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. METHODS: A library of polymers was synthesized and screened by a high-throughput, absorbance-based assay. The lead polymers were selected and characterized for their ability to alter the precipitation kinetics of a mAb in physiologically relevant conditions using two model systems. RESULTS: Biophysical testing via surface tension measurements, isothermal titration calorimetry (ITC), microscale thermophoresis (MST), and intrinsic fluorescence quenching indicated that the polymers delayed onset of mAb precipitation from a combination of surfactant behaviour and interactions with the protein to prevent protein-protein interactions leading to colloidal instability. CONCLUSIONS: The random heteropolymers described are a new class of excipients that may enable development of SC mAb formulations previously inaccessible to patients.

Ultrafast interligand electron transfer in cis-[Ru(4,4'-dicarboxylate-2,2'-bipyridine)2(NCS)2]4- 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 (cis-[Ru(dcb)2(NCS)2]4-, 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-1, assigned to a symmetric carboxylate stretch, showed significant anisotropy. A rapid anisotropy increase (τ 1 ≈ 2 ps) was tentatively assigned to vibrational and solvent relaxation, considering the excess energy available after the excited singlet-triplet conversion. Thereafter, the anisotropy decayed to zero with a time constant τ 2 ≈ 240 ps, which was assigned to the rotational correlation time of the complex in deuterated acetonitrile. No other distinctive changes to the anisotropy were observed and the amplitude of the slow component at time zero agrees well with that predicted for a random mixture of MLCT localization on either of the two dcb ligands. The results therefore suggest that MLCT randomization over the two dcb ligands occurs on the sub-ps time scale. This is much faster than proposed by previous reports on the related N3 complex [Benkö et al., J. Phys. Chem. B, 2004, 108, 2862, and Waterland et al., J. Phys. Chem. A, 2001, 105, 4019], but in agreement with that found by Wallin and co-workers [J. Phys. Chem. A, 2005, 109, 4697] for the [Ru(bpy)3]2+ (bpy = 2,2'-bipyridine) complex. This suggests that electron injection from the excited dye into TiO2 in dye-sensitized solar cells is not limited by ILET.

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. Although no lysozyme adsorption is observed on the polystyrene surface in low solution pH condition, the mAb can be strongly absorbed on the polystyrene surface with a layer thickness of ≈145 Å. The mAb concentration near the surface is about 135 mg/ml significantly larger than the bulk protein concentration. The differences in adsorption behavior are attributed to different protein interactions with a hydrophobic surface. Further, both surfactants and proteins adsorbed on the polystyrene surfaces can not be rinsed off using pure water.

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. Second, a tricarbonyl (η(5)-cyclopentadienyl) rhenium(I) probe modified with an alkynyl linkage is coupled via the Click reaction. We demonstrate these steps using the C-terminal domain of the ribosomal protein L9 as a model system. An overall incorporation level of 92% was obtained at residue 109, which is a surface-exposed residue. Incorporation of the probe into a surface site is shown not to perturb the stability or structure of the target protein. Metal carbonyls are known to be sensitive to solvation and protein electrostatics through vibrational lifetimes and frequency shifts. We report that the frequencies and lifetimes of this probe also depend on the isotopic composition of the solvent. Comparison of the lifetimes measured in H2O versus D2O provides a probe of solvent accessibility. The metal carbonyl probe reported here provides an easy and robust method to label very large proteins with an amino-acid-specific tag that is both environmentally sensitive and a very strong absorber.

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. For the antimicrobial peptide ovispirin bound to membrane bilayers, we find that the amide II peak decays with a biexponential with rate constants of 0.54 ± 0.02 and 0.12 ± 0.01 min(-1), which is a measure of the overall HDX in the peptide. The cross peaks between Ile-10-labeled ovispirin and the amide II mode, which specifically monitor HDX kinetics at Ile-10, decay with a single rate constant of 0.36 ± 0.1 min(-1). Comparing this exchange rate to theoretically determined exchange rates of Ile-10 for ovispirin in a solution random coil configuration, the exchange rate at Ile-10 is at least 100 times slower, consistent with the known α-helix structure of ovispirin in bilayers. Because backbone isotope labels produce only a very small shift of the amide II band, site-specific HDX cannot be measured with FTIR spectroscopy, which is why 2D IR spectroscopy is needed for these measurements.

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. The frequency range spans 10 cm(-1), and the variation in lifetimes ranges from 6 to 19 ps, indicating that these probes are very sensitive to their environments. Also, we attached probes with -(CH2)-, -(CH2)3-, and -(CH2)4- linkers to ubiquitin at positions 6 and 63 and collected spectra in aqueous buffer. The frequencies and lifetimes were correlated for 3C and 4C linkers, as they were in the solvents, but did not correlate for the 1C linker. We conclude that lifetime measures solvation, whereas frequency reflects the electrostatics of the environment, which in the case of the 1C linker is a measure of the protein electrostatic field. We also labeled V71C α-synuclein in buffer and membrane-bound. Unlike most other infrared labels, this label has extremely strong cross sections and thus can be measured with 2D IR spectroscopy at sub-millimolar concentrations. We expect that these labels will find use in studying the structure and dynamics of membrane-bound, aggregated, and kinetically evolving proteins for which high signal-to-noise at low protein concentrations is imperative.

Parallel ß-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 ß-sheets. Using a synthetic macrocycle that enforces a two stranded parallel ß-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. The frequencies of the labels ranged from 1585 to 1595 cm(-1), with the largest frequency shift occurring for in-register amino acids. The 2D IR spectra of the coupled isotope labels were calculated from molecular dynamics simulations of a series of macrocycle structures generated from replica exchange dynamics to fully sample the conformational distribution. The models used to simulate the spectra include through-space coupling, through-bond coupling, and local frequency shifts caused by environment electrostatics and hydrogen bonding. The calculated spectra predict the line widths and frequencies nearly quantitatively. Historically, the characteristic features of ß-sheet infrared spectra have been attributed to through-space couplings such as transition dipole coupling. We find that frequency shifts of the local carbonyl groups due to nearest neighbor couplings and environmental factors are more important, while the through-space couplings dictate the spectral intensities. As a result, the characteristic absorption spectra empirically used for decades to assign parallel ß-sheet secondary structure arises because of a redistribution of oscillator strength, but the through-space couplings do not themselves dramatically alter the frequency distribution of eigenstates much more than already exists in random coil structures. Moreover, solvent exposed residues have amide I bands with >20 cm(-1) line width. Narrower line widths indicate that the amide I backbone is solvent protected inside the macrocycle. This work provides calculated and experimentally verified couplings for parallel ß-sheets that can be used in structure-based models to simulate and interpret the infrared spectra of ß-sheet containing proteins and protein assemblies, such as amyloid fibers.

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 ß-sheet, but at 8 h after mixing, rat amylin blocks the N-terminal ß-sheet instead. At 24 h after mixing, rat amylin blocks neither ß-sheet and forms its own ß-sheet, most probably on the outside of the human fibrils. This is striking, because rat amylin is natively disordered and not previously known to form amyloid ß-sheets. The results show that even seemingly intuitive inhibitors may function by unforeseen and complex structural processes.

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 ß-sheets, whereas the N-terminal domain becomes extremely disordered but lies in close proximity to the ß-sheets. Two-dimensional IR kinetics experiments show that fibril nucleation and extension occur exclusively in the C-terminal domain. These results are unexpected because the N-terminal domain is less stable in the monomer form. Isotope dilution experiments reveal that each C-terminal domain contributes two or fewer adjacent ß-strands to each ß-sheet. From these observations, we propose an initial structural model for γD-crystallin amyloid fibrils. Because only 1 µg of protein is required for a 2D IR spectrum, even poorly expressing proteins can be studied under many conditions using this approach. Thus, we believe that 2D IR and protein ligation will be useful for structural and kinetic studies of many protein systems for which IR spectroscopy can be straightforwardly applied, such as membrane and amyloidogenic proteins.

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. TEM analysis shows the peptide forms normal amyloid (abstract image).

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. Our rapid-scan technology relies on a mid-IR pulse shaper that computer generates the pulse shapes, much like in an NMR spectrometer. With this device, data collection is faster, easier, and more accurate. We describe our 2D IR spectrometer, as well as protocols for (13)C(18)O isotope labeling, and then illustrate the technique with an application to the aggregation of the human islet amyloid polypeptide implicated in type 2 diabetes.

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. A comparison of the line shapes reveals an oscillation in the inhomogeneous line width that has a period equal to that of an alpha-helix (3.6 amino acids). The periodic trend is caused by the asymmetric environment of the membrane bilayer that exposes one face of the alpha-helix to much stronger environmental electrostatic forces than the other. We compare our experimental results to 2D IR line shapes calculated using the lowest free energy structure identified from molecular dynamics simulations. These simulations predict a periodic trend similar to the experiment and lead us to conclude that ovispirin lies in the membrane just below the headgroups, is tilted, and may be kinked. Besides providing insight into the antibiotic mechanism of ovispirin, our procedure provides an infrared method for studying peptide and protein structures that relies on the natural vibrational modes of the backbone. It is a complementary method to other techniques that utilize line shapes, such as fluorescence, NMR, and ESR spectroscopies, because it does not require mutations, the spectra can be quantitatively simulated using molecular dynamics, and the technique can be applied to difficult-to-study systems like ion channels, aggregated proteins, and kinetically evolving systems.