Effect of Temperature on Separation and Characterization of Monoclonal Antibody
Using Asymmetrical Field-Flow Fractionation
aGenentech
Shiang Gwee*a, Soheyl Tadjiki*b, Evelin Moldenhauerc and Jun Liua
Inc., South San Francisco, CA 94080, USA, bpostnova analytics, Inc., Salt Lake City, UT 84102, USA, cpostnova analytics, Landsberg, Lech 86899, Germany
*Co-first author
Introduction:
Introduction:
Results:
Results:
350
0
5
10
15
20
25
30
35
Method
Method and
and materials:
materials:
250
200
150
100
0
5
10
15
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30
35
40
40
Figure 4:
5
10
15
20
25
30
35
d
SLS 90o response
SLS 90o response
0
20oC
40oC
60oC
80oC
0
40
50
60
70
80
90
100
Temperature (deg C)
Retention time (min)
c
1
2
3
4
50
0
40
20oC
40oC
60oC
80oC
b
SLS 90o response
20oC
40oC
60oC
80oC
Excess Molar Heat Capacity
(kCal/mol/deg C)
a
Retention time (min)
System: AF2000 MT Mid temperature AF2000 system (Postnova Analytics)
Sample
Sample
Sample
Sample
300
SLS 90o response
Thermal stability of a protein drug is an important property for
formulation development. Traditionally, this has been done by differential
scanning calorimetry (DSC) and long term storage at elevated
temperatures. In this study, we have used the new postnova analytics
AF2000 MT system to study the thermal stability of several monoclonal
antibody (MAb) formulations at four different temperatures. Sample
stability was monitored in terms of size distribution of aggregates. The
results were compared with those from the conventional DSC method.
DSC Thermogram of monoclonal antibody samples
20oC
40oC
60oC
80oC
5
10
Retention time (min)
15
20
25
30
35
Discussion:
Discussion:
40
Retention time (min)
• MAb product in lyophilized formulation is more stable under thermal
stressed conditions than the reconstituted liquid formulation.
Figure 2: Fractograms of monoclonal antibody samples run at 20, 40, 60 and 80 oC
a) sample 1, b) sample 2, c) sample 3 and d) sample 4
• Aggregates with broader distribution are formed by the thermal
stressed reconstituted liquid products (samples 2 and 4)
Detector
sample 3
sample 4
0
waste
5
10
Temperature control
conduit
15
20
25
30
35
sample 2
sample 4
0
5
10
15
20
25
30
35
Retention time (min)
40
Conclusion:
Conclusion:
Channel
Table1: Description of monoclonal antibody samples analyzed by AF2000 MT system
Sample
Description
1
MAb Lyo control
2
Reconstituted MAb Lyo stored at 60oC for 1 month
3
MAb Lyo stored at 60oC for 1 month
4
MAb Lyo stored at 60oC for 1 month, then reconstituted and. stored at 60oC for another
month
d
sample 2
SLS 90o response
Samples:
sample 1
sample 1
c
sample 3
sample 4
0
5
10
15
20
25
Retention time (min)
30
35
• Thermal stressed and no stressed samples exhibit different
thermograms, but not on transition midpoint (Tm).
40
Retention time (min)
Figure 1: Picture and schematic of the AF2000 MT system
• Significant changes on size distribution of aggregates were observed
for thermal stressed liquid product even at 20oC, whereas the
lyophilized form exhibits no significant changes up to 80oC.
sample 3
sample 2
SLS 90o response
TIP pump
sample 2
sample 1
b
SLS 90o response
Cross flow pump
waste
FOCUS pump
a
SLS 90o response
Temperature control module
sample 1
40
sample 3
sample 4
• AF2000 MT is an excellent analytical tool in characterization of protein
and protein aggregates at physiological temperature.
• The data shows that thermal stress forms different types of
aggregates from the lyophilized and reconstituted MAb products.
0
5
10
15
20
25
30
35
Retention time (min)
Figure 3: Comparison of fractograms of monoclonal antibody samples run at different
temperatures, a) 20 oC, b) 40 oC, c) 60 oC and d) 80 oC
40
• The data also demonstrates different degradation mechanisms for
aggregates formed by the lyophilized and liquid MAb products.
• AF2000 MT provides more useful information about thermal stability
of a protein formulation than the conventional DSC method.