Bulletin 1913

3/10/99 8:19 AM

Page 1Developing and Scaling Up A Purification Procedure
With Two New Ion Exchange Supports
M. Navvab, M. Abouelezz, T. Tisch, E. Scheinpflug, R. Frost, P. Tunón
Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547

0Separation of Rattlesnake
Venom

The study was started at 2 mg per ml of support in an analytical 2 ml Bio-Scale Q2 column(Figure 2 A), and then step wise transferred tolarger columns with increasing sample loads(Figure 2 B and C). The last column, packed
with 60 ml of the 50 µm Macro-Prep high Q
support, was used to separate 1 gram of sample (Figure 2 D).

Gradient [NaCl]

OD280

0VolumeVolumeSample: 105 mg
Column: 20 ml of 50 µm Macro-Prep highS support, I.D. 1.5 cm
Flow Rate: 78 cm/h (2.3 ml/min)Gradient time: 195.6 min
Gradient volume: 450 ml
Buffer A: 20 mM sodium acetate, pH 4.8
Buffer B: A + 1.0 M NaCl
Gradient:
0% B in 2.5 column volumes
0-100% B in 22.5 column volumes

Sample: 20 mg
Column: Bio-Scale S5 column (5 ml of 10 µm
Macro-Prep high S support, I.D. 1.0 cm)
Flow Rate: 78 cm/h (1.02 ml/min)Gradient time: 112.5 minutes
Gradient volume: 112.5 ml
Buffer A: 20 mM sodium acetate, pH 4.8
Buffer B: A + 1.0 M NaCl
Gradient:
0% B in 2.5 column volumes
0-100% B in 22.5 column volumes

Sample: 1 gram
Column: 57 ml of 50 µm Macro-Prep highS support, I.D. 2.5 cm.
Flow Rate: 25 ml/min (306 cm/h)
Gradient time: 217 min
Gradient volume: 1,358 min
Buffer A: 20 mM sodium acetate, pH 4.8
Buffer B: A + 1.0 M NaCl
Gradient:0% B in 2.5 column volumes
0-100% B in 22.5 column volumes

0VolumeSample: 4 mg
Column: Bio-Scale Q2 column (2 ml of 10 µm
Macro-Prep high Q support, I.D. 0.7 cm)
Flow Rate: 78 cm/h (0.5 ml/min)Gradient time: 90 min
Gradient volume: 45 ml
Buffer A: 50 mM Tris-HCl, pH 8.3
Buffer B: A + 1.0 M NaCl
Gradient:
0% B in 2.5 column volumes
0-100% B in 22.5 column volumes

0VolumeSample: 20 mg
Column: Bio-Scale Q5 column (5 ml of 10 µm
Macro-Prep high Q support, I.D. 1.0 cm)
Flow Rate: 78 cm/h (1.02 ml/min)Gradient time: 112.5 min
Gradient volume: 112.5 ml
Buffer A: 50 mM Tris-HCl, pH 8.3
Buffer B: A + 1.0 M NaCl
Gradient:0% B in 2.5 column volumes
0-100% B in 22.5 column volumes

0.500
VolumeVolumeSample: 105 mg
Column: 20 ml of 50 µm Macro-Prephigh Q support
Flow Rate: 78 cm/h (2.3 ml/min)Gradient time: 195.6 min
Gradient volume: 450 ml
Buffer A: 50 mM Tris-HCl, pH 8.3
Buffer B: A + 1.0 M NaCl
Gradient:0% B in 2.5 column volumes
0-100% B in 22.5 column volumes

Sample: 1 gram
Column: 60 ml of 50 µm Macro-Prephigh Q support
Flow Rate: 78 cm/h (6.4 ml/min)Gradient time: 217 min
Gradient volume: 1,358 ml
Buffer A: 50 mM Tris-HCl, pH 8.3
Buffer B: A + 1.0 M NaCl
Gradient:
0% B in 2.5 column volumes
0-100% B in 22.5 column volumes

Materials and Methods

Discussion

References

Both samples were obtained from Sigma
Chemical Company (St. Louis, MO). The
snake venom (catalog number V-7000) wasdissolved in 20 mM sodium acetate, pH 4.8,
and filtered prior to application. The yeast
extract (catalog number Y-3000) was dissolvedin 50 mM Tris-HCl, pH 8.3, and filtered prior toapplication.

Developing methods using the high resolution
Bio-Scale columns allows rapid development
and optimization, using minimal amounts ofbuffer and sample. Once the method is developed, it is easily transferred to the larger 50 µm
Macro-Prep bead.

1. Faure, G., et al. , European Journal of
Biochemistry, 214 (2), 491-496 (1993).

All separations were performed using a Bio-Rad
HPLC system. The O.D.280 detection was doneusing a Bio-Dimension® UV/Vis monitor withthe standard analytical cell.

Gradient [NaCl]

OD280

0.51.0D
Gradient [NaCl]

0.5

1.0COD280

OD280

Gradient [NaCl]

0.5

1.0

B

Gradient [NaCl]

1.0

A

All chemicals were of analytical grade. Allbuffers were filtered and degassed prior to use.

Bulletin 1913 US/EG REV B

0.50Volume

Separation of Yeast Enzyme
Extract
Genetically engineered yeast (Saccharomyces
cerevisiae ) is emerging as an important
expression system for recombinant proteins.2
The advantages of yeast over E. coli expression systems include the post-translational
modifications of proteins. Figure 2 illustrates
how a crude extract of enzymes from yeast is
separated using Macro-Prep supports.

1.0DFig. 2. Separation of yeast extract.

OD280

The study was started at 2 mg per ml of support in an analytical 2 ml Bio-Scale S2 column
packed with 10 µm material (Figure 1 A), andthen step wise transferred to larger columns
with increasing sample loads (Figures 1 B andC). The last column, packed with 57 ml of 50µm Macro-Prep high S support, was used toseparate 1 gram of sample (Figure 1 D).

0.50Volume
Sample: 4 mg
Column: Bio-Scale S2 column (2 ml of 10 µm
Macro-Prep high S support, I.D. 0.7 cm)
Flow Rate: 78 cm/h (0.5 ml/min)Gradient time: 90 minutes
Gradient volume: 45 ml
Buffer A: 20 mM sodium acetate, pH 4.8
Buffer B: A + 1.0 M NaCl
Gradient:0% B in 2.5 column volumes
0-100% B in 22.5 column volumes

This poster demonstrates scale up from analytical HPLC (2 ml and 5 ml columns, 10 µmbeads) to a 60 ml preparative column utilizing
50 µm beads.

Snake venoms are a rich source of pharmacologically-active substances including proteinsand peptides.1 Figures 1 A to D show the scaling up of a separation of venom from theDiamondback rattlesnake (Crotalus atrox).

0.5

1.0

C

Gradient [NaCl]

The new 10 µm support provides a tool fordevelopment of separation methods on an
analytical and semi-preparative scale.
Methods developed using the Bio-Scalecolumns can then easily be transferred to production scale, without modifications necessary
when going from one type of bead to another.

1.0OD280

0.5

B

Gradient [NaCl]

1.0OD280

AGradient [NaCl]

Fig. 1. Separation of venom from the Diamondback rattlesnake.

OD280

Introduction
A new 10 µm Macro-Prep® support has beendeveloped to complement the 50 µm MacroPrep chromatography support. The new 10 µm
support is based on the same macro-porous
methacrylate copolymer in the 50 µm material,
albeit with a higher degree of cross linking.
The material is available in pre-packed BioScale columns in different dimensions ranging
from 2 to 20 ml. The 50 µm Macro-Prep material is available in packages ranging from 100
ml to 10 liter.

2. Manivasakam, P., and Shiestl, R., H.,
Nucleic Acids Research, 21, 18, 4414-4415(1993).

Through utilizing the same methacrylate copolymer in both the 10 and the 50 µm material,
the problems associated with transferring amethod from a small synthetic bead to a larger
bead which is based on natural polymers, canbe avoided.
We believe that the new Bio-Scale columns
will provide a valuable tool for process development and analytical chromatographic work.

95-0278a 0495