Journal of Virological Methods 85 (2000) 23–34

A method for the preparation of highly purifiedadeno-associated virus using affinity column

chromatography, protease digestion and solvent extraction

Robert Anderson *, Ian Macdonald, Tim Corbett, Alistair Whiteway,H. Grant Prentice

Bone Marrow Transplant Programme, Department of Haematology,Royal Free and Uni6ersity College Medical School of Uni6ersity College London, Rowland Hill Street, Hampstead,

London NW 3 2PF, UK

Received 31 March 1999; received in revised form 29 September 1999; accepted 29 September 1999

Abstract

Recombinant adeno-associated virus (AAV) is becoming the vector of choice for many gene therapy protocols.There has been much recent progress made toward increasing AAV titres but a continuing problem in using AAV hasbeen that it is relatively difficult to concentrate and purify. Traditional methods, such as caesium chloride (CsCl)gradients, have drawbacks, notably extended purification times and the ability to process only limited volumes. Wherethe target cells of interest require a high multiplicity of infection (MOI), or to complete in vivo experiments, there isa requirement for both the production of high titre and a large volume of virus. This is laborious to obtain usingtraditional methods. A simple technique is described here for purifying AAV, involving affinity chromatography,protease digestion and solvent extraction that retains both the high yields and titres obtained using CsCl gradients.In addition, this technique displays a fast throughput and may be used to purify AAV from larger volumes than CsClgradients. The high yield and purity of these virus preparations has allowed us to achieve good levels of expressionin the target cell types tested. The purification technique described here will be applicable to any protocol thatrequires high titre, high purity recombinant AAV (rAAV). © 2000 Elsevier Science B.V. All rights reserved.

Keywords: Adeno-associated virus; Purification; Solvent extraction; Protease digestion

www.elsevier.com/locate/jviromet

1. Introduction

A vector that is showing promise for genetherapy is the replication defective, non-patho-genic parvovirus adeno-associated virus (AAV).AAV has a single stranded DNA genome of 4.7kB which encodes both structural (Cap) and non-

* Corresponding author. Tel.: +44-171-794-0500; fax: +44-171-794-0645.

E-mail address: r.anderson@rfc.ucl.ac.uk (R. Anderson)

0166-0934/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.

PII: S 0166 -0934 (99 )00150 -0

R. Anderson et al. / Journal of Virological Methods 85 (2000) 23–3424

structural (Rep) proteins and a pair of 145 bpinverted terminal repeats (ITRs) which, are theonly cis acting elements required for replicationand packaging (Laughlin et al., 1983; Srivastavaet al., 1983).

Traditionally, recombinant AAV (rAAV) isproduced by transfection of a permissive cell thathas been previously infected with an appropriatehelper virus, usually adenovirus (Ad). These cellsare then transfected with a mix of two plasmids,the first supplying the AAV functions in trans (i.e.containing only the AAV rep and cap genes), thesecond containing the gene(s) of interest betweenviral ITRs (Hermonat and Muzyczka, 1984). Af-ter 3–4 days, rAAV is produced in the cell nu-cleus of the infected cells. These infected cells arethen pelleted and layered onto CsCl gradientswhere the mature rAAV is separated from emptycapsids and other cellular contaminants.

There are, however, drawbacks to this purifica-tion technique. Despite being able to provide thehighest purity of recombinant virus, purificationon CsCl gradients is both laborious and timeconsuming. However, perhaps the biggest disad-vantage of this technique is the limited amount oflysed pellet that can be processed on each run.Additionally, due to the limited volume that canbe processed by CsCl gradients, this technique isunable to purify rAAV that may have been re-leased into the cell supernatant.

Recent work has identified the cellular recep-tors of AAV. Foremost amongst these is heparansulphate proteoglycan (HSPG) which has beenshown to be essential in the binding stage of AAVinfection (Summerford and Samulski, 1998).More recently, both human fibroblast growth fac-tor receptor 1 (FGFR1) and the integrin aVb5have been shown to be important in mediatingAAV infection (Summerford et al., 1999) and(Qing et al., 1999).

It was reasoned then, that as AAV binds toHSPG in the course of an infection it should bepossible to purify AAV using a heparin sulphate(HS) affinity column. The relatively specific na-ture of the HS affinity column should allow ahigh level of purification relative to the tissueculture starting material.

We describe the use of an HS affinity column,which allows the concentration and purification ofrAAV from large volumes of tissue culture start-ing material. It is shown that the HS affinitycolumn is able to concentrate and partially purifyrAAV from a crude cell lysate but this partialpurification is unable to remove all additionalproteins that remain present as contaminants.Therefore, the use of protease digestion and sol-vent extraction was investigated as additionalsteps to the HS affinity column protocol to in-crease the purity of our rAAV preparations.These additional steps preserve the integrity of therAAV in the sample whilst removing the vastmajority of contaminating protein.

2. Materials and methods

2.1. Cloning and plasmid construction

Enhanced green fluorescent protein (EGFPClonetech) was PCR cloned into the expressionplasmid pBK-CMV (Stratagene) using standardtechniques (Sambrook et al., 1989). The GFPcassette, including an approximately 2.0 Kbp ofnon-coding sequence, was cloned into the AAV-2derivative pD-10 (Wang et al., 1997) to yieldpAAV-GFP. The rep/cap trans plasmid was cre-ated from pD-10 by removing both ITRs to yieldpAAV-Helper. AAV-2 was the only serotype usedin these experiments.

2.2. Preparation of rAAV containing cell lysate

To produce the rAAV, 293 cells were estab-lished in Dulbeccos Modified Eagle Medium(DMEM; Life Technologies) with 10% fetal calfserum at 37°C under 5% CO2. Cells 5×105 wereplated and allowed to achieve 50–60% confluencein a 90-mm petri dish. Cells were twice washed inserum free Opti-MEM (Life Technologies). Onehour prior to transfection, the cells were infectedwith wild type Ad type 5 (m.o.i.=3–5) and 25 mgof a 1:3 mixture of pAAV-GFP and pAAV-Helper was transfected into the cells using Lipo-fectin (Life Technologies) in Opti-MEM followingthe manufacturer’s instructions. After 5 h incuba-

R. Anderson et al. / Journal of Virological Methods 85 (2000) 23–34 25

tion at 37°C, the DNA/Lipofectin/Opti-MEMmixture was removed and replaced with freshsupplemented DMEM. Transfection efficiencywas not assessed. Cultures were left for 4 daysafter transfection when the supernatant and thesmall remaining cell pellet were harvested. Alllysates were cleared of contaminating Ad by incu-bation at 56°C for 1 h prior to any subsequenttreatments.

The supernatants were set aside and the cellpellets were resuspended in 2 ml Dulbeccosmodified PBS pH 7.4 (PBS). These pellets werethen frozen and thawed three times followed by30 s of sonication using an MSE Benchtop sonica-tor on full power. Cell debris was pelleted bycentrifugation at 2000×g and the supernatantfrom here added to the initial supernatant. Thisprocess was repeated up to two times on anyremaining cell debris.

2.3. HS affinity column preparation and rAAVconcentration

A POROS HE/P 4.6×50 mm heparin affinitycolumn (Boehringer Mannheim) was equilibratedwith 50 column volumes of 3 M NaCl at a flowrate of 2 ml/min using a FPLC machine (Pharma-cia). Prior to rAAV purification, the HS affinitycolumn was washed using 50 column volumes ofPBS such that the background absorbance at 280nm (A280) was less than 0.01. Supernatant con-taining rAAV was filtered through a 0.20-mmdisposable filter (Millipore) before being appliedto the HS affinity column at a flow rate of 6ml/min. After extensive washing with PBS, at aflow rate of 6 ml/min, continuing until the A280

reading returned to its pre-application level,rAAV was eluted in 10×1 ml aliquots using a0–100% gradient of PBS containing a final con-centration of 1 M NaCl. After each chromato-graphic run, the HS affinity column was washedwith 50 column volumes of 8 M urea at a flowrate of 2 ml/min followed by 50 column volumesof 3 M NaCl at a flow rate of 2 ml/min and storedin the same buffer.

Aliquots containing protein, as detected by in-creased A280 over background, were either incu-bated at 37°C for 1 h or incubated with a final

concentration of trypsin of 0.02% for 1 h at 37°Cor incubated with trypsin as described and thenextracted with two equal volumes of 1,1,2-trichlorotrifluoroethane (Arklone; BDH). Afterprotease and solvent treatment, some aliquotswere desalted using Pharmacia PD-10 desaltcolumns, following the manufacturers instruc-tions, and reapplied to the HS affinity column asdescribed above.

2.4. Polymerase chain reaction analysis

All reactions were carried out as described withthe following primer pairs. The CMV primerswere 5% ATT ACG GGG TCA TTA GTT CA 3%and 5% AAT GGG GCG GAG TTG TTA CG 3%.Reactions were carried out for 28 cycles annealingat 60°C giving a 497-bp fragment.

Prior to all PCR reactions, each sample wastreated with DNase to remove any contaminatingDNA from the virus sample. Samples were incu-bated with 10 U of DNase 1 (Promega) in abuffer containing 50 mM Tris HCl, (pH 8.0), 10mM NaCl, 6 mM MgCl2 for 30 min at 37°C. TheDNase was heat inactivated at 95°C for 5 min.The samples were then used directly in a PCRreaction as described above.

2.5. Viral titration

This was carried out using two separate assays:1. Dot blot quantitation of the virus was per-

formed as described (Chiorini et al., 1995;Anderson et al., 1997) using a 497-bp probegenerated by PCR against the CMV promoterregion of the virus, described above. Prior tothe dot-blots all samples were incubated with10 U of DNase 1 (Promega) at 37°C for 2 h todegrade any unencapsidated DNA. Titres weredetermined by comparing the densitometricsignal strength of the unknown viral fraction,to that obtained from a diluted known stan-dard plasmid.

2. Replicative centre assay of the virus was per-formed essentially as described (Einerhand etal., 1995), however the adenovirus used wasadenovirus type 5 (m.o.i.=15–20) and wtAAV (m.o.i.=5).

R. Anderson et al. / Journal of Virological Methods 85 (2000) 23–3426

2.6. Protein assays and blots

Total protein concentration was assessed usinga Bio-Rad Protein Assay. Polyacrylamide gelsand Western blots were carried out as described(Anderson et al., 1997) but using 4–12% Bis-Tris gels (Novex Technologies). Total proteingels were stained using Coomassie Brilliant BlueR-250 and the gels destained with CoomassieDestain solution (Bio-Rad), Western blots wereprobed using anti-AAV Cap antibodies (ProgenClone B1; Wistuba et al., 1995) following thepreviously published protocol (Anderson et al.,1997). Blots were stripped with a solution con-taining 50 mM Tris pH 6.7, 2% SDS and 0.75%b-mercaptoethanol at 50°C for 30 min. Blotswere then rinsed in PBST buffer (PBS contain-ing 0.05% Tween 20 (v/v) and 5% dried milk

(w/v)) before being re-probed using anti-actinantibodies (Sigma).

2.7. Viral infections and flow cytometry

Prior to infection of the target cells, allaliquots of rAAV were desalted using PharmaciaPD-10 desalt columns. Aliquots of rAAV pre-pared with the various modifications describedabove, were added to cells at a multiplicity ofinfection (MOI) of 10. Prior to incubation withrAAV, adherent target cells were removed fromthe culture flask with either trypsin or non-enzy-matic cell dissociation fluid as indicated. Afterremoval, cells were then washed in Hanks bal-anced salt solution (HBSS) prior to incubationwith rAAV as above. rAAV was diluted inDMEM and allowed to incubate with cells for 1h at 37°C, after which the medium was removedand replaced with fresh DMEM. Expression ofGFP was assessed 48 h post-infection using afluorescent activated cell scanner (FACS; FAC-Scan-Becton Dickinson) machine. Briefly, cellswere harvested, washed twice in HBSS and thenresuspended in FACSFlow (Becton Dickinson)prior to flow cytometric analysis. 10 000 viablecells as determined by light scatter wereanalysed from each sample. The negative controlsamples were mock infected.

3. Results

3.1. PCR analysis of the eluted aliquots

To assess the ability of the HS affinitycolumn to purify rAAV, an aliquot of AAV-GFP was prepared as described in Section 2.After loading, washing and eluting from the HSaffinity column, an aliquot of each of the frac-tions displaying an increase in A280 above back-ground was analysed by PCR for the presenceof the virus. These fractions correspond to elu-tion conditions of 0–0.7 M NaCl. As well asbeing present in the starting fraction, the PCRresult demonstrates that rAAV was present inthe fractions collected between 0.2 and 0.7 MNaCl. This is shown in Fig. 1.

Fig. 1. PCR analysis of the fractions eluted from the HSaffinity column. Using CMV primers it was possible to showthat the fractions with an A280 peak above background con-tained a specific 497-bp band indicating the presence of rAAV.Twenty-five microlitres aliquots eluted from the HS affinitycolumn were incubated with DNase 1 and then subjected toPCR as described in Section 2. Lane plan left to right: (1)molecular weight marker; (2) 0–0.1 M NaCl; (3) 0.1–0.2 MNaCl; (4) 0.2–0.3 M NaCl; (5) 0.3–0.4 M NaCl; (6) 0.4–0.5M NaCl; (7) 0.5–0.6 M NaCl; (8) 0.6–0.7 M NaCl; (9)starting culture; (10) tissue culture medium; (11) positive con-trol; (12) negative control; and (13) molecular weight marker.Molecular weights are 1500, 1200, 1000, 900, 800, 700, 600,500, 400, 300, 200 and 100 bp.

R. Anderson et al. / Journal of Virological Methods 85 (2000) 23–34 27

Fig. 2. Polyacrylamide gel of fractions eluted from the HSaffinity column stained for total protein. Twenty-five mi-crolitres aliquots eluted from the HS affinity column wereseparated by SDS-PAGE under reducing conditions using a4–12% Bis-Tris gels. The gel was stained with CoomassieBrilliant Blue and destained with Destain solution (Bio-Rad).Lane plan left to right: (1) 0–0.1 M NaCl; (2) 0.1–0.2 MNaCl; (3) 0.2–0.3 M NaCl; (4) 0.3–0.4 M NaCl; (5) 0.4–0.5M NaCl; (6) 0.5–0.6 M NaCl; (7) 0.6–0.7 M NaCl; (8)starting culture; and (9) tissue culture medium. Molecularweights are in kDa.

3.2. Determination of purity of eluted HS affinitycolumn fractions

In order to assess the purity of the fractions thatcontained AAV, polyacrylamide gel electrophore-sis was carried out with an aliquot of each fraction.The gel was then stained with Coomassie BrilliantBlue for total protein, shown in Fig. 2.

Fig. 2 lanes 1 and 2 show that no detectableprotein was eluted from the HS affinity columnusing a maximum of 0.2 M NaCl. It was notpossible to detect the presence of rAAV from thesefractions using PCR.

The presence of rAAV was detected by PCR inthe fractions corresponding to 0.2–0.7 M NaCland these fractions contain the most protein(Lanes 3–7). The major protein peak was eluted atapproximately 0.35 M NaCl (data not shown).

The total amount of protein present in eachsample post elution, is less than that present in thestarting sample. This indicated that the HS affinitycolumn is able to increase the purity of rAAV inthe samples with respect to protein concentration.This is shown in Table 1. However, a pure aliquotof AAV should yield only three protein species of90, 72 and 60 kDa in the ratio of 1:1:10 corre-sponding to VP1, VP2 and VP3 (Ruffing et al.,1992). It is clear that despite the apparent specific-ity of the HS affinity column, each of these frac-tions shows the presence of many differentmolecular weight protein contaminants.

3.3. Impro6ing the purity of the HS affinitycolumn eluate

Due to the high level of stability of AAV to bothphysical and chemical agents (Bachmann et al.,1979), we considered that it might be possible todegrade the additional contaminants seen in theabove fractions whilst retaining the integrity of therAAV.

An aliquot of each sample run in Fig. 2 wasdigested with trypsin and extracted with Arkloneas described in Section 2, re-run on a polyacry-lamide gel and stained with Coomassie BrilliantBlue for total protein, shown in Fig. 3. The resultof these treatments was the removal of a signifi-

Fig. 3. Polyacrylamide gel of treated aliquots eluted from theHS affinity column after incubation with trypsin and extrac-tion with Arklone and stained for total protein. Twenty-fivemicrolitres aliquots eluted from the HS affinity column andpost trypsin and Arklone treatment were separated by SDS-PAGE under reducing conditions using a 4–12% Bis-Tris gels.The gel was stained with Coomassie Brilliant Blue anddestained with Destain solution (Bio-Rad). Lane plan left toright: (1) 0–0.1 M NaCl; (2) 0.1–0.2 M NaCl; (3) 0.2–0.3 MNaCl; (4) 0.3–0.4 M NaCl; (5) 0.4–0.5 M NaCl; (6) 0.5–0.6M NaCl; (7) 0.6–0.7 M NaCl; (8) starting culture; and (9)tissue culture medium. Molecular weights are in kDa.

R. Anderson et al. / Journal of Virological Methods 85 (2000) 23–3428

Tab

le1

Are

pres

enta

tive

assa

yof

prot

ein

amou

nts

and

conc

entr

atio

nsat

vari

ous

stag

esof

the

puri

ficat

ion

proc

ess

Star

ting

prot

ein

End

prot

ein

Star

ting

amou

ntE

ndam

ount

End

volu

me

Star

ting

volu

me

prot

ein

(mg)

(ml)

conc

.(m

g/ml

)(m

l)pr

otei

n(m

g)co

nc.

(mg/

ml)

435

Star

tto

post

colu

mn

112

000

5000

2.77

0.08

733

240

880

0.2

348

0.08

744

00P

ost

colu

mn

1to

post

colu

mn+

tryp

sin

4000

0.04

225

0050

010

525

000.

2P

ost

colu

mn+

tryp

sin

topo

stco

lum

n+tr

ypsi

n+ar

klon

e0.

042

0.00

994

.536

2250

Pos

tco

lum

n+tr

ypsi

n+ar

klon

eto

post

4000

colu

mn

2

R. Anderson et al. / Journal of Virological Methods 85 (2000) 23–34 29

cant amount of most protein species. Indeed onlytwo protein species are apparent after trypsin andArklone treatment in Lane 3. The major species inLane 3 is approximately 70 kDa and the minorspecies is approximately 60 kDa in weight. Thesemay be AAV capsid proteins as AAV has threecapsid proteins of 90, 72 and 60 kDa (Ruffing etal., 1992).

The bands present in the lanes at about 30 kDawere not present in the blot in Fig. 2. The molec-ular weight of trypsin is approximately 28 kDaand it may be that these bands are either trypsin

or multimers of trypsin.Treatment with Arklone has not removed these

species which, due to their low molecular weight,are unlikely to be AAV capsid proteins, and sothe fractions were again passed down the HSaffinity column to increase the purity of thesample.

3.4. Analysis of the samples following proteasedigestion and sol6ent extraction and a second HSaffinity column elution

To assess whether the high molecular weightprotein species that remain post trypsin digestionand Arklone extraction are rAAV capsid proteins,a Western blot was performed using an anti-AAVcap antibody (clone B1 (Wistuba et al., 1995))which recognises the three AAV capsid proteinsVP1, VP2 and VP3 (Ruffing et al., 1992). This isshown in Fig. 4. All samples were equally loadedwith respect to DNA concentration as determinedby dot blot analysis.

Fig. 4 Lane 1 shows that the three AAV capsidproteins VP1, VP2 and VP3 in the expected ratioof 1:1:10 are present in the starting material.These proteins are still present in the eluate fromthe HS affinity column, Lane 2. Trypsin treatmentof this sample, Lane 3, shows that the AAVcapsid proteins still remain in the sample. Thisobservation is repeated after extraction withArklone treatment (Lane 4) and after the secondHS affinity column elution, Lane 5. The sample inLane 6 is rAAV purified by CsCl centrifugation.

To verify that the HS affinity column and sub-sequent treatments was removing cellular debris,the blot was stripped and probed with an anti-actin antibody, shown in Fig. 5. Even after over-exposure, actin was only detected in the startingmaterial, Lane 1, and not in any of the purifiedsamples, Lanes 2–5. As expected actin was notpresent in the CsCl purified sample, Lane 6.

3.5. Efficiency of the HS affinity column forpreparing rAAV

At various stages of purification, aliquots weretaken and assayed for total protein and the pres-ence of rAAV genomes, as determined by dot-blot

Fig. 4. Western blot analysis of the untreated and treated HSaffinity column eluates. Samples as indicated below were sepa-rated by SDS-PAGE under reducing conditions using a 4–12% Bis-Tris gels. Polypeptides were transferred tonitrocellulose and probed using an anti cap antibody whichrecognises VP-1 VP-2 and VP-3. Lane plan left to right: (1)starting culture; (2) pooled fractions post HS affinity column;(3) pooled fractions post HS affinity column and trypsinincubation; (4) pooled fractions post HS affinity column,trypsin incubation and Arklone extraction; (5) pooled frac-tions post second HS affinity column pass; and (6) AAVprepared by CsCl centrifugation. Molecular weights are inkDa.

Fig. 5. Western blot analysis of the untreated and treated HSaffinity column eluates. Samples as indicated below were sepa-rated by SDS-PAGE under reducing conditions using a 4–12% Bis-Tris gels. Polypeptides were transferred tonitrocellulose. The membrane was stripped of the anti-AAVcap antibody and re-probed with and anti-actin antibody.Lane plan as Fig. 4.

R. Anderson et al. / Journal of Virological Methods 85 (2000) 23–3430

Table 3Ratios of genomes: infectious particles of the purified rAAV

Infections particles/mlb RatioParticles/mla

3.01×1010Purification run number 1 45.81.38×1012

Purification run number 2 2.58×1011 2.06×1010 12.5Purification run number 3 4.25×10101.56×1012 36.7

1.05×1011 9.49.88×1011Purification run number 4

a Titre measured by dot blot hybridisation.b Titre measured by replicative centre assay.

analysis. The first pass of the samples through theHS affinity column reduced the amount of proteinpresent by 98.7% of the starting amount whilstthe number of rAAV genomes was reduced byonly 3.3%.

The effect of the trypsin treatment was anincrease in protein concentration, attributed tothe trypsin itself and with a slight reduction inAAV genomes. The effect of the Arklone treat-ment was a further reduction of protein by 79% ofthe starting amount but coupled to a 9.7% reduc-tion in rAAV genomes. The effect of passing thesamples through the HS affinity column a secondtime was to reduce the amount of protein presentby a further 61.9% and to reduce the number ofrAAV genomes as compared to the initial startingnumber to 34.7%. These data are presented inTables 1 and 2.

Several different aliquots of the trypsin treatedand Arklone extracted virus were assayed usingthe replicative centre assay. These data are pre-sented in Table 3.

3.6. Functional analysis of purified rAAV 6ectors

Finally using the rAAV that had been purifiedby HS affinity column purification, trypsin diges-tion and Arklone extraction, we infected 293 cellsand monitored for the presence of GFP usingFACS as described above. The results are shownin Fig. 6. The solid histogram shows cells thatwere mock infected, the open rear histogram showthe effect of the addition of AAV-GFP where15.4% of cells are positive for the presence ofGFP after 48 h. This indicates that the virus wasprepared intact after the purification treatmentsand was still able to be internalised by the cells.

The uptake of the virus was demonstrated to bereceptor mediated as 293 cells pre-treated withtrypsin immediately prior to viral addition did notshow expression of GFP (Fig. 6, grey hatchedsecond histogram). Treatment of cells with trypsinhas previously shown to prevent viral uptake(Mizukami et al., 1996). Pre-treatment of the cellsusing a non-enzymatic cell dissociation fluidshowed a slight reduction in viral expression ascompared to untreated cells but there were still anumber of cells (14.1%) which were positive forGFP (Fig. 6, grey diamond filled, thirdhistogram).

4. Discussion

Several recent publications report improvedmethods to increase the amount of rAAV pro-duced by the packaging cell line (Li et al., 1997;Vincent et al., 1997; Inoue and Russell, 1998).However there has been a dearth of publicationsconcerning the purification of the rAAV as thevast majority of rAAV stocks are prepared usingCsCl gradient centrifugation.

Highly purified vector stocks are important forin vivo experiments but there are additional po-tential benefits. It has been suggested that poorlypurified rAAV may be responsible for a phe-nomenon termed pseudotransduction (Alexanderet al., 1997), which occurs when biologically ac-tive peptides that are present in the rAAV vectorstocks are transferred to target cells in a virionindependent manner. Pseudotransduction is par-ticularly noted when reporter genes such as b-galactosidase and alkaline phosphatase areemployed.

R. Anderson et al. / Journal of Virological Methods 85 (2000) 23–34 31

Tab

le2

Are

pres

enta

tive

assa

yof

DN

A(r

AA

V)

amou

nts

and

conc

entr

atio

nsat

vari

ous

stag

esof

the

puri

ficat

ion

proc

ess

Star

trA

AV

End

rAA

VSt

arti

ngnu

mbe

rE

ndnu

mbe

rSt

arti

ngvo

lum

eE

ndvo

lum

ege

nom

es(m

l)(g

enom

es/m

l)(m

l)ge

nom

es(g

enom

es/m

l)

3.84

×10

12

Star

tto

post

colu

mn

112

000

5000

3.31

×10

87.

68×

108

3.97

×10

12

3.02

×10

12

6.87

×10

83.

07×

1012

7.68

×10

844

00P

ost

colu

mn

1to

post

colu

mn+

tryp

sin

4000

6.21

×10

825

001.

72×

1012

1.55

×10

12

2500

6.87

×10

8P

ost

colu

mn+

tryp

sin

topo

stco

lum

n+tr

ypsi

n+ar

klon

e6.

21×

108

3.45

×10

81.

39×

1012

1.38

×10

12

2250

Pos

tco

lum

n+tr

ypsi

n+ar

klon

eto

post

4000

colu

mn

2

R. Anderson et al. / Journal of Virological Methods 85 (2000) 23–3432

The major limitations of the traditional CsClpurification method are the length of the purifica-tion process and that only a small volume ofrAAV can be purified in each run. The purifica-tion of rAAV using double CsCl banding can takeupto 48 h. As a consequence of the limited vol-ume of vector that can be purified in one run,most protocols for rAAV production harvest theproducer cells 3 days after transfection when mostof the rAAV is still cell associated. A recent paper(Chirico and Trempe, 1999), showed that thegreatest titres of rAAV were obtained 4 days aftertransfection. However at this point a substantialtitre of virus was no longer cell associated as itwas released from the lysed cells into the cellsupernatant. This is a disadvantage when purify-ing rAAV using a CsCl gradient.

Recent data (Tamayose et al., 1996) showedthat by using a negatively charged celluloseaffinity medium, Cellufine sulphate, it was possi-ble to overcome many of the difficulties seen withCsCl. The virus binds to the matrix whilst otherproteins and debris are washed off allowing aconcentrated and purified virus fraction to beeluted. Although this technique is more rapid and

able to process much larger quantities of samplethan possible on CsCl gradients, the eluate con-taining the rAAV is contaminated additionalproteins. In their original report, Tamayose et al.(1996) noted that affinity chromatography usingCellufine Sulphate alone was itself unlikely topurify rAAV to high levels. However, the princi-ple of affinity column purification had beenestablished.

The discovery that HSPG mediated AAV cellentry (Summerford and Samulski, 1998) has al-lowed the use of a specific HS affinity column topurify rAAV. Unlike CsCl based purification, thetotal disruption of the producer cells is advanta-geous when using any column purification tech-nique. This total cell lysis ensures that all of therAAV produced is recovered.

Disappointingly however, the purity obtainedfrom the HS affinity column is initially low, ex-cluding the use of HS affinity column chromatog-raphy alone for the preparation of high qualityvirus. We therefore investigated the possibility ofusing trypsin digestion followed by extractionwith an organic solvent to remove any polypep-tides and other contaminants that may have been

Fig. 6. Fluorescent analysis of cells transduced with highly HS affinity column purified AAV-GFP. Cells were either mock infectedor infected with HS affinity column purified, trypsin digested and Arklone extracted AAV at an MOI of 10:1. Cells were analysed48 h post-infection. Results are presented as histograms with fluorescence intensity shown in log10 intervals. The solid histogramshows mock infected 293 cells, the grey second histogram shows the effect of trypsin treatment of the cells abrogates expression, thethird diamond filled histogram shows that non-enzymatic treatment of cells allows expression of AAV-GFP and the final openhistogram shows the effect of addition of AAV-GFP without cell pre-treatment.

R. Anderson et al. / Journal of Virological Methods 85 (2000) 23–34 33

co-purified with the rAAV. rAAV capsids aretrypsin resistant when fully formed but the indi-vidual cap polypeptides are trypsin sensitive (An-derson, unpublished observations). The organicsolvent 1,1,2-trichlorotrifluoroethane (Arklone)has been previously used in the purification of Ad(Precious and Russell, 1985). By introducing theseadditional steps to the HS affinity column proto-col we have been able to remove most proteincontaminants from our viral preparation, whilstretaining the integrity of the viral stock.

As rAAV is known to contain a substantialnumber of empty particles (Laughlin et al., 1979),the addition of a trypsin step could be responsiblefor removing these particles thus reduction in theamount of protein present in the final prepara-tion. The ratio of infectious particles, as assayedby replicative centre assay, and genomes, as as-sayed by dot blots, is consistently less than 100which is in broad agreement with that reportedpreviously (Salvetti et al., 1998).

This purification technique is applicable to allrAAV preparations, retaining the advantages of aHS affinity column purification technique overCsCl gradients such as the speed of the purifica-tion and the ability to process large volumes,whilst overcoming the major disadvantage of theHS affinity column, the impure end product. Weare confident that HS affinity column chromatog-raphy, together with these modifications, will gotoward the replacement of CsCl gradients in thepurification and concentration of large volumes ofrAAV vectors.

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