Capillary isoelectric focusing (cIEF) is widely used in the biopharmaceutical industry to measure the charge distribution of therapeutic proteins. The implementation of this technology has created a new challenge. Capillary volumes are on the order of hundreds of nanoliters and cannot be scaled up for the preparative collection of charge variants. This makes it difficult to identify the charge variants in a cIEF electropherogram. Therefore, preparative IEF methods are needed to fractionate charge variants for characterization. We used free-flow electrophoresis (FFE) to isolate monoclonal antibody charge variants observed in a cIEF electropherogram. The same antibody was also fractionated using the Rotofor and Offgel instruments for comparison. A strategy for purifying the fractionated charge variants and downstream characterization is described. Acidic and basic variants were identified and related back to the analytical cIEF charge profile. This study establishes free-flow isoelectric focusing as a valuable tool for characterizing therapeutic proteins.
Download full-text PDF |
Source |
|---|---|
| http://dx.doi.org/10.1021/acs.analchem.5b03946 | DOI Listing |
Publication Analysis
Top Keywords
Similar Publications
Structure-Function Analysis of CYP105A1 in the Metabolism of Nonsteroidal Anti-inflammatory Drugs.
Biochemistry
January 2025
Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
CYP105A1 exhibits monooxygenase activity to a wide variety of structurally different substrates with regio- and stereospecificity, making its application range broad. Our previous studies have shown that CYP105A1 wild type and its variants metabolize 12 types of nonsteroidal anti-inflammatory drugs (NSAIDs). In particular, the R84A variant exhibited a high activity against many NSAIDs.
View Article and Find Full Text PDFIntegrating multi-layered biological priors to improve genomic prediction accuracy in beef cattle.
Biol Direct
December 2024
Key Laboratory of Animal Genetics Breeding and Reproduction, Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing, 100193, China.
Background: Integrating multi-layered information can enhance the accuracy of genomic prediction for complex traits. However, the improvement and application of effective strategies for genomic prediction (GP) using multi-omics data remains challenging.
Methods: We generated 11 feature sets for sequencing variants from genomics, transcriptomics, metabolomics, and epigenetics data in beef cattle, then we assessed the contribution of functional variants using genomic restricted maximum likelihood (GREML).
Evolution of SARS-CoV-2 spike trimers towards optimized heparan sulfate cross-linking and inter-chain mobility.
Sci Rep
December 2024
Institute of Physiological Chemistry and Pathobiochemistry, University of Münster, Waldeyerstrasse 15, D-48149, Münster, Germany.
The heparan sulfate (HS)-rich extracellular matrix (ECM) serves as an initial interaction site for the homotrimeric spike (S) protein of SARS-CoV-2 to facilitate subsequent docking to angiotensin-converting enzyme 2 (ACE2) receptors and cellular infection. More recent variants, notably Omicron, have evolved by swapping several amino acids to positively charged residues to enhance the interaction of the S-protein trimer with the negatively charged HS. However, these enhanced interactions may reduce Omicron's ability to move through the HS-rich ECM to effectively find ACE2 receptors and infect cells, raising the question of how to mechanistically explain HS-associated viral movement.
View Article and Find Full Text PDFMechanistic modeling of anti-Langmuirian to Langmuirian behavior of Fc-fusion proteins in cation exchange chromatography.
J Chromatogr A
December 2024
Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Zhejiang Key Laboratory of Smart Biomaterials, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou 310058, China. Electronic address:
Development of a next-generation chromatographic model, capable of simultaneously meeting academic demands for thermodynamic consistency and industrial requirements in everyday project work, has become a focal point of research. In this study, anti-Langmuirian to Langmuirian (AL-L) elution behavior was observed in cation-exchange chromatographic separation of charge variants of industrial Fc-fusion proteins. To characterize this behavior, the multi-protein Mollerup activity model was integrated into the steric mass action (SMA) model, resulting in a new model named the generalized ion-exchange (nGIEX) isotherm for multi-protein systems.
View Article and Find Full Text PDFCas12e orthologs evolve variable structural elements to facilitate dsDNA cleavage.
Nat Commun
December 2024
Beijing Frontier Research Center for Biological Structure, State Key Laboratory of Membrane Biology, School of Life Sciences, Tsinghua University, Beijing, 100084, China.
Exceptionally diverse type V CRISPR-Cas systems provide numerous RNA-guided nucleases as powerful tools for DNA manipulation. Two known Cas12e nucleases, DpbCas12e and PlmCas12e, are both effective in genome editing. However, many differences exist in their in vitro dsDNA cleavage activities, reflecting the diversity in Cas12e's enzymatic properties.
View Article and Find Full Text PDFWant AI Summaries of new PubMed Abstracts delivered to your In-box?
Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!