![]() ![]() Netsu sokutei (Japan), 38, 9-15.Ģ) Greenfield, N. Satoko Suzuki 1, Taiji Oyama 1, Ai Yamane 1, Yasuo Horiguchi 1, András Micsonai 2, József Kardos 2, Ken-ichi Akao 1ġJASCO Corporation, Hachioji, Tokyo, 192-8537, Japan, 2ELTE NAP Neuroimmunology Research Group, Department of Biochemistry, Institute of Biology, ELTE Eötvös Loránd University, Budapest H-1117, Hungaryġ) Ama, D., Hasegawa, J., Uchiyama, S., and Fukui, K. Poster Session at 14th annual PEGS Europe (Protein & Antibody Engineering Summit, November 14 – 16, 2022, in Barcelona, Spain ) In addition, Spectra Manager™ Ver.2.5 CFR BeStSel allows this detailed protein structural analysis to work in GxP compliant environment. BeStSel is shown to be useful in understanding the detailed mechanisms of structure formation in proteins, including antibody drugs. ![]() At 30 degree C, h-IgG and Herceptin have a similar secondary structure, but it was found that they have different denaturation mechanisms by using BeStSel. There is a significant difference between Herceptin® and h-IgG in changes in the distorted α-helix, left-twisted β-strand, relaxed β-strand and right-twisted β-strand. Detailed secondary structure changes as a function of temperature.Įstimated secondary structure fraction of a) distorted α-helix, b) left-twisted, c) relaxed, and d) right-twisted antiparallel β-strand of Herceptin® (red) and human IgG (blue). 3 shows the detailed secondary structure change as a function of temperature.įigure 3. 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. Melting property of antibodiesĬ) β-strand, d) others fraction of Herceptin® (red) and human IgG (blue) The Tm values for Herceptin® and human IgG estimated from these curves were consistent with previously reported values. The ratio of “others” increased while the ratio of β-strand decreased with heating for both Herceptin® and human IgG (Fig. 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). In order to analyze these structural changes in detail, secondary structure estimation was performed on the spectra as a function of temperature using Spectra Manager™ Ver.2.5 CFR BeStSel to determine the change in the ratio of each secondary structure with temperature. Thermal denaturation spectra for a) Herceptin®, and b) human IgG Both antibodies showed a spectral shape with a minimum around 217 nm, which is characteristic of the β-sheet at 30 degree C, and the peak intensity decreased as the temperature increased.įigure 1-1. The thermal denaturation spectra of Herceptin® and h-IgG are shown (Fig. Here, we report the detailed tracking of heat stress-induced conformational changes of human Immunoglobulin G (h-IgG) and Herceptin® using Spectra Manager™ Ver.2.5 CFR BeStSel. To make BeStSel accessible to biopharma, we developed Spectra Manager™ Ver.2.5 CFR BeStSel as an add-in software for Spectra Manager™, a control and analysis platform for CD spectrometers, which is compatible with GxP. 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. ![]() 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. 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 β-strands and the twist of the antiparallel β-sheets. 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. ![]() Thermodynamic stability of antibodies is generally evaluated by DSC (differential scanning calorimetry) and circular dichroism (CD) spectroscopy. Therefore, the evaluation of structural stability is extremely important in the development and formulation of candidate antibodies. 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. The market for therapeutic antibodies has dramatically expanded over the past decades since their first approval in 1986. ![]()
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