nique. All chromatography experiments were carried out onAKTA FPLC system (Amersham Biosciences) installed withthe Unicorn 4.1 software for data acquisition and processing.The outlet stream was continuously monitored by both a con-ductometer and an UV monitor at 254 nm.

In the purification experiments, the crude pcDNA3 prepa-ration obtained after precipitation with (NH4)2SO4 was usedas the feedstock. Prior to a purification experiment, the col-umn was equilibrated with 10 column volumes (CVs) ofbuffer A (50 mM Tri-HCl, 1 M (NH4)2SO4, 10 mM EDTA,pH 8.0). To scout a proper chromatographic condition, 0.1 mLof the feedstock containing 0.32 mg/mL of pcDNA3 wasapplied at 1 mL/min. After washing of unbound or weaklyretained species with buffer A, the ionic strength [(NH4)2SO4concentration] of the buffer was linearly decreased by thec ME

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G-DNA-PW followed by TSKgel G-6000-PWXL column(Tosoh Corporation, Japan) in series. To prepare a calibrationcurve, plasmid standards of concentrations ranging from 5 to200�g/mL were injected to the column and the area under thepeak of absorbance at 260 nm was plotted against the plasmidconcentration. Samples of unknown plasmid concentrationwere then analyzed by the SE-HPLC, and the concentrationwas determined from the calibration curve. The percentageof plasmid peak area (first peak, see below) in each chro-matogram was thus used as a measure of the sample purity[12].

The concentration of the nucleic acids in the crude plasmidpreparation was also measured by Genequant DNA/RNA cal-culator (Amersham Biosciences). Together with the results ofSE-HPLC for the plasmid analysis, RNA concentration in thecrude feedstock was determined.

Protein content of the plasmid preparation and the frac-tions collected form HIC was determined by Micro BCAprotein assay kit from Pierce (Rochford, USA) accordingto the manufacturer’s instructions. Bovine serum albumin(BSA) was used as the standard in the range of 0.5–20�g/mL.

3. Results and discussion

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hange of buffer A to buffer B (50 mM Tri-HCl, 10 mDTA, pH 8).To detect the breakthrough volume of the impurities (

eins and RNA), 20 mL of the feedstock was loaded to themn through the Superloop 50 mL (Amersham Bioscient 0.5 mL/min. The effluent was collected every 1 minnalyzed by SE-HPLC, agarose gel electrophoresis androtein assay (see below). By the breakthrough volum

he impurities, a proper loading volume of the feedstocketermined for high-throughput purification at high flow rmL/min (20 cm/min).After each run, the column was regenerated with 15

f 0.5 M of sodium hydroxide solution. Then, the coluas equilibrated with buffer A until UV signal reachedaseline.

.6. Analysis

Plasmid DNA and other nucleic acids were analyzelectrophoresis using 0.7% agarose gel in 40 mM Tris b0 mM sodium acetate, 2 mM EDTA (pH 8.3). The el

rophoretic separation was performed at a constant vot room temperature for 1 h. Nucleic acids were stained�g/mL of ethidium bromide solution included in the geThe concentrations of plasmid and RNA in the plas

reparation were measured by size exclusion HPLCPLC) on the Agilent 1100 system (Agilent TechnologE). Injection of 25�L of sample was followed by isoratic elution with 25 mM Tris containing 0.3 M NaCl amM EDTA (pH 7.5) for 70 min at 0.4 mL/min on TSKg

.1. Properties of the biporous matrix

To describe the particle size range, the volumetric dtersd10 andd90 are defined as the points on the size

ribution where, respectively, 10 and 90% by volume ofarticles are smaller than the stated diameter. As a resulty volume of the particles were in the range of 29–54�m, and

he volume-weighted mean diameter of the matrix partas determined to be 41�m.Fig. 1 shows an SEM photograph of the matrix surfa

t can be seen that the matrix contains irregular superp

ig. 1. Scanning electron micrographs of the matrix at a magnificati0,000.


Fig. 2. Volume fractions of pores in different size ranges.

larger than 0.5�m among the nanometer-sized micropores,which are considered to be created by the calcium carbonategranules in the fabrication. In addition to the SEM obser-vation, the pore size distribution of the matrix was mea-sured by mercury intrusion porosimetry.Fig. 2 illustratesthe results. It indicates that the micropores smaller than100 nm took 31% volume of the total pores while the super-pores of 500–7300 nm were about 23%. The results clearlydemonstrate the biporous structure of the matrix. The spe-cific surface area measured by this method was 20.4 m2/g.The phenyl group coupled to the resin was determined to be0.078 mmol/g.

The biporous feature of matrix made it possible for themobile phase to flow convectively through the wide poresinside the particles with reduced flow resistance[26]. Thebackpressure of the column increased linearly with flowvelocity up to 35 cm/min, and the column backpressureincreased more slowly with flow velocity than a columnpacked with the microporous particles did (the microp-orous particles of a similar mean diameter were preparedwithout using calcium carbonate in the reaction mixture asdescribed by Wu et al.[26]) (data not shown). For exam-ple, at 35 cm/min, the backpressure of the biporous columnwas only 1.5 MPa, while the backpressure of the columnpacked with the microporous particles reached 2.7 MPa. Sothe biporous resin showed better flow hydrodynamic prop-e flowh fort y. Ina resina dingo ofm

3

pita-t Aa i-c d the

Fig. 3. Ethidium bromide-stained agarose gel of pcDNA3 solutions. Lane 1,DNA marker; lane 2, pure pcDNA3 prepared with plasmid purification kit;lane 3, crude pcDNA3 (the supernatant after precipitation with (NH4)2SO4).

purified plasmid produced with the plasmid purification kit.It can be seen that there was a great deal of RNA besides plas-mid in the crude preparation. Similar to the purified standard,the plasmid in the crude preparation existed in three forms:supercoiled, relaxed and linear plasmids.

To examine the hydrophobic binding behavior of theplasmid, RNA and proteins, HIC separation of the plasmidpreparation was optimized with mobile phases of differentammonium sulfate concentrations. It was to find a properammonium sulfate concentration that could result in the bind-ing of proteins and RNA when little pDNA was adsorbed. Asa result, 1 M ammonium sulfate was found to reach the pur-pose. This was the reason we used buffer A of 1 M ammoniumsulfate as the regeneration and washing buffer (see Section2.5). Fig. 4 is the chromatographic profile for the separationunder the optimized condition. In the separation, the unboundand weakly retained species were washed out with buffer A,and then the bound species were eluted by linearly decreas-ing ammonium sulfate concentration in the mobile phase bythe change of buffer A to buffer B in 5 min.

The two peaks inFig. 4 were collected and analyzed bySE-HPLC. As shown inFig. 5, the unbound fraction (firstpeak inFig. 4) was consisted of plasmid (first peak in the

F ationa lumnw 00%b

rties than the common microporous medium. Theydrodynamic property of the matrix made it possible

his column to be used for high-speed chromatographddition, the superpores in the hydrophobic biporousre expected to provide a large surface area for the binf high-molecular-mass RNA during the convective flowobile phase through them.

.2. Separation of pcDNA3 by HIC

The feedstock obtained by ammonium sulfate preciion contained 0.32 mg/mL of pcDNA3, 0.42 mg/mL of RNnd 50�g/mL of protein.Fig. 3is the gel electrophoresis indating a comparison between the crude preparation an

ig. 4. Hydrophobic interaction chromatography of the plasmid prepart 1 mL/min. The feedstock in buffer A (0.1 mL) was applied and the coas washed with buffer A for 6 min. Then the linear gradient of 0–1uffer B was accomplished within 2.5 column volume.


Fig. 5. SE-HPLC analyses of the fractions collected from the chromatog-raphy shown inFig. 4. The top shows the SE-HPLC profiles of peak 1 inFig. 4and the bottom shows those of peak 2 inFig. 4.

top panel ofFig. 5) and small molecular mass impurities(eluted in 61 min inFig. 5), while the bound species (sec-ond peak inFig. 4) were eluted as two peaks at 58.5 and61 min, respectively from the SE-HPLC (bottom panel ofFig. 5). Independent experiments by loading EDTA solu-tion to the SE-HPLC column have shown that EDTA waseluted at 61 min, the same as that of the last peak inFig. 5.Because the concentrations of EDTA in buffers A and B wereboth 10 mM, higher than that in the mobile phase for the SE-HPLC analysis (1 mM), it could be therefore generated in theHPLC. In addition, independent experiments have shown thatboth bovine serum albumin (MW = 67 kDa) and lysozyme(MW = 14.3 kDa) were eluted at 58.5 min. Moreover, previ-ous work has shown that RNA was eluted from the SE-HPLCcolumns at approximately the same volume as that of the firstpeak in the bottom panel ofFig. 5 [6]. Hence, it is consideredthat the bound species inFig. 4were composed of mainly pro-teins and RNA, and the unbound plasmid fraction obtainedin Fig. 4 was lack of proteins and RNA but only some lowmolecular mass substances such as EDTA.

From the result shown inFigs. 4 and 5, we can knowthat in buffer A plasmid was little retained while RNA andproteins were done by the HIC column. Thus, the plasmidcan be separated from RNA and proteins under the conditionused inFig. 4.

3

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Fig. 6. HIC purification of pcDNA3 by loading 10 mL of the plasmid prepa-ration. The chromatography was operated at 4 mL/min (20 cm/min). Thecolumn was washed with buffer A for 6 mL. Then the linear gradient of0–100% buffer B was accomplished within four column volumes.

This implies that at the chromatographic condition (flowrate of 0.5 mL/min) 12 mL of the crude feedstock could beapplied to the 2-mL HIC column with little breakthroughof the impurities. As mentioned above, the concentrationof RNA in the feedstock was 0.42 mg/mL, therefore, thedynamic capacity of the column for RNA is calculated tobe (0.42 mg/mL)× (12 mL)/2 mL = 2.5 mg/mL column at theflow rate of 0.5 mL/min (2.5 cm/min). It is considered that thesuperpores provided more binding sites for RNA during theconvective flow of mobile phase through them.

The dynamic capacity of the column for bovine serumalbumin has been determined to be 35 mg/mL column, anddecreased only slightly up to a flow rate of 20 cm/min[26].Hence, we performed pcDNA3 purification by loading 10 mLof the feedstock at 20 cm/min (4 mL/min), which was eighttimes higher than that in the frontal analysis experiment.As displayed inFig. 6, at the elevated flow rate and highfeedstock loading, the chromatographic profile was approx-imately the same as that inFig. 4. Analyzed by SE-HPLC,the unbound fraction (peak 1 inFig. 6) contained 92% of theplasmid (top panel ofFig. 7). Since no protein in the plasmidpool was detected by the BCA assay, the impurity in the plas-mid fraction should be RNA. In addition, little plasmid wasfound in the second peak inFig. 6 (bottom panel ofFig. 7).It indicates that the plasmid was purified in the breakthroughstream with a recovery yield of nearly 100%.

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.3. High-throughput purification

To test the loading capacity of the HIC column, fronnalysis was performed by loading 20 mL of the crude pid preparation at 0.5 mL/min and the effluent pools wnalysed by SE-HPLC at different time intervals (datahown). As a result, the plasmid flowed through the cols a pure component in the first 12-mL loading. ThereaNA and/or protein began to appear in the effluent stre

To improve the purity of the recovered plasmid,educed the feedstock volume to 8 mL. The chromatograrofile was shown inFig. 8. Analyzed by SE-HPLC (Fig. 9),

he unbound fraction (peak 1 inFig. 8) was pure plasmid (anow molecular mass impurities such as EDTA) and none p

id was found in the second peak inFig. 8 (see the bottomanel inFig. 9). So the plasmid was purified in the flo

hrough stream with a recovery yield of about 100%.Besides the SE-HPLC analysis, the plasmid pool colle

n Fig. 8 was also analyzed by agarose gel electrophoFig. 10). It can be observed that pDNA and RNA ineedstock (lane 2) were completely separated, consistenhe results of HPLC shown inFig. 9. In addition, BCA proteinssay detected no proteins in the plasmid pool. Hence,


Fig. 7. SE-HPLC analyses of the fractions collected from the chromatogra-phy shown inFig. 6. The top shows the analysis of peak 1 inFig. 6and thebottom shows the analysis of peak 2 inFig. 6.

Fig. 8. HIC purification of pcDNA3 by loading 8 mL of the plasmid prepa-ration. The chromatography was operated at 4 mL/min (20 cm/min). Thecolumn was washed with buffer A for 5 mL. Then, the linear gradient of0–100% buffer B was accomplished within four column volumes.

Fig. 9. SE-HPLC analyses of the fractions collected from the chromatogra-phy shown inFig. 8. The top shows the analysis of peak 1 inFig. 8and thebottom shows the analysis of peak 2 inFig. 8.

Fig. 10. Electrophoresis of the fractions collected form chromatographyexperiment shown inFig. 8. Lane 1, DNA marker; lane 2, Feedstock; lane3, Peak 1 inFig. 8; lane 4, peak 2 inFig. 8.

be concluded that nearly 100% of plasmid was recovered witha purity of 100%.

It is notable that the HIC purification was carried out ata flow velocity of 20 cm/min, which is a superficial velocity5–10 times higher than normal preparative chromatography[6,13]. At this high flow rate, about 2.6 mg plasmid was puri-fied within 8 min by the 2-mL HIC column. The throughputof the HBiPR was much higher than that of other adsorbentsbased on Sepharose Fast Flow[13]. Moreover, the produc-tivity of the separation process was estimated at 9.75 g/L h[ = 2.6 mg/2 mL/8 min), comparable to the pDNA productiv-ity obtained using the commercial CIM (Convective Inter-action Media) radial flow monoliths in an anion exchangechromatographic mode (8.7 g/L h)[29].

The HBiPR column was repeatedly used for more than10 runs from the optimization experiments (see Section3.2)till the high loading purification. After each run, the col-umn was regenerated with 0.5 M of NaOH solution. Duringthe process, little change of the column backpressure andseparation capacity was observed. This demonstrated the sta-bility and reproducibility of the biporous adsorbent basedon the crosslinked GMA and EDMA. Since the stability ofthe GMA–EDMA matrix, it has been developed to commer-cial monolith columns[29,30]. All the results indicate thatthe customized biporous adsorbent is promising for high-throughput purification of plasmid molecules.

4

withs asmidp bilep niques ofm ncedi pro-v RNAd em,g teda id

. Conclusions

In this work, a phenyl-based hydrophobic adsorbentuperpores larger than 500 nm has been fabricated for plurification. Using 1.0 M ammonium sulfate as the mohase, the adsorbent adsorbed proteins and RNA. The utructure of the biporous matrix allows convective flowobile phase through the wide pores, leading to the enha

ntraparticle mass transport. Moreover, the wide poreside more binding sites for the macromolecules such asuring the convective flow of mobile phase through thiving a dynamic capacity of 2.5 mg/mL for RNA. Operat a flow velocity of 20 cm/min, 8 mL of the crude plasm


preparation containing 2.6 mg pcDNA3 has been purified to100% with nearly 100% recovery on a 2-mL column. It isnotable that the flow velocity is 5–10 times higher than nor-mal preparative chromatography. The results indicate that theHIC with the customized biporous adsorbent is promising forhigh-speed purification of large plasmid molecules. If moreRNA and proteins can be removed in the primary processingsteps, the plasmid purification capacity of the HIC columnmay be further increased.

Acknowledgment

This work was supported by the Natural Science Founda-tion of China (Grant No. 20025617 and No. 20276051).

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