![]() The secondary structure fractions calculated from the CD and FT-IR results show excellent agreement.įigure 1. Secondary structure estimation (SSE) was performed for each concentration of antibody using the amide I peak at 1700-1600 cm -1. The same samples were measured by FT-IR spectroscopy, which is an orthogonal method to CD, to verify the plausibility of the calculated secondary structure fractions (Fig. The MRE spectra for each IgG concentration are consistent, and there is no change in the calculated secondary structure in this concentration range (Figs. Concentration calculation and subsequent conversion to MRE were also automatically conducted by Spectra Manager™ 2.5 BeStSel CFR using a sequence of antibodies. The MRE spectra are obtained from raw CD spectra normalized by concentration. The intensity of the raw CD spectra increases linearly with concentration. 1a and 1b show simultaneously measured CD and absorption spectra for various concentrations of IgG. Here, we describe an expanded application of CD spectroscopy for highly concentrated antibodies (undiluted) and report the results of a detailed secondary structure evaluation of antibody drugs using Spectra Manager™ 2.5 BeStSel CFR.įirst, we examined the validity of measuring highly concentrated samples using short-pathlength cells with IgG from rabbit serum. To make BeStSel accessible to biopharma, we developed Spectra Manager™ 2.5 BeStSel CFR as an add-in software for Spectra Manager™, a control and analysis platform for JASCO CD spectrometers, which is compatible with GxP. While many academic researchers use the BeStSel web server, researchers in biopharma companies 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-strand-rich proteins such as antibodies, 2) provides eight types of secondary structure information, 3) capability to predict protein folding following the CATH classification, and 4) open web server. ![]() developed the BeStSel algorithm that can accurately estimate the secondary structure from a CD spectrum by taking into account parallel-antiparallel orientations of β-strands and the twist of antiparallel β-sheets. ![]() Secondary structure estimation offers a route to evaluate protein higher order structure, but historically has not worked well on proteins with high amounts of β-sheet such as antibodies. Fortunately, evaluation of high concentration solutions can be done via short CD path lengths (~10 mm) or using FTIR with ATR. The ability to perform structural characterization of antibodies without dilution is extremely important because intermolecular interactions and changes in the HOS can lead to the formation of aggregates that cause serious immunogenicity. Under these conditions, the antibody molecules exhibit physicochemical properties that differ from those under dilute conditions. Although CD spectroscopy can provide robust structural information for low-concentration protein solutions (< ≈0.1 mg/mL), antibody drugs are formulated at concentrations higher than 10 mg/mL. 1) The most well-known method for HOS characterization is circular dichroism (CD) spectroscopy. Higher-order structure (HOS) is an important antibody drug physicochemical property.
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