Virus filtration using small pore virus filter in downstream processing of biotherapeutic products: The effect of operating pressure.

Virus filtration is a robust and effective method to remove potential virus contaminants. Planova 20 N, a virus filter form Asahi Kasei Bioprocess, has been widely used in the manufacturing process of biotherapeutics. Previous studies have shown that parvovirus removal by Planova 20 N can be impacted by low operation pressure and depressurization. Therefore, it is critical to define an operating pressure range for robust virus removal. In this work, the effect of pressure combined with depressurization on virus removal by Planova 20 N was investigated. Our studies showed that effective virus removal can be achieved in the pressure range from 0.7 bar to 1.6 bar. The data also suggest that re-starting with higher pressure after depressurization is highly desirable for large-scale manufacture to mitigate virus leakage risk. In addition, skipping buffer flush post mainstream filtration minimizes the likelihood of depressurization.

Selective polypeptide ligand binding to the extracellular surface of the transmembrane domains of the class B GPCRs GLP-1R and GCGR.

Crystal and cryo-EM structures of the glucagon-like peptide-1 receptor (GLP-1R) and glucagon receptor (GCGR) bound with their peptide ligands have been obtained with full-length constructs, indicating that the extracellular domain (ECD) is indispensable for specific ligand binding. This article complements these data with studies of ligand recognition of the two receptors in solution. Paramagnetic NMR relaxation enhancement measurements using dual labeling with fluorine-19 probes on the receptor and nitroxide spin labels on the peptide ligands provided new insights. The glucagon-like peptide-1 (GLP-1) was found to interact with GLP-1R by selective binding to the extracellular surface. The ligand selectivity toward the extracellular surface of the receptor was preserved in the transmembrane domain (TMD) devoid of the ECD. The dual labeling approach further provided evidence of cross-reactivity of GLP-1R and GCGR with glucagon and GLP-1, respectively, which is of interest in the context of medical treatments using combinations of the two polypeptides.

A Genetically Encoded F-19 NMR Probe Reveals the Allosteric Modulation Mechanism of Cannabinoid Receptor 1.

Due to the lack of genetically encoded probes for fluorine-19 nuclear magnetic resonance spectroscopy (19F NMR), its utility for probing eukaryotic membrane protein dynamics is limited. Here we report an efficient method for the genetic incorporation of an unnatural amino acid (UAA), 3'-trifluoromenthyl-phenylalanine (mtfF), into cannabinoid receptor 1 (CB1) in the Baculovirus Expression System. The probe can be inserted at any environmentally sensitive site, while causing minimal structural perturbation to the target protein. Using 19F NMR and X-ray crystallography methods, we discovered that the allosteric modulator Org27569 and agonists synergistically stabilize a previously unrecognized pre-active state. An allosteric modulation model is proposed to explain Org27569's distinct behavior. We demonstrate that our site-specific 19F NMR labeling method is a powerful tool in decoding the mechanism of GPCR allosteric modulation. This new method should be broadly applicable for uncovering conformational states for many important eukaryotic membrane proteins.

Disulfide-Containing Detergents (DCDs) for the Structural Biology of Membrane Proteins.

Disulfide-containing detergents (DCDs) are introduced, which contain a disulfide bond in the hydrophobic tail. DCDs form smaller micelles than corresponding detergents with linear hydrocarbon chains, while providing good solubilization and reconstitution of membrane proteins. The use of this new class of detergents in structural biology is illustrated with solution NMR spectra of the human G protein-coupled receptor A2A AR, which is an α-helical protein, and the ß-barrel protein OmpX from E. coli.