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Detailed structural analysis of thermal denaturation of monoclonal antibodies
using JASCO Spectra ManagerTM Ver.2.5 CFR BeStSel
INTRODUCTION
RESULTS
EXPERIMENTAL
The market for therapeutic antibodies has dramatically expanded over the past decades since their first approval in 1986. Antibodies, which are proteins, form their structure and exert
their activity through a complex series of non-covalent bonds and may lose their activity due to various external stimuli. Therefore, the evaluation of structural stability is extremely important
in the development and formulation of candidate antibodies. Thermodynamic stability of antibodies is generally evaluated by DSC (differential scanning calorimetry) and circular dichroism
(CD) spectroscopy.1, 2) CD spectroscopy is an easy and rapid method for obtaining information on the secondary and tertiary structure of proteins in solution and can be used to directly
evaluate the protein structural change caused by heat. Recently, Micsonai et al. developed the BeStSel algorithm that can accurately estimate the secondary structure composition from the
CD spectrum by taking into account the parallel-antiparallel orientation of the b-strands and the twist of the antiparallel b-sheets. BeStSel has the following features: 1) high estimation
accuracy for a wide range of proteins, including β-structure-rich-proteins such as antibodies, 2) providing eight types of secondary structure information 3) a capability to predict the protein
fold following the CATH classification, and 4) an open web server.3-7) While many academic researchers use the BeStSel web server, researchers in biopharma who need to work in a GxP
environment have not been able to benefit from BeStSel. To make BeStSel accessible to biopharma, we developed Spectra ManagerTM Ver.2.5 CFR BeStSel as an add-in software for
Spectra ManagerTM, a control and analysis platform for CD spectrometers, which is compatible with GxP. Here, we report the detailed tracking of heat stress-induced conformational changes
of human Immunoglobulin G (h-IgG) and Herceptin®using Spectra ManagerTM Ver.2.5 CFR BeStSel.
Satoko Suzuki1, ◯Taiji Oyama1, Ai Yamane1, Yasuo Horiguchi1, András Micsonai2, József Kardos2, Ken-ichi Akao1
1JASCO Corporation, Hachioji, Tokyo, 192-8537, Japan, 2ELTE NAP Neuroimmunology Research Group, Department of Biochemistry, Institute of
Biology, ELTE Eötvös Loránd University, Budapest H-1117, Hungary
taiji.oyama@jasco.co.jp
REFERENCES
1) Ama, D., Hasegawa, J., Uchiyama, S., and Fukui, K. (2010). Netsu sokutei (Japan), 38, 9-15.
2) Greenfield, N. J. (2006). Nat. Protoc, 1, 2733-2741.
3) Micsonai, A., Wien, F., Kernya, L., Lee, Y. H., Goto, Y., Réfrégiers, M., & Kardos, J. (2015). Proc. Natl. Acad. Sci. U.S.A.,112, E3095–E3103.
4) Micsonai, A., Wien, F., Bulyáki, É., Kun, J., Moussong, É., Lee, Y. H., Goto, Y., Réfrégiers, M., & Kardos, J. (2018). Nucleic Acids Res.,46, W315-W322.
5) Micsonai, A., Bulyáki, É., & Kardos, J. (2021). Methods in molecular biology (Clifton, N.J.),2199,175–189.
6) Micsonai, A., Moussong, É., Lee, Y. H., Murvai, N., Tantos, Á., Tőke, O., Réfrégiers, M., Wien, F., & Kardos, J. (2022). Front. mol. biosci,9
7) Micsonai, A., Moussong, É., Wien, F., Boros, E., Vadászi, H., Murvai, N., Lee, Y. H., Molnár, T., Réfrégiers, M., Goto, Y., Tantos, Á., & Kardos, J. (2022). Nucleic Acids Res.,
50, W90-W98.
8) Moro Pérez, L., Rodríguez Taño, A. C., Martín Márquez, L. R., Gómez Pérez, J. A., Valle Garay, A., & Blanco Santana, R. (2019). PloS one,14, e0215442.
9) Moggridge, J., Biggar, K., Dawson, N., & Storey, K. B. (2017). Technology in cancer research & treatment,16,997–1005.
CONCLUSIONS
⚫Spectra ManagerTM Ver.2.5 CFR BeStSel enables the
tracking of detailed structural changes of antibody
drugs.
⚫Spectra ManagerTM Ver.2.5 CFR BeStSel satisfy the
GxP compliant environment and offer CSV, ER/ES, and
DI for practice ALCOA+.
Methods
MaterialsMeasurement system
The thermal denaturation spectra of Herceptin®and h-IgG are shown (Fig. 1a, 1b). Both
antibodies showed a spectral shape with a minimum around 217 nm, which is characteristic
of the β-sheet at 30 C,and the peak intensity decreased as the temperature increased.
In order to analyze these structural changes in detail, secondary structure estimation was
performed on the spectra as a function of temperature using Spectra ManagerTM Ver.2.5
CFR BeStSel to determine the change in the ratio of each secondary structure with
temperature. The secondary structure ratio of β-strands and “others” including random coils
at room temperature was consistent with that for human IgG (PDB ID 1IGT) obtained from
the X-ray crystal structure (data not shown). The ratio of “others” increased while the ratio of
β-strand decreased with heating for both Herceptin®and human IgG (Fig. 1c, 1d). The Tm
values for Herceptin®and human IgG estimated from these curves were consistent with
previously reported values.8, 9)
The advantage of BeStSel is not
only its high accuracy in analyzing β-
structure-rich-proteins, but also its
feature of being able to calculate the
fraction of eight secondary structure
elements, including three types of
twisting in antiparallel β-sheets (Fig. 2).
Fig. 3 shows the detailed
secondary structure change as a
function of temperature.
CD/absorbance simultaneous
measurement with high
accuracy
High throughput and high
sensitivity
Parameter
Bandwidth
1.0 nm
D.I.T.
2 sec
Data pitch
0.1 nm
Scan speed
50 nm/min
Accumulations
4
Pathlength
5 mm
Temp. range
30
–85C
•Herceptin®(trastuzumab, Roche)
powder dissolved in ultrapure water to 0.025 mg/mL
•IgG, from human serum (Sigma Aldrich)
powder dissolved in ultrapure water to 0.025 mg/mL
J-1500 CD Spectrometer
PTC-510 single-position
Peltier cell holder
Temperature range of 0 to
100 C with a measurement
probe that can be placed
inside or adjacent to the cell
and a magnetic stirrer to
eliminate thermal gradients.
NEW
Integrated into Spectra ManagerTM
GxP compliant and satisfying computer system validation
(CSV), electronic record/electronic signature (ER/ES), and
data integrity (DI) for practice ALCOA+.
Table 1. Measurement conditions for far-UV/CD
Figure 1. Melting property of antibodies
Thermal denaturation spectra for a) Herceptin®, and b) human IgG. c) b-strand, d) others fraction of
Herceptin® (red) and human IgG (blue)
30C
85C
Herceptin®Human IgG
b-strand Others
a) b)
c) d)
There is a significant difference between Herceptin®and h-IgG in changes in the
distorted α-helix, left-twisted b-strand, relaxed b-strand and right-twisted b-strand. At 30 C,
h-IgG and Herceptin have a similar secondary structure, but it was found that they have
different denaturation mechanisms by using BeStSel. BeStSel is shown to be useful in
understanding the detailed mechanisms of structure formation in proteins, including
antibody drugs.
In addition, Spectra ManagerTM Ver.2.5 CFR BeStSel allows this detailed protein
structural analysis to work in GxP compliant environment.
Figure 2. Twisting of β-strand
Left-twisted Relaxed Right-twisted
Spectra Manager TM Ver.2.5 CFR BeStSel
Figure 3. Detailed secondary structure changes as a function of temperature.
Estimated secondary structure fraction of a) distorted α-helix, b) left-twisted, c) relaxed, and d) right-
twisted antiparallel b-strand of Herceptin®(red) and human IgG (blue).
Distorted
α-helix Left-twisted
b-strand Relaxed
b-strand Right-twisted
b-strand
a) b) c) d)