The increased interest in using monoclonal antibodies (mAbs) as a platform for biopharmaceuticals has led to the need for new analytical techniques that can precisely assess physicochemical properties of these large and very complex drugs for the purpose of correctly identifying quality attributes (QA). One QA, higher order structure (HOS), is unique to biopharmaceuticals and essential for establishing consistency in biopharmaceutical manufacturing, detecting process-related variations from manufacturing changes and establishing comparability between biologic products. To address this measurement challenge, two-dimensional nuclear magnetic resonance spectroscopy (2D-NMR) methods were introduced that allow for the precise atomic-level comparison of the HOS between two proteins, including mAbs. Here, an inter-laboratory comparison involving 26 industrial, government and academic laboratories worldwide was performed as a benchmark using the NISTmAb, from the National Institute of Standards and Technology (NIST), to facilitate the translation of the 2D-NMR method into routine use for biopharmaceutical product development. Two-dimensional 1H,15N and 1H,13C NMR spectra were acquired with harmonized experimental protocols on the unlabeled Fab domain and a uniformly enriched-15N, 20%-13C-enriched system suitability sample derived from the NISTmAb. Chemometric analyses from over 400 spectral maps acquired on 39 different NMR spectrometers ranging from 500 MHz to 900 MHz demonstrate spectral fingerprints that are fit-for-purpose for the assessment of HOS. The 2D-NMR method is shown to provide the measurement reliability needed to move the technique from an emerging technology to a harmonized, routine measurement that can be generally applied with great confidence to high precision assessments of the HOS of mAb-based biotherapeutics.

The orientation of the substrate camphor in the active site of reduced CO-bound cytochrome P450 cam (CYP101) as a function of reduced putidaredoxin (Pdx r ) addition has been examined by NMR using perdeuterated CYP101 and peredeuterated Pdx as well as isotopically labeled d-camphor. This permits the 1 H resonances of CYP101-bound camphor to be observed without interference from the signals of CYP101 or Pdx, and confirms assignments of the methyl signals of camphor in the bound form. The Cys 4 Fe 2 S 2 ferredoxin Pdx is the physiological redox partner and effector of CYP101. The addition of Pdx to the reduced CYP101-camphor-CO complex results in a conformational selection that is slow on the chemical shift time scale with spectral effects observed primarily at the 8-CH 3 group of the camphor. The camphor signals are ring-current shifted by the heme, and for the 9-and 10-CH 3 resonances, these shifts are reasonably well predicted by ring current calculations from the crystal structure of CO-bound CYP101. However, in the absence of Pdx the 8-CH 3 resonance of CYP101-bound camphor is observed at considerably higher field than predicted. Dynamic simulations using ring current shift restraints generated a structure with low chemical shift violations in which the hydrogen bond between the camphor carbonyl oxygen and the OH of Tyr 96 is lost, and an expansion of the active site takes place that permits reorientation of the camphor within the active site.Cytochrome P450 cam (CYP101) catalyzes the 5-exo-hydroxylation of camphor 1, the first step of catabolism of 1 by the soil bacterium Pseudomonas putida. 1 Two electrons are required for turnover, and the second reduction, of the Fe(II) P450 cam -O 2 -1 ternary complex (CYP-1-O 2 ), is the rate limiting step under physiological conditions. 2 The presence of an effector is required for the formation of hydroxycamphor product. 3 The Fe 2 S 2 ferredoxin putidaredoxin (Pdx) is the biological effector and reductant of CYP101. 3 Structural perturbations have been observed spectroscopically in the CYP101 active site upon binding of Pdx, and it has been proposed that these perturbations are involved in the effector and/or electron transfer activity of Pdx. 4-9The complex between reduced Pdx (Pdx r ) and reduced 1-and CO-bound P450 cam (CYP-1-CO) is often used a model for the catalytically competent Pdx r -CYP-1 -O 2 complex, and is convenient for NMR spectroscopy in that the heme is diamagnetic. Recently, we reported perturbations in the conformational equilibrium of CYP-1-CO as a function of addition of Pdx r.7 These perturbations were monitored by 1 H, 15 N HSQC experiments using sequencespecific resonance assignments in CYP101. In addition to changes that take place in the proposed interface between the two proteins, we identified perturbations in regions of the P450 cam molecule that have been shown to be involved in substrate access and orientation, including the B', F and G helices and portions of the β3 sheet. Based on this, we proposed a dynamic model for the effe...

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Extensive testing of hydrolysates of commercially available organosilanes has identified a number of bifunctional organosiloxane compounds that show potential as therapeutics for treatment of diseases characterized by amyloid deposition such as Alzheimer's disease (AD). All of these compounds protect from and/or reverse the metal-induced aggregation of amyloid Aβ(1-42) peptide in dynamic light scattering (DLS) assays in trifluoroethanol (TFE) solutions, protect from and/or reverse the metal-induced loss of α-helical structure in TFE solutions of amyloid Aβ (1-42) as measured by circular dichroism (CD), and are able to cross blood-brain barrier models at rates above background using Caco-2 and MDCK cell permeation assays. Based on these studies, we conclude that members of this class of bifunctional organosiloxanes are promising candidates for testing in treatment and/or prevention of AD and other diseases characterized by amyloid deposition.