Biopharmaceutical
Formulation
Group
Taylor Dispersion Analysis to Gain an Insight into Formulation Buffer Salts on the Stability and
Aggregation Behaviour of Proteins
Wendy L. Hulse, Robert T. Forbes
Biopharmaceutical Formulation Group, School of Life Sciences, University of Bradford, Bradford UK.
Email: w.l.hulse1@bradford.ac.uk
Objectives
Results
Figure 1 shows the TDA results for different Lysozyme concentrations in phosphate buffer (pH 4.5, 50 mM) which show that increasing lysozyme
concentration results in an increased hydrodynamic radius. All TDA and most DLS data points show that the hydrodynamic radius had increased
after storage. Figure 3 shows the two VP-DSC heating cycles for lysozyme (10mg/ml). No unfolding enthalpy is present in the re-heat cycle indicating
that lysozyme may be irreversibly unfolded by the first heating cycle.
2.10
-0.001
1.90
y = 0.4086Ln(x) + 0.7436
R2 = 0.9932
-0.002
1.80
1.90
Hydrodynamic radius (nm)
TDA analysis was carried out using 60nl of sample injected
using a flow-rate of 2 mm s-1, capillary size was 75:360 ID:OD
with a total length of 143cm.
y = 0.5219Ln(x) + 0.2015
R2 = 0.9992
2.30
Hydrodynamic Radius (nm)
Methods
2.00
2.50
1.70
Phosphate initial
1.50
Phosphate 6 months
1.30
1.10
Phosphate initial
Phosphate 6 months
1.60
-0.004
-0.005
1.50
-0.006
1.40
0.70
30
0.50
40
50
60
70
1.30
0
5
10
15
20
25
30
35
40
45
0
5
10
15
Lysozyme conc. (mg/ml)
20
25
30
35
40
80
90
100
o
Temperature ( C)
45
Lysozyme conc. (mg/ml)
Figure 1. TDA results for Lysozyme hydrodynamic radius in phosphate buffer.
Actipix TDA 200
-0.003
0.90
DLS analysis was carried out using a Malvern Nano-S
system.
Thermal analysis was carried out using a VP-DSC with a
heating and cooling rate of 1°C/min.
1.70
o
To compare the results obtained with those from the
conventional size measuring technique of dynamic light
scattering (DLS) and thermal analysis.
Cp(cal/ C)
To explore the use of Taylor Dispersion Analysis to size
lysozyme in the presence of different formulation buffers and
additives and to detect protein aggregation potential with
time.
Figure 2. DLS results for Lysozyme hydrodynamic radius in phosphate buffer.
Figure 3. VP-DSC results for Lysozyme in phosphate buffer (black trace initial heating
cycle, blue trace second heating cycle).
Figures 4 and 5 show the TDA and DLS results for different lysozyme concentrations in acetate buffer (pH 4.5, 0.1M) which displays a narrower size range in
hydrodynamic radius with increasing lysozyme concentration for TDA. The VP-DSC in Figure 6 does shows that unlike in the phosphate system, lysozyme
shows an unfolding enthalpy in the second heating cycle.
1.80
2.05
0.006
1.60
2.00
0.004
1.90
Acetate 6 months
1.85
1.20
1.00
0.80
-0.008
20
1.75
30
40
50
60
70
80
90
0.60
10
15
20
25
30
35
40
45
0
Lysozyme conc. (mg/ml)
2.50
2.00
y = 0.5219Ln(x) + 0.2015
R2 = 0.9992
1.50
Phosphate initial
Phosphate plus NaCl 2mg/ml
Phosphate plus NaCl 40 mg/ml
Phosphate plus NaCl 100 mg/ml
0.50
0.00
5
10
15
20
15
20
25
30
35
40
o
Temperature ( C)
45
Figure 5. DLS results for Lysozyme hydrodynamic radius in acetate buffer.
3.00
0
10
Lysozyme conc. (mg/ml)
Figure 4. TDA results for Lysozyme hydrodynamic radius in acetate buffer.
1.00
5
25
30
35
Lysozyme conc. (mg/ml)
Figure 7. TDA results for Lysozyme hydrodynamic radius in phosphate buffer
with and without added sodium chloride.
40
Figure 6. VP-DSC results for Lysozyme in acetate buffer (black trace initial heating cycle,
blue trace second heating cycle).
Figure 7 shows the TDA results of lysozyme
(phosphate) with added sodium chloride in differing
concentrations. The data indicates that addition of
NaCl produces a more consistent average
hydrodynamic radius with protein concentration
than without. Secondly, the hydrodynamic radius by
TDA was little influenced by salt level for this system
over 2-100mg/ml.
The VP-DSC thermal profile of lysozyme in the
presence of NaCL (fig. 8) is different to that obtained
without NaCl present (fig. 3), reflecting the
conformational size related changes observed using
TDA.
Conclusions
Buffer salt selection is important and TDA demonstrates that it can notably alter the hydrodynamic
radius of proteins. The TDA is a useful complement to DSC in buffer selection. The inherent, intricate
behaviour of proteins in solution is complicated further by buffer systems. Both the ion and the ionic
strength can affect the proteins behaviour in solution. Additional buffer systems of different salt and
ionic strength are being investigated.
0.012
0.010
0.008
Cp(cal/ C)
5
o
0
Hydrodynamic Radius (nm)
The band is imaged at two points,
the first on entry to and the
second on exit from a loop in the
capillary.
The
hydrodynamic
radius is calculated from the
measured differences between
peak times (first moments) and
variances (second moments) at
the two windows.
Band broadening due to Taylor dispersion is calculated from
absorbance versus time data using the peak centre times at
the first and second window, t1 and t2 respectively, and the
corresponding standard deviations, t1 and t2 (band
broadening).
-0.002
-0.004
TDA200 Coupled to HP CE system
The TDA200 instrument uses UV area imaging and Taylor
dispersion analysis (TDA) for determining diffusion
coefficients and hydrodynamic radii of proteins in solution.
The detector monitors broadening of a band of a therapeutic
protein or small molecule solution injected into a stream of
buffer solution and driven through a fused-silica capillary.
0.000
-0.006
1.80
TDA200 Coupled to PrinCE CE system
Acetate 6 months
Cp(cal/ C)
Acetate initial
0.002
1.40
o
1.95
Hydrodynamic radius (nm)
Hydrodynamic Radius (nm)
Acetate initial
0.006
0.004
0.002
0.000
30
40
50
60
70
80
90
100
o
Temperature ( C)
Figure 8. VP-DSC results for Lysozyme in phosphate buffer with 2mg/ml sodium
chloride added to buffer
(black trace initial heating cycle, blue trace second heating cycle).
Acknowledgements
The authors wish to thank the EPSRC and TSB for
financial support
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