Random Heteropolymer Excipients Improve the Colloidal Stability of a Monoclonal Antibody for Subcutaneous Administration.

Purpose: Developing stable high concentration monoclonal antibody (mAb) formulations is increasingly important to move toward subcutaneous (SC) administration for better patient experience. Challenges stemming from protein-protein interactions in these crowded solutions, such as colloidal instability, limit the feasibility of some formulations because of concerns of safety, product quality, and/or manufacturability. Herein, we report novel random heteropolymer excipients that improve the colloidal stability of a high concentration mAb formulation for SC administration.

Ultrafast interligand electron transfer in -[Ru(4,4'-dicarboxylate-2,2'-bipyridine)(NCS)] and implications for electron injection limitations in dye sensitized solar cells.

Interligand electron transfer (ILET) of the lowest metal-to-ligand charge transfer (MLCT) state of N712 (-[Ru(dcb)(NCS)], where dcb = 4,4'-dicarboxylate-2,2'-bipyridine) in a deuterated acetonitrile solution has been studied by means of femtosecond transient absorption anisotropy in the mid-IR. Time-independent B3LYP density functional calculations were performed to assign vibrational bands and determine their respective transition dipole moments. The transient absorption spectral band at 1327 cm, assigned to a symmetric carboxylate stretch, showed significant anisotropy.

Adsorption of polysorbate 20 and proteins on hydrophobic polystyrene surfaces studied by neutron reflectometry.

Understanding the adsorption of protein and surfactant molecules on hydrophobic surfaces is very important for storage stability and delivery of pharmaceutical liquid formulations as many commonly-used devices, such as drug containers and syringes, have hydrophobic surfaces. Neutron reflectometry is used here to investigate the structure information of the adsorption process of non-ionic surfactant (polysorbate 20) and proteins (monoclonal antibody (mAb) and lysozyme) on polystyrene surfaces. Thickness of adsorbed polysorbate 20 thin film is observed to be ≈21 Å, comparable to the radius of gyration of polysorbate 20 micelles in solution.

General strategy for the bioorthogonal incorporation of strongly absorbing, solvation-sensitive infrared probes into proteins.

A high-sensitivity metal-carbonyl-based IR probe is described that can be incorporated into proteins or other biomolecules in very high yield via Click chemistry. A two-step strategy is demonstrated. First, a methionine auxotroph is used to incorporate the unnatural amino acid azidohomoalanine at high levels.

2D IR cross peaks reveal hydrogen-deuterium exchange with single residue specificity.

A form of chemical exchange, hydrogen-deuterium exchange (HDX), has long been used as a method for studying the secondary and tertiary structure of peptides and proteins using mass spectrometry and NMR spectroscopy. Using two-dimensional infrared (2D IR) spectroscopy, we resolve cross peaks between the amide II band and a (13)C(18)O isotope-labeled amide I band, which we show measures HDX with site-specific resolution. By rapidly scanning 2D IR spectra using mid-IR pulse shaping, we monitor the kinetics of HDX exchange on-the-fly.

A strongly absorbing class of non-natural labels for probing protein electrostatics and solvation with FTIR and 2D IR spectroscopies.

A series of non-natural infrared probes is reported that consist of a metal-tricarbonyl modified with a -(CH2)n- linker and cysteine-specific leaving group. They can be site-specifically attached to proteins using mutagenesis and similar protocols for EPR spin labels, which have the same leaving group. We characterize the label's frequencies and lifetimes using 2D IR spectroscopy in solvents of varying dielectric.

Parallel β-sheet vibrational couplings revealed by 2D IR spectroscopy of an isotopically labeled macrocycle: quantitative benchmark for the interpretation of amyloid and protein infrared spectra.

Infrared spectroscopy is playing an important role in the elucidation of amyloid fiber formation, but the coupling models that link spectra to structure are not well tested for parallel β-sheets. Using a synthetic macrocycle that enforces a two stranded parallel β-sheet conformation, we measured the lifetimes and frequency for six combinations of doubly (13)C═(18)O labeled amide I modes using 2D IR spectroscopy. The average vibrational lifetime of the isotope labeled residues was 550 fs.

Two-dimensional infrared spectroscopy reveals the complex behaviour of an amyloid fibril inhibitor.

Amyloid formation has been implicated in the pathology of over 20 human diseases, but the rational design of amyloid inhibitors is hampered by a lack of structural information about amyloid-inhibitor complexes. We use isotope labelling and two-dimensional infrared spectroscopy to obtain a residue-specific structure for the complex of human amylin (the peptide responsible for islet amyloid formation in type 2 diabetes) with a known inhibitor (rat amylin). Based on its sequence, rat amylin should block formation of the C-terminal β-sheet, but at 8 h after mixing, rat amylin blocks the N-terminal β-sheet instead.

Two-dimensional IR spectroscopy and segmental 13C labeling reveals the domain structure of human γD-crystallin amyloid fibrils.

The structural eye lens protein γD-crystallin is a major component of cataracts, but its conformation when aggregated is unknown. Using expressed protein ligation, we uniformly (13)C labeled one of the two Greek key domains so that they are individually resolved in two-dimensional (2D) IR spectra for structural and kinetic analysis. Upon acid-induced amyloid fibril formation, the 2D IR spectra reveal that the C-terminal domain forms amyloid β-sheets, whereas the N-terminal domain becomes extremely disordered but lies in close proximity to the β-sheets.

2DIR spectroscopy of human amylin fibrils reflects stable β-sheet structure.

The aggregation of human amylin to form amyloid contributes to islet β-cell dysfunction in type 2 diabetes. Studies of amyloid formation have been hindered by the low structural resolution or relatively modest time resolution of standard methods. Two-dimensional infrared (2DIR) spectroscopy, with its sensitivity to protein secondary structures and its intrinsic fast time resolution, is capable of capturing structural changes during the aggregation process.

Efficient microwave-assisted synthesis of human islet amyloid polypeptide designed to facilitate the specific incorporation of labeled amino acids.

A cost-efficient, time-reducing solid-phase synthesis of the amyloidogenic, 37 residue islet amyloid polypeptide (IAPP) is developed using two pseudoprolines (highlighted blue in sequence) in combination with microwave technology. A yield twice that obtained with conventional syntheses is realized. The utility of this protocol is demonstrated by the synthesis of a (13)C(18)O-labeled Ser-20 IAPP variant, a prohibitively expensive and chemically challenging site to label via other protocols.

Residue-specific structural kinetics of proteins through the union of isotope labeling, mid-IR pulse shaping, and coherent 2D IR spectroscopy.

We describe a methodology for studying protein kinetics using a rapid-scan technology for collecting 2D IR spectra. In conjunction with isotope labeling, 2D IR spectroscopy is able to probe the secondary structure and environment of individual residues in polypeptides and proteins. It is particularly useful for membrane and aggregate proteins.

2D IR line shapes probe ovispirin peptide conformation and depth in lipid bilayers.

We report a structural study on the membrane binding of ovispirin using 2D IR line shape analysis, isotope labeling, and molecular dynamics simulations. Ovispirin is an antibiotic polypeptide that binds to the surfaces of membranes as an alpha-helix. By resolving individual backbone vibrational modes (amide I) using 1-(13)C=(18)O labeling, we measured the 2D IR line shapes for 15 of the 18 residues in this peptide.