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Immunoglobulins: Structure and diversity
Published in Gabriel Virella, Medical Immunology, 2019
Early studies of the characteristics of antibodies demonstrated that in serum electrophoresis, the γ-globulin fraction contained the majority of the proteins with antibody activity (Figure 5.1) and that two major types of antibodies could be defined by their sedimentation coefficient determined by analytical ultracentrifugation (7S and 19S antibodies), reflective of the molecular weight of monomeric and polymeric immunoglobulins. As protein fractionation techniques became available, complete immunoglobulins and their fragments were isolated in large amounts, particularly from the serum and urine of patients with multiple myeloma. These proteins were used both for studies of chemical structure and for immunological studies allowed to identify antigenic differences between proteins from different patients; this was the basis for the initial identification of the different classes and subclasses of immunoglobulins and the different types of light chains.
Genosomes (DNA−Lipid Complexes)
Published in Danilo D. Lasic, LIPOSOMES in GENE DELIVERY, 2019
Another method for size characterization is gel chromatography. However, in positively charged systems it is normally very difficult to elute particles. When one wants to suppress electrostatic attraction with high salt, the complex often precipitates and clogs the column. Heterogeneous samples also reduce the applicability of analytical ultracentrifugation. This method, however, can yield useful data on the distribution of the complexes with respect to their density, size and shape (friction coefficient), and molecular weight. Size and shape of the particles can be determined by EM. Cryo-EM is undoubtedly the least prone to artifacts.
Proteoglycans of the Intervertebral Disc
Published in Peter Ghosh, The Biology of the Intervertebral Disc, 2019
Canine and human89,96,97 annulus fibrosus proteoglycans contained a higher proportion of aggregable proteoglycans than that of nucleus pulposus. The center of the nucleus pulposus contains the higher proportion of nonaggregable proteoglycans.121 This low proportion of nucleus aggregates is not an artifactual degradative phenomenon, because labeled cartilage proteoglycans do not lose their capacity to aggregate when introduced into the extraction milieu.92 Moreover, the lower proportion of aggregates is demonstrable by different techniques, such as gel chromatography,90 analytical ultracentrifugation,90 and electron microscopy.28 Biosynthetic studies (see below) confirm that the genesis of nonaggregable proteoglycans is an event that occurs in vivo.
The impact of forced degradation conditions on mAb dimer formation and subsequent influence on aggregation propensity
Published in mAbs, 2022
Michael J. Knight, Léontine Floret, Nisha Patel, John O’Hara, Elizabeth Rodriguez
Spectroscopic analysis revealed that there were some differences in secondary and tertiary structure between all of the samples. We next decided to characterize the tertiary and quaternary structure of the dimer samples. The samples were thus analyzed by sedimentation velocity analytical ultracentrifugation (SV-AUC). Analytical ultracentrifugation is used to observe macromolecular sedimentation under high centrifugal force in order to determine their masses and shapes.36 The main advantage of ultracentrifugation is the detection of different species in a sample from the peaks in a 2-dimensional spectrum analysis (2DSA).37 The SV-AUC data revealed several dimer species with different sedimentation coefficients for each of the dimer samples. These are shown in Table 2. The dimer species in the native dimer and 37°C dimer samples had an average sedimentation coefficient of 9.02, whereas the dimer species present in the 50°C dimer sample had a lower average sedimentation coefficient of 8.92. This is indicative of the dimers in the 50°C dimer sample adopting a more elongated conformation. The heterogeneity of the dimers appeared to decrease from native dimers to 50°C dimer, indicating a shift to a more homogeneous elongated conformation.
Analytical comparability study of recombinant monoclonal antibody therapeutics
Published in mAbs, 2018
Alexandre Ambrogelly, Stephen Gozo, Amit Katiyar, Shara Dellatore, Yune Kune, Ram Bhat, Joanne Sun, Ning Li, Dongdong Wang, Christine Nowak, Alyssa Neill, Gomathinayagam Ponniah, Cory King, Bruce Mason, Alain Beck, Hongcheng Liu
Extended characterization assays are either orthogonal to release methods or state-of-the-art technology with the capability to detect subtle differences. In contrast to release methods that need to be appropriately qualified or validated according to the phase of development, extended characterization methods need not be validated, but should be scientifically sound and appropriately controlled, with the underlying science qualifying these methods for detection of quality attributes that could be challenging by release methods. For example, analytical ultracentrifugation (AUC) is orthogonal to size-exclusion chromatography for determination of the percentage of aggregates, monomer, and fragments in solution. Additionally, AUC determines the hydrodynamic size distribution of the molecules, which is a reflection of the molecule's conformation. Molecular weight measurements using mass spectrometry can provide not only the accurate molecular weight, but also the relative percentage of PTMs. Circular dichroism is a method that measures the secondary and tertiary structures of the mAb molecule, information that is not typically obtained by release assays.
Rituximab biosimilar CT-P10 for the treatment of rheumatoid arthritis
Published in Expert Opinion on Biological Therapy, 2019
Ju-Yang Jung, Ji-Won Kim, Hyoun-Ah Kim, Chang-Hee Suh
Target concentration of CT-P10 was designed to be the same as those of EU- and US-rituximab, and this was confirmed by the UV-Vis spectrophotometric method. Size-exclusion chromatography-multi angle light scattering showed prominent monomer peaks and highly similar molecular weights of monomers in all three products. Sedimentation velocity analytical ultracentrifugation analysis showed that the relative content and sedimentation coefficients of monomers and dimers were highly similar. Isoelectric focusing and ion-exchange chromatography (IEC) showed seven charge variants and revealed that the acidic peaks were slightly higher and the basic peaks were slightly lower in CT-P10 than in the other two products.