Peak

The
The

Volume 7, Issue 2
A TOSOHAAS Newsletter
Preparative Scale-Up of Size Exclusion
Chromatography
Size exclusion chromatography (SEC) is often used as
a preliminary purification method particularly in analyt-
ical laboratories. Initial scale-up considerations usually
employ SEC for purification of material so that it can be
subjected to amino acid analysis, amino acid
sequencing, initial validation of biological assays, or for
generating enough material to initiate immunological
experiments.
Of course, with any product, the usual constraints are
placed on the chromatographic separation, and these
include yield, purity, cycle time and mobile phases.
Yield is important because the cost of the raw material
may be prohibitive in the early stages, especially if the
expression or synthesis of the product has not been
optimized. Purity is important to obtain accurate amino
acid analysis or amino acid sequencing (Edman
Degradation). The generation of antibodies for ELISA
or other immunological characterization requires rela-
tively pure product. Cycle time is considered particu-
larly critical if the product of interest is labile or if the
time constraints placed on the generation of the
product are very short. Mobile phases are particularly
important because of the product lability or if the
mobile phases are necessary for solubility, stability, etc.
Included here would be the need for organic solvents
as well, and whether these solvents are environmen-
tally friendly.
This article assumes that an analytical size exclusion
chromatography column has been successfully
employed to purify small amounts of the target protein.
This includes optimizing the separation by investigating
column length, flow rate, pore size and particle size.
Also, this article assumes that the mobile phase has
been optimized to some extent for this particular
product. TosoHaas offers a wide range of size exclusion
chromatography columns which utilize silica as well as
polymer based media. There is also a full line of gel
filtration products which can be successfully used for
non-aqueous applications.
As we begin this discussion, it is apparent that there
may be several ways to approach the problem
described in this article. Here we discuss one way that
has been successfully employed in the laboratories at
TosoHaas.
The basic variables used to calculate a size exclusion
chromatography scale-up are listed in Table I on the
following page.
continued on page 2

TOSOHAAS Contents
156 Keystone Drive GmbH UK:
Montgomeryville, PA 18936 Zettachring 6 Phone: +44-1223-890098 ■ Prepara- ■ Prepara- ■ One-Step ■ One-Step ■ One-Step
Phone: 215 283-5000 D-70567 Stuttgart Fax: +44-1223-893783 tive Scale- tive Scale- Oligonu- Oligonu- Oligonu-
Fax: 215 283-5035 Germany France: Up... (cont.) Up... (cont.) cleotide cleotide cleotide
Orders and Service: Phone: +49-711-13257-0 Phone: +33-3-21252007 Purification Purification Purification
800 366-4875 Fax: +49-711-13257-89 Fax: +33-3-21252008 (cont.) (cont.)
■ Confer-
ences
Or reach us via the Internet:

2 3 4 5 6
World Wide Web Address: http://www.tosohaas.com
Orders and Sales E-Mail Address: tosohaas_direct@rohmhaas.com
Technical Service E-Mail Address: tosohaas_tech@rohmhaas.com
 Preparative Scale-Up (cont.)
diameter of the preparative column, dc2:
Table I dc2
Variables Used in the A2 = a 5 A1 = 5 π (Eq. 2)
2
Semi-Preparative Scale-Up of SEC
It is necessary to redefine the column diameter using a
a Scale-up factor dimensionless number
commercially available column with its diameter closest
dp1 Particle Diameter µm
to the calculated value:
dc1 Column Diameter cm
Z1 Column Length cm dc2' ≈ dc2
A1 Column Cross Sectional Area cm2 (corrected to the closest available column diameter)
Vt1 Column Volume cm3 or ml and then using dc2' recalculate the cross sectional area
F1 Volumetric Flow Rate (ml/min) to account for this change in diameter:
1 Empty Column Linear Velocity (ml/hr/cm2) or (cm/hr)
dc2'
X C1 Sample Volume ml A 2' = 5 π (Eq. 3)
X O1 Relative Sample Volume dimensionless number
2
P1 Product Produced mg/cycle The preparative column length, Z 2, is also calculated
Ts1 Cycle Time min using the scale-up factor:
Many of these variables are self explanatory, however a Vt2 = a 5 Vt1 = A 2' 5 Z1 (Eq. 4)
few need further discussion. The cycle time is not a
and
true cycle time, but an estimate of when the first peaks
might appear on the chromatogram. The actual cycle Vt2
Z2 = (Eq. 5)
time will include the time it takes between the void A 2'
volume and the included volume of the column. For
TSK-GEL® size exclusion columns, the void volume is It is again necessary to redefine the column length
approximately 40% of the empty column volume and using a commercially available column with its length
the included volume is estimated to be about 80% of closest to the calculated value, Z 2, which is now repre-
the empty column volume. In addition, there is no sented as Z 2'. The relative sample size is calculated by
accounting for any column cleaning between runs. All the following ratios:
of these will increase the actual cycle time of the purifi- X C1 X C2
cation. The Relative Sample Volume is a fraction of the X O1 = ≈ X O2 = (Eq. 6)
sample injected to the total column volume Vt1 Vt2
X C1 In general, as one scales up most SEC applications,
represented by X O1 = . The ratio X O1 should not
Vt1 there are mechanical limitations present. These are
exceed 0.25 (25%), or the resolution of the product manifested in the pressure/flow characteristics of the
from the contaminants will be severely impaired. The chromatographic system. The primary way to alleviate
amount of the product of interest produced is P. In these limitations is to increase the particle size of the
some cases one may desire to purify the entire amount media. If the particle size remains constant, the
of P in one experiment, whereas it may be more problem is simplified. However, as is the case with most
economical and efficient to produce the product of SEC scale-up methods, we must account for the
interest by pooling the samples from several column change in particle size. The relationship between
runs. column length, linear flow rate, and particle size
between two columns is represented as follows:
The first step in this process is to determine the scale-
up ratio, a, which is defined as: Z2 Z1
= (Eq. 7)
P2 2
(dp2 )(µ2) (dp12)(µ1)
a= (Eq. 1)
P1
The linear velocity of the preparative column is deter-
where P refers to the production rate of the analytical mined by rearranging Equation 7:
(subscript 1) and scaled-up (subscript 2) chromato- Z2 dp12
graphic steps. Using the scale-up factor, a, calculate µ2 = µ1 5 5 (Eq. 8)
Z1 dp22
the new column cross sectional area, A 2, and then the

continued on page 3

The Peak A TOSOHAAS Newsletter Page two

 Preparative Scale-Up (cont.)
Finally, calculate and then double check that the cycle lating the diameter of the scaled-up column using
time is realistic for the desired product and the time Equation 2:
constraints of the particular laboratory. In addition, dc2
confirm that the relative sample volume, X O1, is real- A2 = a 5 A1 = 5 π (Eq. 2)
istic and within the limitations of the sample itself and 2
the column selected. The scaled-up column diameter, dc2, is calculated to
A practical example of this strategy employs a 30-fold be:
scale up of a proprietary molecule on the TSKgel dc2 = 11.8 c m
G3000SW column. Table II lists the sample data from
the laboratory scale experiments and Table III lists the As indicated above, use the commercially available
requirements of the larger scale process. column with the closest diameter. Using TSK-GEL
columns as a guide, the nearest column diameter is
Table II dc2' = 10.8 cm. Use scale up factor and the analytical
Sample Data from column volume to calculate the new column length:
Laboratory Scale Separation Vt2 = Vt1 5 a = (30)(217.8) = 6,534 ml = A 2 5 Z 2
Sample: proprietary remembering that A 2' is the corrected cross sectional
Sorbant: TSKgel G3000SW (13 µm) area using the commercially available column with the
Column: 21.5 mm ID x 60 cm L diameter dc2' = 10.8 cm, and then solving for Z 2:
(TosoHaas PN 05147)
Flow Rate: 7.5 ml/min (25°C) Z 2 = 71 c m
Sample Loading: 150 mg/cycle in a 1% Solution The closest commercially available column length is
60 cm = Z 2' and the final column volume is recalcu-
lated:
Table III
Scale-Up Requirements Vt2 = A 2' 5 Z 2' = 5,946 ml

Sample Loading: 4,500 mg/cycle The column dimensions are now defined. All that
Operation: 1 cycle/day (8 hrs.) remains is to calculate the operating conditions for the
Conditions: 25°C preparative column packed with the larger particle size
Must use 20 µm resin
media. Using Equation 8:
Z2 dp12
The first step is to calculate the cross sectional area µ2 = µ1 (Eq. 8)
Z1 dp22
A 1, the volume, Vt1, the linear flow rate, µ1, and the
sample volume, X C1, of the analytical column: the linear flow rate of the preparative column is deter-
21.5 c m 2 mined:
A1 = π = 3.63 c m2 60 c m (13 c m)2
2 µ2 = 124 cm/hr = 124 cm/hr
2 3
60 c m (20 c m)2
Vt1 = A 1 5 Z 1 = 3.63 c m 5 60 c m = 217.8 c m = 217.8 ml
µ1 = (7.5 ml/min) 5 (60 min/3.63 c m2) = 124 cm/hr which is converted to a volumetric flow rate:
0.150 g/cycle F 2 = µ2 5 A 2 = (52.4 cm/hr) 5 (91.6 c m2) =
X C1 = = 15 ml/cycle 4800 ml/hr or 80 ml/min
(1 g/100 ml)
The cycle time, Ts2, is calculated as follows:
Using Equation 1, determine the scale-up factor:
Z 2' 60 c m
(4,500 mg/cycle) Ts2 = = = 1.14 hr or 68 min
a= = 30 µ2 52.4 cm/hr
(150 mg/cycle)
and lastly the sample loading is assessed:
Calculate the relative sample volume using the ratios
described in Equation 6: X C2 =(30)(15) =(450 ml (of a 1% solution).
X C1 15 ml X C1 X C2
X O1 = = or 6.9% X O1 = ≈ X O2 = = 0.086 or 8.6%
Vt1 217.8 ml
Vt1 Vt2
Now calculate the column dimensions by first calcu-
continued on page 4

The Peak A TOSOHAAS Newsletter Page three

 Preparative Scale-Up (cont.)
These results demonstrate that scaling-up of size
exclusion chromatography is relatively simple if the Table IV
required variables are identified and a sound strategy is Comparison of Analytical and
followed. The preparative column (108 mm ID x 60 cm Preparative Scale Columns
L TSKgel G3000SW column with 20 µm particle size) is
not listed in the TosoHaas catalog, however, it is avail- Analytical Preparative
able by special order. Scale Scale

If there are any questions about scaling-up SEC appli- Particle Size of Media (µm) dp 13 20
cations and the availability of large HPLC columns not
Column Diameter (cm) dc 2.15 10.8
listed in the TosoHaas catalog, please call our expert
Technical Service group at 1-800-366-4875 or e-mail Column Length (cm) Z 60 60
your questions to tosohaas_tech@rohmhaas.com. In
Column Volume (ml) Vt 218 5494
addition, a rather extensive compilation of size exclu-
sion chromatography applications and products are Volumetric Flow Rate (ml/min) F 7.5 80
listed in a recently published book Column Handbook
Sample Loading (ml/cycle) XC 150 4500
for Size Exclusion Chromatography edited by Chi-san
Wu and published by Academic Press in April, 1999. Relative Sample Volume XO 6.9% 8.6%
Look for Chapter 4 which reviews all of the commer-
Cycle Time (min) Ts 29 68
cially available size exclusion products provided by
TosoHaas. Turn to the Fax Back page to request litera-
ture on size exclusion chromatography products.

Conferences
Look for TosoHaas at the following upcoming conferences. We look forward to meeting you and hearing about your needs.
Conference Location Dates Booth # Conference Location Dates Booth #
PREP ‘99 San Francisco, CA May 23-26 17 BioPharm West San Francisco, CA June 9-11 21
HPLC ‘99 Granada, Spain May 30-June 4 59 BioPharm East Boston, MA June 16-18 29
ASGT Washington, DC June 9-13 209 Biotop ‘99 Brussels, Belgium July 12-15 N/A

One Step Oligonucleotide Purification With

AmberchromTM Reversed Phase Resin
INTRODUCTION
The use of synthetic oligonucleotides for primers, diag-
nostics, and therapeutics has increased dramatically in
the past ten years. The increased need for high purity
oligonucleotides means that purification of these
compounds must be done economically, simply, and
reproducibly.
The objective of this study is to develop a chromato-
graphic purification process for a crude, synthetic,
DMT-on, phosphodiester oligonucleotide. The target
compound is a 25 base phosphodiester oligonu-
cleotide. By modifying the reversed phase purification
method originally developed by Somers1, a separation
process was developed on a 1.0 cm ID x 6.2 cm L,
lab-scale column, using an experimental 15 µm, poly-
meric reversed phase resin with a triethyl ammonium
acetate/acetonitrile mobile phase. The experimental
resin was styrene/divinylbenzene with an average pore
diameter of 300 Å. The separation was also performed
using the same column configuration and
Amberchrom® CG-300s, a commercially available
35 µm styrene/divinylbenzene resin also with an
average pore diameter of 300 Å . The process effec-
tively combined removal of failure sequences, on-
column detritylation, and elution of a high-purity
oligonucleotide. The process was able to deliver purity
levels >93% with yields >50% at laboratory scale. This
work is significant because it demonstrates that larger
quantities of oligonucleotides can be successfully puri-
fied in a one-step chromatographic process for thera-
peutic purposes.
GOALS AND CHALLENGES
The target compound is a 25 base phosphodiester
oligonucleotide (25-mer product). The goal is to
continued on page 5

The Peak A TOSOHAAS Newsletter Page four

 One Step Oligonucleotide Purification (cont.)
perform on-column detritylation and obtain product
which is >93% pure with a yield >50%. Table I
Results - Initial Experiments
The key challenges are to obtain optimal detritylation,
obtaining separation of the 25-mer product from the n-1 Amberchrom CG-300 experimental
(24-mer) impurity and from any tritylated species Fraction No. Purity Yield
remaining after the detritylation step. 4 58.3% 10%
5 88.9% 85%
SYNTHESIS AND CLEAVAGE
6 52.4% 2%
The oligonucleotide was synthesized using a Amberchrom CG-300s
Pharmacia OligoPilot II with a 20 x 20 mm (6.28 ml) Fraction No. Purity Yield
column reactor. Phosphoramidites were purchased 4 4.9% 0.1%
from Pharmacia. The support was cleaved from the 5 87.3% 101%
oligonucleotide using the following method: 6 17.3% 0.5%
Approximately 70-80 mg of oligonucleotide (attached to
synthetic support) were weighed out into a 1.0 oz. Anion exchange chromatography using a DEAE phase
glass vial. Then 2-3 ml of concentrated ammonium was employed to determine product purity and yields.
hydroxide were added to the vial, and the contents
were heated at 60°C overnight. The oligonucleotide INITIAL CONDITIONS
was washed with 3 ml of aqueous ethanol 50/50 (v/v), As evident in Table I, with the initial conditions the
and the final volume was adjusted to 10 ml using addi- purity goal of >93% was not met. This is due, in part, to
tional concentrated ammonium hydroxide. The sample the presence of the n-1 impurity which can be
was then filtered with a 0.45 µm filter. observed to co-elute with the product peak. It is repre-
BASIC PURIFICATION SCHEME sented in the chromatogram as a “fronting” of the
product peak, R.T. ≈ 25 minutes (see Figure 1). This
The basic purification scheme, based on a process can be addressed by calculating the approximate
developed by Somers1 was as follows: acetonitrile concentration during elution of the target
• Loading of the oligonucleotide in 0.1M triethyl compound, and changing the eluent profile during the
ammonium acetate (TEAA) gradient step by making the gradient more “shallow”.
• Elution of trityl-off failures in 15% acetonitrile/85% Additionally, at approximately 30 minutes the trityl-on
0.1M TEAA material is represented by a significant response
• Detritylation using 0.5% trifluoroacetic acid (TFA) (Figure 1). This indicates that there is a significant
quantity of trityl-on material remaining after the detrity-
• Elution of trityl-off oligo product using acetonitrile
lation step. This can be remedied by increasing the
gradient
contact time of the detritylating agent through an
• Wash with 100% acetonitrile adjustment in flow rate.

Figure 1
Results - Initial Experiments
Column: 1.0 cm I.D. x 6.2 cm L
Injection Volume: 100µl
Flow Rate: 5.0 ml/min. (382 cm/hr) Amberchrom CG-300 Experimental Amberchrom CG-300s
Mobile Phase:
A: 0.1M triethyl ammonium acetate (TEAA)
B: Acetonitrile mau mau
C: 0.5% trifluoroacetic acid (TFA) 100 100
Detection: UV@254nm, w/ 2AUFS 90
90
Eluent Profile:
80 80
1 CV A - Loading
70 70
2 CV 15%B - Elution of trityl-off failures
10 CV C - Detritylation 60 60
3 CV A - Equilibration 50 50
10 CV 0→30% B - Elution of trityl-off product 40 40
Fraction Volume: 2.5 ml 5 10 15 20 25 30 min 5 10 15 20 25 30 min

continued on page 6
The Peak A TOSOHAAS Newsletter Page five
 One Step Oligonucleotide Purification (cont.)

Figure 2
Results - Amberchrom CG-300s, Optimized
Column: 1.0 cm ID x 6.2 cm L
Injection Volume: 100 µl
Flow Rate: 0.5 ml/min (38 cm/hr) mau
Mobile Phase:
100
A: 0.1 M triethyl ammonium acetate (TEAA)
B: Acetonitrile 90
C: 0.5% trifluoroacetic acid (TFA)
80
Detection: UV@254 nm, w/ 2AUFS
Eluent Profile: 70
1 CV A - Loading
60
2 CV 15%B - Elution of trityl-off failures
10 CV C - Detritylation 50
3 CV A - Equilibration
10 CV 0→30% B - Elution of trityl-off product 40
Fraction Volume: 0.5 ml 0 20 40 60 80 100 120 min

OPTIMIZATION OPPORTUNITIES CONCLUSION

• “Shallow” the gradient A reversed phase resin, Amberchrom CG-300, was
• Increase detritylation time successfully employed for a one step oligonucleotide
purification, which included on-column detritylation.
• Optimize fraction collection volumes
• Decrease linear velocity By manipulating the gradient, linear velocity, detrityla-
tion time, and fraction volume, our initial yield and
OPTIMIZED CONDITIONS purity targets were met.
As the result of the changes made during optimization, REFERENCES:
the resolution of the 25-mer product from the n-1 impu-
1 Somers, R., “A One Step, Reverse Phase Purification Scheme for
rity was significantly enhanced (Figure 2). Rather than the Purification of DMT-on Oligonucleotides”, Poster presented at
appearing as a “front” on the product peak, two distinct PrepTech ’95, East Rutherford, NJ, February 13-15, 1995.
peaks are present. The quantity of trityl-on material
present has been effectively reduced.
Table II
As evident from Figure 3, we can now readily obtain Results - Optimized Conditions
our goal of product which is >93% pure with a yield
>50%, by combining the appropriate fractions. In fact a Amberchrom CG-300s, optimized conditions
product was obtained which is ≈97% pure with a yield Fraction No. Purity Yield
of ≈74% by combining fractions 4→8. 1 42.3% 1%
2 81.6% 5%
3 92.3% 12%
Figure 3
4 96.3% 16%
Results - Optimized Conditions
Yield Curve for Amberchrom CG-300s, 1.0 cm ID x 6.2 cm L 5 97.5% 16%
18 6 98.0% 16%
16 7 98.0% 14%
25mer product
14 n-1 impurity 8 97.3% 12%
12 9 95.0% 9%
% Yield

10
10 91.9% 5%
8
11 85.1% 2%
6
12 69% 1%
4
2
For additional information, turn to the Fax Back form and
0
1 2 3 4 5 6 7 8 9 10 11 12 check Literature 27A21 and Literature 27A32.
Fraction Number
TSK-GEL is a registered trademark of Tosoh Corporation. Amberchrom is a
trademark of Rohm and Haas Company.

The Peak A TOSOHAAS Newsletter Page six

 Fax Back Photocopy this page and fax to
TOSOHAAS
North America fax#: 215-283-5035 Europe fax#: +49-711-13257-89

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Request for Literature:

Fax this page or request this literature from the TosoHaas Web site
http://www.tosohaas.com/freeinfo.html#form
Size Exclusion Chromatography: Oligonucleotide Purification:
28A07 - Toyopearl HW Media for Literature 27A21 - A One Step Reversed
Scale-Up Size Exclusion Chromatography Phase Purification Scheme for the
of Nucleic Acids and Proteins Purification of DMT-on Oligonucleotides
28B12 - TSK-GEL SW Series Literature 27A32 - Development,
28B73 - TSKgel Super SW2000 and Optimization, and Scales-Up of a
TSKgel Super SW3000 Chromatographic Purification for a
Synthetic Oligonucleotide
TosoHaas Catalog
1999 TosoHaas Date Planner
TosoHaas 1999 Price List

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