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Biopharmaceuticals such as monoclonal antibodies are required to be rigorously characterized using a wide range of analytical methods. Various material properties must be characterized and well controlled to assure that clinically relevant features and critical quality attributes are maintained. A thorough understanding of analytical method performance metrics, particularly emerging methods designed to address measurement gaps, is required to assure methods are appropriate for their intended use in assuring drug safety, stability, and functional activity. To this end, a series of interlaboratory studies have been conducted using NISTmAb, a biopharmaceutical-representative and publicly available monoclonal antibody test material, to report on state-of-the-art method performance, harmonize best practices, and inform on potential gaps in the analytical measurement infrastructure. Reported here is a summary of the study designs, results, and future perspectives revealed from these interlaboratory studies which focused on primary structure, post-translational modifications, and higher order structure measurements currently employed during biopharmaceutical development.

1. Summary Monoclonal antibody (mAb) pharmaceuticals account for the emergence of safer, targeted therapeutics currently addressing cancer [1], autoimmune conditions [2-4], osteoporosis, [5] macular degeneration [6], migraines [7], and infectious [...]

1. Summary The spreadsheet file reported herein provides centroid data, descriptive of deuterium uptake, for the Fab Fragment of NISTmAb (PDB: 5K8A) reference material, as measured by the bottom-up hydrogen-deuterium [...]

Alginate is a polysaccharide produced by certain seaweeds and bacteria that consists of mannuronic acid and guluronic acid residues. Seaweed alginate is used in food and industrial chemical processes, while the biosynthesis of bacterial alginate is associated with pathogenic Pseudomonas aeruginosa. Alginate lyases cleave this polysaccharide into short oligo-uronates and thus have the potential to be utilized for both industrial and medicinal applications. An alginate lyase gene, algMsp, from Microbulbifer sp. 6532A, was synthesized as an E.coli codon-optimized clone. The resulting 37 kDa recombinant protein, AlgMsp, was expressed, purified and characterized. The alginate lyase displayed highest activity at pH 8 and 0.2 M NaCl. Activity of the alginate lyase was greatest at 50°C; however the enzyme was not stable over time when incubated at 50°C. The alginate lyase was still highly active at 25°C and displayed little or no loss of activity after 24 hours at 25°C. The activity of AlgMsp was not dependent on the presence of divalent cations. Comparing activity of the lyase against polymannuronic acid and polyguluronic acid substrates showed a higher turnover rate for polymannuronic acid. However, AlgMSP exhibited greater catalytic efficiency with the polyguluronic acid substrate. Prolonged AlgMsp-mediated degradation of alginate produced dimer, trimer, tetramer, and pentamer oligo-uronates.

Resonance-enhanced multiphoton ionization spectroscopy and ab initio calculations are used to further investigate the transient molecule BC1, the study includes details of two new electronic states of BC1.

The reaction rate coefficients, themochemistry and mechanism applicable to the addition of a chlorine atom to allene have been studied by using cavity ring-down spectroscopy and ab initio calculations.

Experiments have been conducted concerning the mechanism that governs the addition reactions of chlorine and bromine atoms with propargyl halides. Ultraviolet spectra of radical adducts C (sub)3 H (sub)3 Cl (sub)2, C (sub)3 H (sub)3 ClBr and C (sub)3 H (sub)3 Br (sub)2 have been observed.

A study on the reactions of atmospheric iodine compound radicals investigated the use of cavity ring-down spectroscopy to determine rates of reaction of iodine monoxide (IO). The experiment focused on the reactions of IO + IO and IO + NO. It furnishes a direct comparison of reaction rate coefficients measured both for detection of IO through a sharply structured, rotationally resolved spectrum and through a diffuse, unstructured absorption spectrum. Experimental methods and findings are discussed.

The resonance enhanced multiphoton ionization spectrum of the free radical SnF has been recorded using excitation wavelengths in the range 540 to 360 nm. The previously documented C 2Δ, D, E 2Π, and F 2Σ+ states and two hitherto unidentified excited states, the H and I 2[uppercase_phi_synonym] states, were all observed as two-photon resonances. Band contour simulations yielded spectroscopic constants for the E, F, and I states. © 1996 American Institute of Physics. [ABSTRACT FROM AUTHOR]

Spectra of fluoromethylene radicals, HCF and DCF, have been observed between 304 and 325 nm using mass-resolved, resonance enhanced multiphoton ionization (REMPI). Singlet HCF is generated by sequential reactions of atomic fluorine in either methane or methyl fluoride. The spectra arise from two-photon resonances between the ground state X 1A′ and the previously unobserved E 1A′ (3p) Rydberg state. The band origins lie at T0=62154±2 cm-1 (321.69±0.01 nm) and T0=62175±2 cm-1 (321.58±0.01 nm) (2σ) above the ground states for HCF and DCF, respectively. Vibrational resolution and partial rotational resolution are obtained, with ν2′=1128±4 cm-1 and ν3′=1614±4 cm-1 for HCF and ν1′=2095±5 cm-1, ν2′=872±3 cm-1, and ν3′=1582±4 cm-1 for DCF (2σ). Under typical experimental conditions, the strongest peak for HCF is due to the 210310 resonance at 308.23±0.04 nm and the strongest peak for DCF is due to the 220 resonance at 312.85±0.01 nm. The results of vibrational and rotational analyses are consistent with predictions from ab initio calculations, which also yield the adiabatic ionization potential IPa=10.06±0.05 eV (2σ). [ABSTRACT FROM AUTHOR]

AlCl was detected and characterized with 1+2, 2+1, 2+2, 3+1, and 3+2 resonance-enhanced multiphoton ionization spectroscopy using laser light between 332 and 570 nm. AlCl was generated in the gas phase with high temperature reaction of Al with InCl3, or by the reaction of Al with AgCl. New states residing between 52 000 and 66 000 cm-1 were identified. Vibrational intervals of the new Rydberg states are about 20% greater than those of AlCl (X 1Σ+), with most lying between 565–595 cm-1. [ABSTRACT FROM AUTHOR]

The 3p 2B1←←X 2B1 bands of CH2D and CHD2 radicals were observed between 305 and 335 nm by mass resolved, 2+1 resonance enhanced multiphoton ionization spectroscopy. Spectroscopic constants were found for the 3p 2B1 Rydberg state of the CH2D radical (ν00=59 940 cm-1, ν1a1 CH2 stretch=2995 cm-1, ν2a1 CD stretch=2220 cm-1, ν4b1 out-of-plane large amplitude (OPLA)=1260 cm-1, ν5b2 CH2 asymmetric stretch=3055 cm-1, ν6b2 CD bend=1115 cm-1) and of the CHD2 radical (ν00=59 920 cm-1, ν1a1 CH stretch=3040 cm-1, ν2a1 CD2 stretch=2150 cm-1, ν4b1 OPLA=1165 cm-1, ν6b2 CH bend=1210 cm-1). Vibrational frequencies calculated by ab initio theory agree well with the experimental data. [ABSTRACT FROM AUTHOR]

The structures and optical spectroscopy of the CHFCl radical and cation were studied by ab initio molecular orbital calculations and by experiment. Ab initio calculations at the MP2/ 6–311++G** theory level found that the optimum structure of the CHFCl+ (X 1A’) cation is planar with r(C–H)=1.092 Å, r(C–F)=1.254 Å, r(C–Cl)=1.599 Å, /_ H–C–F=116.85°, and /_ H–C–Cl=122.14°. CHFCl (X 2A’) radical is nonplanar with r(C–H)=1.083 Å, r(C–F)=1.335 Å, r(C–Cl)=1.705 Å, /_ H–C–F=113.49°, /_ H–C–Cl=116.68°, and /_ F–C–Cl=114.44°. The ab initio angle between the F–C–Cl plane and the C–H bond is [uppercase_phi_synonym]e=38° and the inversion barrier is Binv=1190 cm-1. Using isogyric reactions to obtain empirical corrections, we calculate IPa(CHFCl)=8.37±0.05 eV. Ab initio vibrational frequencies are reported. The electronic spectrum of the CHFCl radicals was observed between 340–420 nm using one color, mass resolved, 2+1 resonance enhanced multiphoton ionization (REMPI) spectroscopy. The spectrum arises from two-photon resonances with the planar F(3p) [λlaser=406.7 nm, ν00=49 160(20) cm-1] and J(3d) [λlaser=361.9 nm, ν00=55 250(20) cm-1] Rydberg states. A third laser photon ionized the radicals. Both states produced the same vibrational constants: ν2 (C–H deformation)=1280(30) cm-1, ν4 (C–Cl stretch)=910(30) cm-1, ν5 (CFCl scissors)=440(30) cm-1, ν6 (OPLA)=980(30) cm-1. The REMPI spectrum exhibited v‘6 = 1–6 hot bands of the CHFCl (X 2A) radical. Modeling of these hot bands with a double-well potential gives the inversion barrier, Binv=1180 cm-1, and [uppercase_phi_synonym]e=42°. [ABSTRACT FROM AUTHOR]

Five new triplet excited states of the ND radical (three in the case of NH) in the wave-number range 85 000–91 000 cm-1 have been identified through analysis of the two-photon resonance enhancements they provide to the wavelength-resolved multiphoton ionization spectrum of X 3Σ- state NH(ND) radicals. The lowest energy of these, the B 3Π state, is found to be a ‘‘regular’’ Rydberg state which, on the basis of its observed quantum defect and its deduced rotational and spin–orbit coupling constant, is surmised to be the 3Π state derived from a 3pσ electron built on the 2Π ground-state ion core. Perturbations are evident in the B 3Π–X 3Σ- origin bands of both NH and ND. In the case of ND the perturbing state provides its own resonance enhancements, the analysis of which enables its definitive identification as the C 3Σ- state. The very small spin–orbit splitting found for the D 3Π state is taken to indicate that (at least in the Franck–Condon region) its wave function is dominated by the configuration involving one 3pπ Rydberg electron and a 4Σ- ion core. To still higher wave number we identify two more 3Σ- excited states, the upper of which (the F 3Σ- state) has a very small rotational constant which we take to imply that it has substantial valence character. Further indications that the F 3Σ- (and C 3Σ-) states possess significant valence character is provided by the observation that both parent and daughter (N+) ions contribute to the overall ion yield when the multiphoton ionization proceeds via these two states. Daughter-ion formation is considered to occur via an overall four-photon excitation process in which the coherent two-photon excitation to the 3Σ- state of interest is followed by a one-photon excitation to a ‘‘superexcited’’ state of the neutral. This is then presumed to absorb a... [ABSTRACT FROM AUTHOR]

The electronic spectra of silyl radicals, SiH3 and SiD3, were observed between 450 and 610 nm (49 200-65 200 cm-1 ) by resonance enhanced multiphoton ionization (REMPI) spectroscopy. The spectra were produced through a 3+1 REMPI mechanism. Spectra of four new planar Rydberg states were observed and assigned. In SiH3 the observed states and spectroscopic constants are D 2A’1(3d)[larger_closed_square] T0=49 787(30), ω2=810(31) cm-1; I’ (4d): T0=56 253(30), ω2=814(25) cm-1; J’ (4d): T0=57 726(30), ω2=835(26) cm-1; and L (5d): T0=59 615(30) cm-1, ω2=839(26) cm-1. In SiD3 the observed states and spectroscopic constants are D 2A1(3d)[ATOTHER]@B:[/ATOTHER]T0=49 685(30), ω2=600(28) cm-1; I’ (4d):T0=56 205(30), ω2=600(17) cm-1; and J’ (4d):T0=57 840(30), ω2=603(20) cm-1. The differences between 2+1 and 3+1 REMPI spectra are explained by a Δl rule, where l is the electronic orbital angular momentum. The H, K, and N states observed in the 2+1 REMPI spectrum of SiD3 spectrum and reported in J. Chem. Phys. 91, 3340 (1989) are reassigned as the H 2E’ (4p), K 2E’ (4f), and N 2E’ (5f). [ABSTRACT FROM AUTHOR]

We study the preferred conformation of the glycosidic linkage of methyl-α-mannopyranoside in the gas phase and in aqueous solution. Results obtained utilizing Car–Parrinello molecular dynamics (CPMD) simulations are compared to those obtained from classical molecular dynamics (MD) simulations. We describe classical simulations performed with various water potential functions to study the impact of the chosen water potential on the predicted conformational preference of the glycosidic linkage of the carbohydrate in aqueous solution. In agreement with our recent studies, we find that results obtained with CPMD simulations differ from those obtained from classical simulations. In particular, this study shows that the trans (t) orientation of the glycosidic linkage of methyl-α-mannopyranoside is preferred over its gauche anticlockwise (g-) orientation in aqueous solution. CPMD simulations indicate that this preference is due to intermolecular hydrogen bonding with surrounding water molecules, whereas no such information could be demonstrated by classical MD simulations. This study emphasizes the importance of ab initio MD simulations for studying the structural properties of carbohydrates in aqueous solution. [ABSTRACT FROM AUTHOR]