chromatographytech note 2946

Plasmid DNA Adsorption Behavior on CHT™ Ceramic
Hydroxyapatite Type II Support
Cheryl Aberin and Samuel Franklin, Bio-Rad Laboratories, Inc.,
Hercules, CA 94547 USA

Introduction
Plasmid DNA adsorbs to the surface of most commerciallyavailable chromatographic supports. These chromatographic
supports were designed and developed for protein purificationand thus have relatively small pore dimensions. The small
pores lead to low adsorption capacity of plasmid DNA on thesupport; in fact, binding capacities for plasmid DNA average
50 times less than those for protein (Ferreira et al. 2000).
There is thus a need to optimize the adsorption capacity
for plasmid DNA on most of the chromatographic supports
currently available. Previously we demonstrated thatchromatography on CHT ceramic hydroxyapatite Type II
yielded plasmid of near pharmaceutical grade in a single step
(Aberin and Franklin 2002). Here we report on the effect ofsize and type of plasmid DNA, as well as the nature of thebuffer system, on the dynamic binding capacity (DBC) andrecovery on CHT Type II support.
Methods and Results
CHT Type II support (20 μm particle size) was used to screen
plasmid DNA to determine adsorption behavior. The threedifferent types of pure plasmid DNA were: pS3 (10 kb), pS5(9.48 kb), and pUC19 (2.6 kb) obtained from an outside vendor
and loaded to saturation onto a 1 ml CHT column (EconoPac ® prepacked CHT-II cartridge, Bio-Rad Laboratories, Inc.)
DNA concentration was determined spectrophotometricallyat 260 nm. Table 1 shows the adsorption behavior of thedifferent types of plasmid DNA loaded onto a CHT column
using 10 mM sodium phosphate, pH 7.0, 1 mM EDTA as
the binding buffer. The plasmid DNA was eluted with 0.4 M
sodium phosphate, pH 7.0, 1 mM EDTA. Fractions collected
were analyzed spectrophotometrically at 260 nm to determine
DNA concentration. The linear flow rate used to bind and elutethe samples was 311 cm/hr. Both DBC and yield varied withplasmid type.
We next evaluated the effect of different buffer systems on theadsorption behavior of different plasmids on CHT. Buffers usedfor screening were 0.01 M sodium phosphate, pH 7.0 and 7.5;
0.02 M borate, pH 7.5; and these buffers containing differentadditives (NaCl and CaCl2). Plasmid was eluted with 0.4 M
sodium phosphate, pH 7.0.

Table 1. Adsorption capacity of different plasmids on CHT Type II support.					pS3 (10 kb)
DNA loaded, µg*
Unbound, µg
Total bound, µg
Bound/eluted% Yield
DBC, µg/ml

PlasmidpS5 (9.48 kb)

pUC19 (2.6 kb)

1,390
1,197
193
151
78
151

2,950
2,574
376213
57
213

2,148
1,735
413
227
55227

*Samples were loaded in pH 7.0 phosphate buffer.

The yield and DBC were again similar for pS3 and pUC19 inphosphate buffer (Table 2). These values increased for pUC19
but decreased for pS5 in borate.
Table 2. Effect of buffer on plasmid DNA adsorption capacity on
CHT Type II support.
Plasmid in Phosphate
Buffer, pH 7.5
DNA loaded, µg
Unbound, µg
Total bound, µg
Bound/eluted% Yield
DBC, µg/ml

Plasmid in Borate
Buffer, pH 7.5

pUC19

pS3

pS5

pUC19		

pS5

2,950
2,574
376
213
57
213

2,148
1,735
413
227
55
227

1,390
1,197
193
151
78
151

2,800		2,300		500		358		72		358		1,370
1,219
151
835583

Addition of 0.3 M NaCl to the phosphate and borate buffers
significantly increased the DBC for pS5 (Table 3) but did not
improve the yield.
Table 3. Effect of 0.3 M NaCl on pS5 adsorption capacity on CHT in
pH 7.5 phosphate and borate buffers.			Phosphate Buffer		Borate Buffer		

Control		 0.3 M NaCl

Control		 0.3 M NaCl

DNA loaded, µg
Unbound, µg
Total bound, µg
Bound/eluted% Yield
DBC, µg/ml

1,390		1,197		193		151		78		151		

1,370		1,219		151		
83		55		
83		

1,380
803
577
398
69398

1,310
96834227480274

Table 4. Effect of different additives on pS5 adsorption capacity.
[NaCl]DNA loaded, µg*
Unbound DNA, µg
Total bound DNA, µg
Bound/eluted% Yield
DBC, µg/ml[CaCl 2]Control

0.3 M

0.6 M

1.0 M

1 mM

3 mM

5.5 mM

10 mM

1,370
1,219
151
83
55
83

1,310
968
342
274
80
274

1,320
701
619
540
87
540

1,310
698
612
518
85
518

1,480
1,301
179
136
76
136

1,400
814
586
357
61
357

1,350
779
571
485
85
485

1,280
542
738
651
88651

*Samples were loaded in 20 mM borate, pH 7.5 containing 1 mM EDTA.

Plasmid pS5 adsorption capacity on the CHT Type II support
was found to increase with sodium chloride up to 0.6 M in
borate buffer and then remained constant (Table 4). However,with calcium chloride, the adsorption capacity continued
to increase with concentration. At 5.5 mM and higher, theeffluent was turbid due to precipitation of calcium phosphate.
Adding EDTA to the effluent cleared the solution.
Discussion
The adsorption behavior and recovery of plasmid DNA onCHT varied with the size and type of the plasmid as well aswith the buffer systems and additives used. This is consistent
with observations that size, base composition, and secondary
structure affect plasmid binding to ion exchange supports(Ferreira et al. 2000). Plasmids prepared by different lysis
procedures may be nicked or denatured to various degrees,
also affecting binding capacities and recoveries (Ferreiraet al. 2000).

CHT adsorption capacity for plasmid DNA was enhanced
using sodium chloride. DNA swells at low ionic strength.
In the presence of sodium chloride, the plasmid DNA ismore tightly constricted, which may allow further penetration
into the pores of the CHT, resulting in increased binding.
The effects of an increase in calcium chloride concentration
may be due to bridging of the plasmids to each other or to
the phosphate residues of the CHT.
Use of the buffer systems described here, or further
modifications thereof, may significantly increase the DBC,
recovery — or both — of many other types of plasmid on
CHT and other chromatography media.
References
Aberin CS and Franklin SG (2002). Plasmid purification using CHT ceramichydroxyapatite support. Bio-Rad bulletin 2731.
Ferreira GN et al. (2000). Downstream processing of plasmid DNA for genetherapy and DNA vaccine applications. Trends Biotechnol 18, 380 – 388.
Information in this tech note was current as of the date of writing (2003) andnot necessarily the date this version (Rev A, 2009) was published.

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Life ScienceGroup

Bulletin 2946

Rev A

US/EG

09-03130509

Sig 0308