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Order Picornavirales
Published in Paul Pumpens, Peter Pushko, Philippe Le Mercier, Virus-Like Particles, 2022
Paul Pumpens, Peter Pushko, Philippe Le Mercier
Somasundaram et al. (2016) presented obstacles for the enhanced production of both EV71 and CVA16 VLPs, comparing the VLP yields in Sf9 and HighFive cells. The authors used high-resolution asymmetric flow field-flow fractionation couple with multiangle light scattering (AF4-MALS) for the first time to characterize the EV71 and CVA16 VLPs, displaying an average root mean square radius of 15 ± 1 nm and 15.3 ± 5.8 nm, respectively.
Nonclinical Safety Evaluation of Drugs
Published in Pritam S. Sahota, James A. Popp, Jerry F. Hardisty, Chirukandath Gopinath, Page R. Bouchard, Toxicologic Pathology, 2018
Thomas M. Monticello, Jeanine L. Bussiere
The assays for immune complex (IC) detection rely on specific platforms and reagents that can detect both entities of the ADA–drug complex, i.e., the therapeutic antibody and the species-specific ADA (Stubenrauch et al. 2010). Nonclinical methods like size exclusion multi-angle light scattering (SEC-MALS) combined with high-performance liquid chromatography (HPLC) can be used to separate complexes based on size (Moxness et al. 2005; Goncalves et al. 2012). Another approach to detect species-specific ADA is by capturing with the therapeutic protein and detecting with a species-specific antibody. Similarly, the other component of the IC, the therapeutic protein or drug, can be detected using the ligand binding PK assays (Stubenrauch et al. 2010).
Experimental Strategies
Published in Clive R. Bagshaw, Biomolecular Kinetics, 2017
Historically, proteins were usually first characterized by means of some functional characteristic, such as catalytic or binding activity, and subsequently, their primary, secondary, and higher-order structures were determined, which greatly aided the understanding behind the mechanism of their activity. Nowadays, proteins are frequently expressed from a known DNA sequence and they may fold into some compact form assumed to be the native state. Checking for secondary structure using circular dichroism (Section 7.6.3) provides evidence for folding but does not prove that the sample is functional. It is also useful to determine the oligomeric state of the protein using methods which are sensitive to the native molecular weight, such as ultracentrifugation and gel-permeation chromatography. The latter is particularly powerful when combined with multiangle light scattering [319].
Isolation and characterization of a monoclonal antibody containing an extra heavy-light chain Fab arm
Published in mAbs, 2018
Dan Boyd, Arpa Ebrahimi, Sarah Ronan, Brian Mickus, Matthew Schenauer, Jenny Wang, Darren Brown, Alexandre Ambrogelly
Analysis by size exclusion HPLC (SEC-HPLC) of purification intermediates during process development showed the presence of a well-resolved peak eluting between the peaks typically attributed to the mAb in its monomeric and dimeric forms (Fig. 1A). Analysis of the sample by in-line multi-angle light scattering (MALS) after separation by SEC-HPLC confirmed the monomer and dimer peak assignments (Fig. 1B). It also attributed an approximately 200 kDa molecular mass to the additional peak (Fig. 1B). In order to characterize this unexpected species, we fractionated the sample into three pools: Main peak (monomer), 200 kDa fraction, and a pool including the mAb dimers and larger molecular weight species. The fractions were 99%, 96% and 88% pure, respectively, after reinjection on the SEC-HPLC column (Fig. 1C).
Productive common light chain libraries yield diverse panels of high affinity bispecific antibodies
Published in mAbs, 2018
Thomas Van Blarcom, Kevin Lindquist, Zea Melton, Wai Ling Cheung, Chris Wagstrom, Dan McDonough, Cendy Valle Oseguera, Sheng Ding, Andrea Rossi, Shobha Potluri, Purnima Sundar, Steven Pitts, Marina Sirota, Meri Galindo Casas, Yu Yan, Jeffrey Jones, Zygy Roe-Zurz, Surabhi Srivatsa Srinivasan, Wenwu Zhai, Jaume Pons, Arvind Rajpal, Javier Chaparro-Riggers
The molecular weights were determined using multi-angle light scattering and the thermal stability determined using differential scanning calorimetry as previously described.16 Antibody binding kinetics and affinities were determined by SPR using a Biacore 4000 biosensor (GE Lifesciences) similar to a manner previously described.64 Antibody epitope binning was performed using a previously described sandwiching technique44 with the exception of modifications for antibody capture and regeneration and a combination of SPR and BLI was used. An automated mouse 4-1bb signaling assay was based on a previously described method.53 Full details are described in in Supplemental materials.
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.