and C - Waters

BIOMOLECULE SEPARATIONS BY UPLC-SEC
Edouard S. P. Bouvier, Susan Serpa, Nadya Brady, Kevin D. Wyndham, Nicole L. Lawrence, Paula Hong, Uwe D. Neue, Thomas H. Walter
Waters Corporation, 5 Technology Drive, Milford, MA 01757
80
H (µm)
Figures 1-2. H-u curves for (1) Waters ACQUITY UPLC®
BEH200 SEC, 1.7µm, (2) Prototype “D”. Column dimensions were 4.6x150mm. Flow rate range 0.1-0.7 mL/min.
Other conditions as described in Methods Section.
40
BEH200 SEC
1.7 µm, 203Å
20
Uracil
Ribonuclease A
Lysozyme
pI
N/A
8.7
11
B
Ovalbumin
4.6
44000
BSA
5.8
67000
IgG
6.7
150000
17000
Thyroglobulin
4.6
669000
Blue Dextran
N/A
2000000
1.7
297
239
139
191
311
86
108
182
135
217
1.6
193
207
1.16
H
2.8
213
225
1.42
BEH200
Biosuite
TM
250
1.7
4
203
233
217
218
229
2.00
Ribonuclease A
3.12
Ovalbumin monomer 15.92
Ovalbumin dimer
30.86
A
0.9982
r2
C
5.38
Prototype "A"
2.5 µm, 297Å
6.00
A
1.36
0.9886
4.33
6.99
1.74
0.9741
13.30
11.81
0.9991 11.36 25.43 0.9980
4.23
0.9199
19.19
35.82
0.9949
N.D.
0.9963
N.D.
5.32
3.83
0.9749
13.93
37.45
0.9973 12.09
0.9978
10.08
IgG2 monomer
6.80
6.66
0.9953
14.77
43.19
0.9979 19.65 29.17 0.9998
15.16
47.82 13.40 0.9335
24.18
111.91
N.D.
N.D.
0.005
60
BEH200 SEC
40
Ribonuclease A
N.D.
8.88
N.D.
0.9991
N.D.
N.D.
N.D.
Ovalbumin dimer
0
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
2.00
ui (cm/s)
TO DOWNLOAD A COPY OF THIS POSTER, VISIT WWW.WATERS.COM/POSTERS
14
Prototype "D"
Prototype "B"
5
15
Time (minutes)
10
20
25
A/dp
C/dp2
A/dp
C/dp2
A/dp
C/dp2
2.082
0.607
1.505
0.842
1.873
0.728
1.893
2.038
0.773
4.619
1.424
3.959
3.087
3.882
7.825
20.571
1.879
6.663
4.319
N.D.
N.D.
N.D.
N.D.
IgG1 monomer
7.177
1.702
4.836
4.515
4.212
1.196
3.589
3.160
IgG2 monomer
4.534
2.960
5.128
5.207
6.848
3.541
5.395
8.780
IgG2 dimer
31.879
5.955
8.396
13.492
N.D.
N.D.
N.D.
N.D.
7
10
Prototype C
300
200
BioSuite 250 - dimer
BioSuite 250 - monomer
150
BEH200 - dimer
BEH200 - monomer
100
50
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Flow rate (mL/min)
3.0
Dextran Blue
Prototype D
Prototype F
Thyroglobulin
Prototype G
Prototype H
IgG
5
10
Prototype I
BSA
Ovalbumin
BioSuite 250
Cytochrome c
3
10
2
10
Uracil
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
J
G
BEH200
BioSuite
2.0
4
0.12
0.12
0.08
0.08
2
30°C
40°C 1
50°C
BEH200 SEC 1.7 µm
4.6x150 mm
3
5
0.04
0.04
0.00
0.00
2.50
2.50
3.00
3.0
0.100
0.100
30°C
0.075
0.075
40°C
0.050
0.050
50°C
3.5
3.50
4.0
4.00
4.5
4.50
5.00
5.0
Minutes
5.5
5.50
6.0
6.00
4
2
3
6.5
6.50
7.0
7.00
7.5
BioSuite 250 UHR SEC 4 µm
4.6x300 mm
5
1
0.025
0.025
0.000
0.000
H
5.00
5
6.00
6
7.00
7
8.00
8
9.00
9
10.00
10
Minutes
11.00
11
12.00
12
13.00
13
14.00
14
15
Time (minutes)
1.5
0
0.2
0.4
0.6
0.8
1
Flow Rate (mL/min)
F
D
C
REFERENCES
Normalized Retention Volume, V /V
R C
Pore Volume (cm3/g)
Figure 3. Normalized calibration curves for columns packed with
materials as described in Table I. Chromatographic conditions as
described in Methods Section. Slopes were obtained from the
linear region of each curve.
Figures 7 & 8. Effect
of flow rate on plate
height and resolution
for separation of
monoclonal antibody
monomer and dimer.
Columns: (1) BEH200
SEC, 1.7µm, 4.6x150,
and (2) BioSuite 250
4µm UHR, 4.6x300.
Other conditions as
described in Methods
Section.
Figure 4. Plot of normalized slope vs. Specific Pore Volume
obtained for each curve shown in Figure 3. Slopes were
determined for the linear range of each curve.
1. D Held, G Reinhold and P Kilz, “U-GPC? Making GPC/SEC Faster,” The
Column (April 6, 2010) 10-14.
In general, a decrease in retention was observed to follow
the Hofmeister series, with the notable exception of sodium
chloride. In this case, retention was greater than with other
analytes. In addition, peaks were significantly broader.
Sodium Sulfate
0.04
0.02
0.00
0.04
Sodium Phosphate
100mM
0.02
0.00
0.04
Sodium Acetate
0.02
0.00
0.04
Sodium Chloride
0.02
0.00
0.04
Sodium Perchlorate
0.02
0.00
2
3
4
5
Time (minutes)
6
7
8
0.04
Ammonium Chloride
0.02
0.00
0.04
Potassium Chloride
0.02
0.00
0.04
Sodium Chloride
0.02
0.00
2
3
4
5
Time (Minutes)
6
7
8
Figures 10 & 11. Effect of salt type on retention of analytes.
Mobile phase consisted of 10 mM sodium phosphate, pH 6.8 with
200 mM of given salt. In the case of phosphate, salt
concentration was 100 mM. Analytes same as in Figure 9. Other
chromatographic conditions as described in Methods Section.
250
0
I
30
Figures 5 & 6. Effect of flow rate on the separation of a monoclonal antibody monomer and dimer. Top: Waters ACQUITY UPLC
BEH200 SEC, 1.7µm, 4.6x150mm. Bottom: Waters Biosuite
4µm, 4.6x300mm.
Prototype "A"
C/dp2
16
0.4 mL/min
2.5
4
10
12
BioSuite 250 UHR SEC 4 µm
4.6x300mm
0
Retention times for proteins were determined for columns packed with Materials A-I, which varied in total pore volume from 0.68-1.63
cm3/g, as shown in Table II. A plot of retention volume vs. log MW was plotted for each of the materials (Figure 3). The slope of the each
of the curves (in the representative linear region) was determined for each column; Figure 4 shows a plot of the pore volume vs. the slope.
As can be seen, a good correlation is obtained between these two factors.
6
10
10
0.00
EFFECT OF PORE VOLUME ON SELECTIVITY
1.27
Chromatographic Conditions (unless otherwise noted):
®
• Instrument: ACQUITY UPLC System with UV detection @
280 nm
• Temperature: 30˚C
• Mobile phase: 100 mM sodium phosphate, pH 6.8
• Flow rate: 0.3 mL/min
• Injection type: Full loop, 2 µL
• Column dimensions: 4.6x150 mm, except for the Waters
BioSuiteTM 250 4µm UHR column, which was 4.6x300 mm.
8
Time (minutes)
0.1 mL/min
0.9982
24.67 0.9982
A/dp
Ovalbumin monomer 10.611
20
1.30
METHODS
6
0.2 mL/min
r2
1.63
1.52
4
21.99 0.9960
N.D.
10.77
N.D.
5.73
10.91
IgG1 monomer
0.9937
9.85
N.D.
C
2
Table IV. A/dp and C/dp obtained from Figures 1 & 2.
0.73
G
201
1.80
r2
C
0.1 mL/min
0.8 mL/min
0.72
1.12
2.0
1.60
(2) Prototype “D”
dp=2.60µm, MPD=191Å
0.69
112
209
1.40
Prototype "B"
2.6 µm, 239Å
80
0.69
348
259
1.20
IgG2 dimer
0.68
1.6
2.4
1.00
100
Mean Pore Specific Surface Total Pore
Dia (Å)
Area (m2/g) Vol. (cm3/g)
F
J
6.8, 7.2
2.6
E
112
Myoglobin
1.8
D
I
14400
2.6
C
MW
13700
2.5
0.80
A
0.2 mL/min
0.00
Resolution
Analyte
A
0.60
120
Table II. Properties of chromatographic
materials used in this study.
dp (µm)
0.40
r2
C
Normalized Slope from Calibration Curve, β
Δ(log MW)/Δ(VR/Vc)
Table I. Test probes used for characterization of SEC columns.
Stationary phase particles were synthesized with different total
pore volume, mean pore size, surface area, and mean particle
size. All materials were diol-bonded to provide a stable chemical
surface that exhibited low protein binding.
Material
0.20
ui (cm/s)
H (µm)
The general resolution equation in chromatography is typically
defined as Rs≡ΔVR/4σv, where ΔVR is the difference in retention
volume between two peaks, and σv is the mean standard
deviation of the two peaks in volume units. In SEC, the
numerator is representative of the selectivity, while the
denominator is a function of the chromatographic efficiency.
For a given packing material, a calibration curve can be
generated that correlates the retention volume to the molecular
weight. A linear relation is obtained for a portion of the curve.
We can use the experimentally-determined slope of the curve to
quantitate the selectivity of a given chromatographic material.
As will be shown, the slope of the calibration curve is directly
related to the pore volume of the stationary phase. The
chromatographic efficiency is a function of the particle size and
packing efficiency.
MATERIALS PROPERTIES
0.00
Molecular Weight (Da)
FACTORS AFFECTING
RESOLUTION IN SEC
A
0
Prototype "D"
2.6 µm, 191Å
0.4 mL/min
0
Table III. A- and C- terms obtained from Figures 1 & 2. Note large
A-terms, which may be a result from partial resolution of multiple
components in the peak.
60
0.7 mL/min
where ΔH˚ and ΔS˚ are the respective standard enthalpies and
entropies, R is the molar gas constant, T is the absolute temperature and φ is the phase ratio of the column.
In adsorption chromatography, the entropy term can usually be
neglected, as it is negligible relative to the enthalpy term. However, in SEC mode, the opposite is true. Thus in an entropicallydriven separation such as SEC, retention should be primarily independent of temperature.
However, chromatographic materials may have ionic or other
functional groups on the surface that may induce binding. Ideally,
the stationary phase and the chromatographic conditions are optimized to prevent adsorption interactions.
Examining retention at different temperatures will provide information as to whether adsorption is occurring. Other factors
which may contribute to temperature-induced changes in retention, such as changes in conformation or changes in the hydration
layer, should also be considered.
The chromatograms below show the effect of temperature on
the retention of a Protein Test Mix. The top chromatogram was
obtained using a BEH-based ACQUITY BEH200 SEC column, while
the bottom chromatogram was from a silica-based BioSuite 250
column. Significantly greater retention change is observed for
proteins in the latter case. This is attributed to the greater acidity
of silanols at the stationary phase surface, which can interact with
and adsorb proteins.
Note in both cases, uracil retention is affected by temperature.
We believe that this is due to H-bond interactions. Further work
is being done to better understand this behavior.
Absobance @ 280 nm (AU)
100
This presentation will discuss the benefits in speed and resolution that can be obtained with UPLC-SEC for biomolecule separations. The effect of particle size, pore volume and pore size distribution on chromatographic resolution will be evaluated, as
well as the effect of temperature on SEC performance. Examples will be given of size-based separations, including separations
of monoclonal antibody monomers from aggregates.
(1) ACQUITY BEH200 SEC
dp=1.7µm, MPD=211Å
IgG2 dimer
IgG2
IgG1 dimer
IgG1
Ovalbumin
Ribonuclease A
Uracil
ln k = ΔH˚/RT - ΔS˚/R + ln (φ)
The effect of different salt additives to the mobile phase
was explored. Both retention and peak shape were found to be
influenced by salt type.
Chromatograms are shown below, following the sequence
of the Hofmeister Series for anions and cations:
SO42- > PO42- > OAc- > Cl- > ClO4NH4+ > K+ >Na+
Absorbance @ 280 nm (AU)
120
AU @ 280 nm
Another important attribute of SEC packing materials is that they interact minimally with the analytes. For traditional silicabased SEC materials, a high ionic strength mobile phase is typically employed to mitigate silanol interactions that may result in
adsorption. The reduced silanol acidity on BEH particles compared to traditional silica results in decreased secondary interactions for charged analytes.
The impact of temperature on a chromatographic separation
is determined thermodynamically by the expression:
BEH200 SEC 1.7 µm
4.6x150mm
0.01
Plate height (µm)
SEC separations typically require long analysis times. This is primarily because the slow diffusion of large molecular weight analytes within the porous media requires operation at low linear velocities to achieve optimum resolution. Mass transfer can be
enhanced greatly by using smaller chromatographic particles, resulting in benefits in speed and resolution. We have recently
expanded the scope of the ethylene-bridged hybrid (BEH) technology by re-engineering our particle-making process to create
porous particles with almost a two-fold increase in pore volume compared with the original BEH particle. This newly designed
chromatographic media (patent pending) has been shown to maintain the mechanical strength requirements necessary of a 1.7
micron particle for UPLC applications.
The effect of flow rate on the resolution of a monoclonal antibody
0.03
monomer and dimer was compared for an ACQUITY BEH200 SEC
1.7 µm, 4.6x150mm and a BioSuite 250 UHR 4µm, 4.6x300mm
column. Despite the shorter column length of the former column,
we0.02
found comparable resolution for the two columns when operated at identical flow rates. The smaller particles in the BEH200
provided for even shorter run times by enabling operation at
faster flow rates, while still achieving the requisite resolution.
AU @AU
280 nm
The introduction of sub 2 micron particles and Ultra-Performance Liquid Chromatography instrumentation has led to significant
improvements in chromatographic efficiency and throughput compared to traditional HPLC separations for reversed-phase
chromatography. However, to-date, similar benefits have not been demonstrated for other modes of chromatography, such as
size-exclusion chromatography. A recent publication by Held et al (1) states that such benefits cannot be achieved with columns packed
with sub-two micron particles for SEC.
We evaluated the effect of flow rate on the chromatographic separation of various proteins on columns packed with particles of a
range of pore sizes and particle sizes. Retention time and peak width data were first corrected for system effects. Plots of plate height
vs. interstitial linear velocity (measured at 50% peak height) are shown in Figures 1 & 2 below. Results show significantly improved
performance for the column with 1.7 µm particles compared to 2.6 µm particles. For proteins, the flow rates used were significantly higher
than the diffusion-limited region, so data were fit to the linear equation, H=A + C•u. Theory predicts that the A- and C- terms are
proportional to 1/dp and 1/dp2, respectively. The experimental data fits well to this expectation. We also found that pore size has limited
impact on chromatographic performance as a function of flow rate.
INFLUENCE OF SALTS
INFLUENCE OF TEMPERATURE
AU
Size exclusion chromatography is one of the primary analytical techniques used today to provide a rapid and simple means to
assess protein aggregation. Aggregate impurities are carefully monitored throughout development of a protein therapeutic because of their correlation to inducing immunogenicity.
MAb MONOMER/AGGREGATE
SEPARATION
AU @ 280 nm
EFFECT OF PARTICLE SIZE AND PORE DIAMETER ON EFFICIENCY
AU @ 280 nm
INTRODUCTION
Figure 9. Effect of temperature on the separation of (1) Thyroglobulin, (2) IgG1, (3) BSA, (4) Myoglobin, (5) Uracil. Top:
Waters ACQUITY UPLC BEH200 SEC, 1.7 µm, 4.6x150 mm.
Bottom: Waters BioSuite 4 µm, 4.6x300 mm.
CONCLUSION
We have evaluated columns packed with stationary
phases containing a range of mean pore sizes, particle sizes
and pore volumes. We have found particle size has a direct
impact on chromatographic performance, and correlates well
with chromatographic theory. We have also found that the
total pore volume correlates well with the slope of the calibration curve.
By reducing particle size, SEC throughput can be increased significantly.
In the case of monoclonal
monomer/aggregate separations, analysis time can be decreased dramatically compared to traditional SEC packing
materials.
Operating temperature was found to have minimal impact
on chromatographic retention times for proteins tested on
the BEH200 SEC column. On the other hand, retention times
were affected by temperature for the BioSuite 250 UHR
column, which is silica-based. We attribute this to interactions with exposed silanols on the silica surface. Uracil retention is affected by temperature for both columns, which we
attribute to hydrogen bonding effects.
SEC separations using various buffer types were explored.
Salt type was found to affect both retention times and chromatographic efficiency.
©2010 Waters Corporation