Comprehensive characterization of charge variants in a mAb population is crucial, because these variants can affect the safety and efficacy of the biotherapeutics. However, there are several challenges using traditional reversed-phase LC-MS for charge variant analysis of intact mAbs. A single peak is usually observed and the MS spectrum and deconvoluted MS spectra are dominated by the major species, so minor variants with critical post translational modifications (PTMs) cannot be differentiated and identified in the LC-MS workflow. Here, we introduce a single workflow that not only provides separation and allows direct identity assignment of charge variants peaks, but also allows detection of low molecular weight (LMW) fragment impurities in the sample. The high resolving power and sensitivity of this CESI-MS workflow for charge variants analysis of mAbs facilitates the separation of basic and acidic variants as well as the identification of major glycan species for each variant peak.
Monoclonal antibodies (mAbs) are complex macromolecules that undergo a wide range of post-translational modifications that can result in charge heterogeneity. Ion exchange (IEX) chromatography is considered the gold standard for characterization of charge variants. This approach separates charge variants and allows for the collection of isoforms for protein identification by mass spectrometry (MS). However, the collection of multiple fractions often needs to be combined with top-down and bottom-up characterization methods, which is both resource and time consuming.. In a recent study by LeBlanc et al., (2019) from LFB Biotechnologies, a newly developed cation exchange column technology was evaluated for its ability to separate charge variants under native conditions with direct coupling to MS. In total, three different mAbs with high basicisoelectric points (pI) and a monoclonal antibody reference material from NIST were analyzed in both their native and proteolyzed forms. The fragments were ...
Learn about ProPac ion exchange chromatography columns for HPLC separation of proteins, monoclonal antibodies, biosimilars, and their charge variants.
The objective of this application note is to demonstrate the efficiency and robustness of Auto•Blend Plus Technology for optimization of an IEX method for charge variant separations.
From sample prep, to analytical instruments, to columns, software, and service, Agilent provides you with the tools and support you need to increase efficiency and reduce risk in your workflow for key biopharma applications such as Aggregate Analysis, Charge Variant Analysis and Peptide Mapping.
Study on Structure Development of Polymer Blends under the Continuous Change of Quench DepthStudy on Structure Development of Polymer Blends under the Continuous Change of Quench Depth ...
References for Abcams Recombinant Human CutA protein (ab95345). Please let us know if you have used this product in your publication
This study shows that the renin inhibitor VTP-27999 acts differently from aliskiren. First, VTP-27999 binding to renin increases renin immunoreactivity in 2 different renin IRMAs. Second, in contrast to aliskiren (and to other renin inhibitors, including remikiren),5,6 VTP-27999 does not induce prorenin unfolding. It does, however, bind to acid-activated, open prorenin, and by doing so, it keeps prorenin in this open conformation and then again increases the level of renin immunoreactivity when measuring this open prorenin in a renin IRMA.. Adding VTP-27999 at concentrations of ≥10 nmol/L during the renin IRMA of plasma samples increased the detected amount of immunoreactive renin by ≥30%. Similar increases were observed when measuring recombinant human renin in buffer. Aliskiren was capable of preventing the VTP-27999 effect in a concentration-dependent manner. This demonstrates that VTP-27999 and aliskiren compete for the same binding site, but that only VTP-27999 binding produces a ...
It is biochemically appropriate to consider that MUC13 consists of 2 distinct subunits, an extracellular α-subunit (consisting of the TR domain, 1 EGF-like domain, and a portion of the SEA domain) and a β-subunit (consisting of a portion of the SEA domain, 2 EGF-like domains, the TM domain, and the cytoplasmic tail, shown in Fig. 1). As described earlier, the SEA domain is predicted to contain a cleavage site and, although the amino acid sequence is unknown, there is biochemical evidence that MUC13 undergoes cleavage (9, 17). Williams and colleagues carried out a comprehensive Western blot analysis of MUC13 protein expressed in 2 cancer cell lines by using a polyclonal MUC13 antibody (9). In this experiment, under nonreducing conditions, MUC13 migrated as a 47-kDa band plus a 93-kDa band homodimer. In contrast, under reducing conditions, MUC13 appeared as a 58-kDa single band. The size difference observed with different conditions can be explained by the denaturation of MUC13 in the reducing ...
单分子力谱 的翻译结果:single-molecule force spectroscopy;single molecule force spectroscopy;single molecular force spectroscopy||双语例句|英文例句|相关文摘
Single-molecule force traces recorded in different protein spots on a single chip with a single cantilever.(a-d) Four proteins of interest, anchored between the
Sturgeon (Acipenser stellatus; A.g ldenst dti) and salmon (Oncorhynchus gorbuscha, Salmo salar) gonadotropic hormone (GTH) , its dimeric structure and the functional role of individual subunits as the specific components of the dimeric molecule has been investigated since 1975. For the first time chromatographically purified sturgeon and salmon GTH was isolated and it was shown that the pituitary gland of the fishes produces molecular charge variants or isoforms of GTH with different properties. It was shown that the quaternary structure of fish GTH is an asymmetric heterodimer, built like a mammalian GTH-s, consisting of two chemically heterologic subunits which differ functionally and are mutually bound noncovalently.. Also, structure-functional significance of some side chain radicals and sugar moiety were investigated using selective chemical modifications of both dimeric GTH and individual subunits.. The chemical structure of carbohydrate chains of the fish GTH and its subunits has been ...
Single-molecule force spectroscopy of bimolecular reactions: system homology in the mechanical activation of ligand substitution reactions.s profile, publications, research topics, and co-authors
Growth cones motility under time modulated tension. (a) Two contacting growth cones of a differentiating rat hippocampal neuron (7 DIV) with an attached poly-D-lysine coated bead. The white arrow indicates the constant force applied on the trapped bead. (b) Traces of the x, y, z coordinates of the tracked bead (in blue, red and green, respectively). Axes origin is at the left upper corner of the field of view in a. Traces are sampled at 2 KHz (kx,y = 4 fN/nm, kz = 2 fN/nm). The blue shadow indicates when the applied force in the x direction is time modulated. Power at the sample, 3.8 mW. Bars, 8 μm. (c) Inset of the traces showed in b. The black line represent the tracked reference point in the x direction indicated in pN on the left side of the plot (oscillating frequency 0.3 Hz ...
Yvonne Carts-Powell. Researchers at the Technical University of Munich (Germany) used single-molecule force spectroscopy with ultrastable optical tweezers to measure the length of a single protein and the force required to cause a change in the folding state.. ...
The versatility of chemical peptide synthesis combined with the high sensitivity of AFM single-molecule force spectroscopy allows us to investigate, quantify, and control molecular recognition processes (molecular nanotechnology), offering a tremendous potential in chemical biology. Single-molecule force spectroscopy experiments are able to detect fast intermediate transition states, details of the energy landscape, and structural changes, while avoiding multiple binding events that can occur under ensemble conditions. Dynamic force spectroscopy (DFS) is even able to provide data on the complex lifetime. This minireview outlines the biophysical methodology, discusses different experimental set-ups, and presents representative results in the form of two case studies, both dealing with DNA-binding peptides. They may serve as model systems, e.g., for transcription factors or gene transfection agents. Copyright (c) 2006 European Peptide Society and John Wiley & Sons, Ltd ...
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