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Resin Screening to Optimize Chromatographic Separations

Article  in  Lc Gc North America · June 2001

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Anurag Rathore
Indian Institute of Technology Delhi
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616 LCGC VOLUME 19 NUMBER 6 JUNE 2001 www.chromatographyonline.com

Resin Screening to Optimize

Chromatographic Separations

The author presents an approach to screen resins for chromatographic

optimization in the purification process of a protein drug substance.
The protocol uses three steps to facilitate a rapid and efficient comparison
of different resins. In the first step, studies examine the binding between
the resin and the target molecule. Next, an identical shallow salt gradient
is used with all resins to generate information about the selectivity and
interactions between the product and the resin. The third step takes data
from preceding steps to provide a fair comparison of the various resins’
performance. The author used this approach with an anion-exchange
chromatography column in a typical protein purification process and was
able to identify an optimal resin.

Q uality and safety guidelines for

Anurag S. Rathore
Bioprocess and Formulation
Technology, Pharmacia Corp.,
Mail Stop GG3K,
700 Chesterfield Parkway North,
Chesterfield, Missouri 63198,
e-mail Anurag.S.Rathore@
pharmacia.com
biotechnology products have
grown increasingly stringent
and have made chromatogra-
phy one of the most important and com-
monly used operations in downstream
processing. Although any number of chro-
matography resins can be suitable for a par-
ticular separation, physicochemical differ-
ences between the resins can significantly
affect their performance for a given problem.
Resin selection is an integral part of chro-
matographic optimization in a purification
process, and it significantly affects the yield
and purity of unit operation (1–6). Further-
more, resin cost generally is one of the
largest contributors to purification costs
(7,8). Few articles in the literature, however,
address the issue of efficient chromatogra-
phy resin screening (5,6,9).
Screening chromatographic media is com-
plicated by the numerous products available
as well as the large number of variables that
users must consider. Comparison based on
full optimization of all conditions for each
resin is resource-intensive and impractical in
industrial environments. For this reason,
investigators typically will work with a lim-
ited number of resins such as those with
which they are familiar or those that are rec-
ommended in the literature. Analysts will
select the first resin that delivers satisfactory
performance and incorporate it into their
processes. In the absence of a systematic
approach, investigators may end up with a
suboptimal resin or spend significant effort
to arrive at a decision.
Alternatively, they might screen chro-
matographic media under a chosen set
of operating conditions. Although this
method allows for rapid screening, it suffers
from a major drawback: because the interac-
tions of a stationary phase with the various
sample components are very specific, the
optimal elution conditions for different
resins fall in a broad range. Thus, if separa-
tions are performed using different resins
under identical operating conditions, the
resulting variations in elution profiles can
make it difficult to compare resin perfor-
mances. Figure 1 shows the change in selec-
tivity during a gradient elution separation
after changing the slope of the gradient. An
identical ion-exchange chromatography gra-
dient for salt starting with a concentration of
0 and going to 2 is performed with
columns packed with resins A and B. Resin
A appears to provide a better separation;
however, as Figure 1 shows, resin B would
yield better resolution if the gradient slope
were halved.
The approach described in the following
sections bridges the gap between the two
extremes mentioned above and provides a
rapid and fair comparison of chromato-
graphic media from different vendors for a
618 LCGC VOLUME 19 NUMBER 6 JUNE 2001 www.chromatographyonline.com

Resin A Resin B Resin B Mode of chromatography

Prepare list of 5–10 resin candidates

based on experience, literature
precedents, and vendor input

Perform resin screening

0 x 2x 0 x 2x 0 x/2 x
Salt concentration Optimize operating conditions

Figure 1: Effect of gradient slope on selectivity. Figure 2: Optimization of a chromato-

graphic separation.

given separation problem. The primary
objective of my study is to select a resin that
yields the best selectivity for a particular sep-
aration. In practice, other issues such as the
physical and chemical stability of the pack-
ing and resin lifetime should be considered
as well.
too strongly (indicated by the absence of
product either in the flowthrough or the elu-
ent), the resin should be rejected from fur-
ther consideration.
In the next step, I pack columns separately
with resins that are still under consideration
and perform separations using an identical
test gradient of salt concentration (cA to c B)
in D column volumes. I choose a broad,
shallow gradient to ensure product elution
with all resins and magnify selectivity differ-
ences. I keep other operating parameters —
such as column dimensions, column vol-
ume, buffer, protein loading, and linear

Procedure
Optimization of chromatographic separa-
tion: Figure 2 illustrates my strategy for opti-
mizing a chromatographic separation. After Pick pH and buffer
selecting the mode of chromatography, I
prepare a list of 5–10 resin candidates based Pick a Step 1: Batch experiment
upon past experience with the molecule, lit- resin for checking binding
erature precedents, and input from chro-
matography media vendors. I screen the
identified resins using the protocol defined Is binding
appropriate? No
in the next section. Finally, I optimize the
operating parameters — buffer conditions End
Yes
and protein loading — for each chromatog- Remove resin
raphy step. Step 2: Run a test gradient
from further
Resin screening protocol: Figure 3 illus- consideration
trates the three-step resin screening strategy.
Although the procedure was designed for Is selectivity No
ion-exchange chromatography, it is applica- adequate?
ble to other modes of chromatography that End
use a gradient system such as hydrophobic Yes
Remove resin
interaction and reversed-phase chromatogra- from further
phy. Furthermore, the product in my study Yes Evaluate ce and tw consideration
was eluted using a salt gradient, as is typical
for protein products. The same philosophy Step 3: Run a comparison gradient
can be used to design a procedure that
addresses pH-change-induced elution in
Calculate recovery Economic
ion-exchange chromatography.
and pool purity considerations
The first step involves batch experiments
with all resins to compare their binding
characteristics with the product. The buffer Any resins
No Final decision
selection is based upon past experience with remaining?
the molecule and literature precedents. If the
product binds too weakly with the resin
(indicated by product in the flowthrough) or Figure 3: Resin screening protocol.
620 LCGC VOLUME 19 NUMBER 6 JUNE 2001
velocity — constant. I then analyze peak
fractions using the appropriate analytical
method to obtain the product and impurity
profiles. These profiles provide useful infor-
mation about the selectivity of the station-
ary phase; that is, the ability of the resin to
differentiate between the product and the
impurities. Based upon these data, I elimi-
nate from further consideration resins that
show little or no selectivity.
For resins that show acceptable selectivity,
I use data from the test gradient to deter-
mine the ionic strength corresponding to
the elution of the product from the resin
(ce ) and the peak width at 20% height from
the baseline (tw in minutes).
In the third step, I pack columns with
resins that are still under evaluation and per-
form separations using a comparison gradi-
ent. In a comparison gradient, the product
peak is eluted in the center of an n column
volume gradient with the product peak
eluted in approximately m column volumes,
where m and n are variables chosen depend-
ing upon the purpose of the column. Elu-
tion profiles obtained under these gradients
would produce very similar elution profiles
and thus provide a fair comparison of per-
formance of the various resins. The com-
parison gradient is defined to start at one
concentration (c1) and go to another con-
centration (c2 ) in n column volumes in the
following manner:
c B  c A tw v
c2  ce  n [1]
2mD V
and
c B  c A tw v
c1  ce  n [2]
2mD V
such that if c1  0, then c1  0
where ce and tw are obtained under the test
gradient (cA to c B in D column volumes), V
is the column volume in milliliters, and v is
the flow velocity in milliliters per minute. I
use two parameters — product recovery and
pool purity — to evaluate resin perfor-
mance. Product recovery is defined as the
sum of product peak areas in milliab-
sorbance units of the pooled fractions per
milliliter of injected sample. Pool purity is
defined as the purity of the total pool
formed by combining the fractions that
meet the pooling criteria. Although the
product recovery measures the quantity of
product that can be recovered with desig-
nated purity, the pool purity describes the
quality of the pool. After I have determined
the product recovery and pool purity for
each resin, I can use these criteria along with
economic considerations to make a more
accurate choice of the optimal resin.
Case Study
Resin screening for an anion-exchange
chromatography column: This case study
presents data obtained during the optimiza-
tion of an anion-exchange chromatography
column used in the purification process of
a microbial-fermentation-derived protein
molecule.
I chose to screen nine anion-exchange
resins: Bio-Rad High Q and Bio-Rad
DEAE (Bio-Rad Laboratories, Hercules,
California); Pharmacia DEAE FF, Pharma-
cia Q FF, and Pharmacia Q HP (Amersham
Pharmacia Biotech Inc., Piscataway, New
Jersey); Whatman Q, Whatman QA52, and
Whatman DE53 (Whatman Inc., Clifton,
New Jersey); and TosoHaas Q650M (Tosoh
Biosep, Montgomeryville, Pennsylvania).
I performed all chromatography experi-
ments using an Åkta Explorer chromatog-
raphy system (Amersham Pharmacia
Biotech). I chose the buffer and other oper-
ating conditions based upon prior experi-
ence with the protein molecule: I used a
pre-equilibration buffer of 1 M Tris (pH
8.5), an equilibration buffer of 50 mM Tris
(pH 8.5), and protein loading at 10 mg/mL
resin. Because the objective of this article is
to present an efficient resin screening proto-
col — not to recommend a particular resin
— I will refer to these resins as resins 1 to 9
(not in the same order as listed above). I
expected that the optimum resin would vary
with the separation problem.
I packed the columns with 1 mL of resin
and equilibrated them for 30 min with the
Table I: Final comparison of resins considered
Resins* Binding Selectivity
Resin 1 Unacceptable — —
Resin 2 Satisfactory Unacceptable
Resin 3 Satisfactory Unacceptable
Resin 4 Satisfactory Unacceptable
Resin 5 Satisfactory Unacceptable
Resin 6 Satisfactory Satisfactory
Resin 7 Satisfactory Satisfactory
Resin 8 Satisfactory Satisfactory
Resin 9 Satisfactory Satisfactory
* Resins 1 to 9 (not in order) include Bio-Rad DEAE, Bio-Rad High Q, TosoHaas Q650M, Whatman Q, What-
man QA52, Whatman DE53, Pharmacia DEAE FF, Pharmacia Q FF, and Pharmacia Q HP.
† Based on measurements by anion-exchange HPLC (anion-exchange HPLC).
www.chromatographyonline.com
equilibration buffer. After equilibration, I
loaded a protein solution that contained
1–2 times the intended protein loading for
the respective column in milligrams protein
per milliliter resin). After 30 min, I washed
the columns with 5 mL of equilibration
buffer and collected the flowthrough
stream. The protein was eluted with 10 mL
of elution buffer (1 M sodium chloride in
the equilibration buffer), and the protein
was collected separately. I analyzed the
flowthroughs and the eluents for protein by
UV absorbance at 280 nm and for its purity
by anion-exchange high performance liquid
chromatography (HPLC). As Table I shows,
most resins showed satisfactory binding
characteristics with the product. Only resin
1 showed anomalous behavior in that the
product was not retained under these con-
ditions, so resin 1 was removed from further
consideration.
Next, I packed columns with 10 mL of
the remaining eight resins and performed
separations using an identical test gradient
of 0–500 mM sodium chloride in 20 col-
umn volumes of equilibration buffer. The
peak fractions were analyzed by anion-
exchange HPLC. Figure 4 illustrates the
performance of resins 3–5 using a test gra-
dient of 0–500 mM sodium chloride in 20
column volumes. The y axis in Figures 4
and 5 denotes the peak area obtained upon
analysis by anion-exchange HPLC. The
flow velocity and the fraction sizes are listed
in the figure captions. It is evident that run-
ning identical gradients with different resins
leads to widely varying elution profiles in
terms of the peak width and peak position
in the overall gradient. The poor selectivity
obtained with resins 3–5 led to their elimi-
nation from further consideration.
As listed in Table I, only resins 6–9
showed satisfactory selectivity between the
Recovery† Pool Purity† Final
(mAU/mL) (%) Decision
— —
— — —
— — —
— — —
— — —
Unacceptable — —
75.4 96.5 Unacceptable
25.3 100 Unacceptable
81.5 97.7 Satisfactory
622 LCGC VOLUME 19 NUMBER 6 JUNE 2001 www.chromatographyonline.com

product and the impurity. Because resin 9
exhibited better resolution than resin 6 and
they had identical matrix and ligand chem-
istry, the former was chosen over the latter
for further consideration.
I calculated comparison gradients for
resins 7–9 according to the procedure
described above. Product recovery was
defined as the sum of product peak areas in
milliabsorbance units in the pooled frac-
tions (having greater than 90% purity by
Table II: Calculation of the comparison gradients*
anion-exchange HPLC) per milliliter of
injected sample. Pool purity was defined as
the purity of the total pool formed by mix-
ing the fractions that meet the pooling cri-
teria. Table II shows the calculation of the
comparison gradient for these three resins,
and Figure 5 illustrates the protein and
impurity profiles obtained after fraction
analysis by anion-exchange HPLC. The fig-
ure shows the product recovery and pool
purity values for the experiment.
Figure 5 reinforces the understanding
that performing separations with designed
comparison gradients yields similar elution
profiles with different resins and enables fair
comparison of resin performance. Figure 5
also illustrates that resin 8 showed good
purity but poor recovery. Resins 7 and 9
showed comparable recovery and pool
purity. Because of better selectivity, I chose
resin 9 as the resin for this purification
process and for further optimization of
buffer pH, protein loading, feed flow rate,
elution flow rate, gradient slope, and col-
umn length.

tw ce cA cB V v c1 c2 Summary
Resins† (min) (mM) (mM) (mM) D‡ (mL) (mL/min) (mM) (mM) Table I summarizes the results I obtained
Resin 7 5.21 110 100 200 20 10 1.7 90 130 using this resin screening strategy. I screened
Resin 8 6.61 109 100 200 20 10 0.9 80 130 a total of nine resins. Based upon my expe-
Resin 9 7.35 146 100 300 20 10 1.7 100 200 rience, this protocol presents a method for
efficient screening of resins and allows me to
* Symbols used in Table II are described in the text.
† Same as Table I.
screen 10 resins in a month. Because resins
‡ Number of column volumes. differ in their physicochemical interactions
with the various feed components, using
screening techniques allows analysts to opti-
mize the chromatographic process.

Acknowledgments
(a) (a) Pool purity: 96% The author would like to thank J.O.
Protein Polazzi, R.A. Heeren, G.V. Johnson, and
Injection (mAU/L)

Protein
Injection (mAU/L)

200 anion-exchange HPLC 70

anion-exchange HPLC B.K. Matthews of Pharmacia Corp. (Ches-
60
150 Impurity
50 Impurity
anion-exchange HPLC anion-exchange HPLC terfield, Missouri) for their help in experi-
100 40
30
mentation and M.E. Gustafson, D.E.
50
20 Steinmeyer, and J.K. McLaughlin, also of
0
10 Pharmacia, for their review of the manu-
0 script and helpful discussions.
(b) 350
(b)
300 References
Injection (mAU/L)

Injection (mAU/L)

Pool purity: 100% (1) J.-C. Janson and P. Hedman, Adv. Biochem.
250 40
Eng. 25, 43–99 (1982).
200 30
20 (2) S. Fulton and T. Londo, in Bioseparations and
150 Bioprocessing, G. Subramanian, Ed. (Wiley-
10
100 0 VCH, New York, 1998), pp. 41–64.
50 (3) G. Sofer and L. Hagel, Handbook of Process
0 Chromatography (Academic Press, New York,
(c) 80 Pool purity: 98% 1997), p. 54.
(4) P. Hedman, J.C. Janson, B. Arve, and J.G.
Injection (mAU/L)

(c) 70
60 Gustafsson, in Eighth International Biotechnol-
Injection (mAU/L)

50 ogy Symposium, G. Durand, L. Bobichon, and J.

150 40 Florent, Eds. (Société Mycologique de France,
100 30 Paris, 1988), pp. 623–643.
50 20 (5) R. Wisniewski, E. Boschetti, and A. Jungbauer,
0 10 in Biotechnology and Biopharmaceutical Manu-
0 facturing, Processing and Preservation, K.E. Avis
0 2 4 6 8 10 12
0 5 10 15 and V.L. Wu, Eds. (Interpharm Press, Engle-
Fraction number wood, Colorado, 1996), pp. 61–198.
Fraction number
(6) G. Sofer and C. Munson, Bio/Technology 5,
239–244 (1987).
Figure 4: Performance under test gradients. Figure 5: Performance under comparison (7) T.C. Ransohoff, M.K. Murphy, and H.L.
(a) Resin 3: 100 cm/h, 0–500 mM sodium chlo- gradients. (a) Resin 7: 50 cm/h, 90–130 mM Levine, BioPharm 3(3), 20–26 (1990).
ride in 20 column volumes, 3-mL fractions. (b) sodium chloride in 11 column volumes, 75- (8) A.P. Peskin and S.R. Rudge, App. Biochem.
Resin 4: 100 cm/h, 0–500 mM sodium chloride in mAU/mL recovery. (b) Resin 8: 50 cm/h, 80–130 Biotech. 34/35, 49–59 (1992).
20 column volumes, 5-mL fractions. (c) Resin 5: mM sodium chloride in 11 column volumes, 25- (9) P.R. Levison, C. Mumford, M. Streater, A.
50 cm/h, 0–500 mM sodium chloride in 20 col- mAU/mL recovery. (c) Resin 9: 50 cm/h, 100–200 Brandt-Nielsen, N.D. Pathirana, and S.E. Bad-
umn volumes, 2-mL fractions. mM sodium chloride in 11 column volumes, 82- ger, J. Chromatogr. A 760, 151–158 (1997). 
mAU/mL recovery.

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