A Reliable Quantification of Cholesterol and 25-Hydroxycholesterol in Liposomal Adjuvant Formulation by Liquid Chromatography High-Resolution Tandem Mass Spectrometry.

Cholesterol, as one of the major components of liposomes, plays a critical role in modulating membrane bilayer permeability, fluidity, and structural stability. Controlling these quality attributes is essential to maintaining the efficacy and fitness of the liposomes in various applications. However, during the manufacture and storage of liposomes, cholesterol has a propensity to undergo oxidative degradation. Hence, an analytical tool that is capable of determining not only the identity and quantity of cholesterol but also its associated degradants is a prerequisite to effective process control and product quality and safety assessments. In this view, a new liquid chromatography electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method with parallel reaction monitoring (PRM) was developed and qualified to accurately quantify cholesterol and monitor the formation of 25-hydroxycholesterol degradant in liposomal drug formulations without the use of an isotopic internal standard (IS). The method was qualified according to the FDA Quality Guidance for Industry: Q2(R1). Study results showed that the method presents good specificity for cholesterol and 25-hydroxycholesterol detection in the liposomal matrix, good sensitivity characterized by LOD/LOQ in the nanomolar range, and accuracy within the range of 80 to 120%. The described method enables accurate evaluation of in-process and product release samples of Army Liposome Formulation with QS21 (ALFQ).

QS21-Initiated Fusion of Liposomal Small Unilamellar Vesicles to Form ALFQ Results in Concentration of Most of the Monophosphoryl Lipid A, QS21, and Cholesterol in Giant Unilamellar Vesicles.

Army Liposome Formulation with QS21 (ALFQ), a vaccine adjuvant preparation, comprises liposomes containing saturated phospholipids, with 55 mol% cholesterol relative to the phospholipids, and two adjuvants, monophosphoryl lipid A (MPLA) and QS21 saponin. A unique feature of ALFQ is the formation of giant unilamellar vesicles (GUVs) having diameters >1.0 µm, due to a remarkable fusion event initiated during the addition of QS21 to nanoliposomes containing MPLA and 55 mol% cholesterol relative to the total phospholipids. This results in a polydisperse size distribution of ALFQ particles, with diameters ranging from ~50 nm to ~30,000 nm. The purpose of this work was to gain insights into the unique fusion reaction of nanovesicles leading to GUVs induced by QS21. This fusion reaction was probed by comparing the lipid compositions and structures of vesicles purified from ALFQ, which were >1 µm (i.e., GUVs) and the smaller vesicles with diameter <1 µm. Here, we demonstrate that after differential centrifugation, cholesterol, MPLA, and QS21 in the liposomal phospholipid bilayers were present mainly in GUVs (in the pellet). Presumably, this occurred by rapid lateral diffusion during the transition from nanosize to microsize particles. While liposomal phospholipid recoveries by weight in the pellet and supernatant were 44% and 36%, respectively, higher percentages by weight of the cholesterol (~88%), MPLA (94%), and QS21 (96%) were recovered in the pellet containing GUVs, and ≤10% of these individual liposomal constituents were recovered in the supernatant. Despite the polydispersity of ALFQ, most of the cholesterol, and almost all of the adjuvant molecules, were present in the GUVs. We hypothesize that the binding of QS21 to cholesterol caused new structural nanodomains, and subsequent interleaflet coupling in the lipid bilayer might have initiated the fusion process, leading to creation of GUVs. However, the polar regions of MPLA and QS21 together have a "sugar lawn" of ten sugars, the hydrophilicity of which might have provided a driving force for rapid lateral diffusion and concentration of the MPLA and QS21 in the GUVs.

Crystal structural investigations of heme protein derivatives resulting from reactions of aryl- and alkylhydroxylamines with human hemoglobin.

Phenylhydroxylamine (PhNHOH) and nitrosobenzene (PhNO) interact with human tetrameric hemoglobin (Hb) to form the nitrosobenzene adduct Hb(PhNO). These interactions also frequently lead to methemoglobin formation in red blood cells. We utilize UV-vis spectroscopy and X-ray crystallography to identify the primary and secondary products that form when PhNHOH and related alkylhydroxylamines (RNHOH; R = Me, t-Bu) react with human ferric Hb. We show that with MeNHOH, the primary product is Hb[α-FeIII(H2O)][ß-FeII(MeNO)], in which nitrosomethane is bound to the ß subunit but not the α subunit. Attempts to isolate a nitrosochloramphenicol (CAMNO) adduct resulted in our isolation of a Hb[α-FeII][ß-FeII-cySOx]{CAMNO} product (cySOx = oxidized cysteine) in which CAMNO was located outside of the protein in the solvent region between the ß2 and α2 subunits of the same tetramer. We also observed that the ßcys93 residue had been oxidized. In the case of t-BuNHOH, we demonstrate that the isolated product is the ß-hemichrome Hb[α-FeIII(H2O)][ß-FeIII(His)2]{t-BuNHOH}, in which the ß heme has slipped ∼4.4 Å towards the solvent exterior to accommodate the bis-His heme coordination. When PhNHOH is used, a similar ß-hemichrome Hb[α-FeIII(H2O)][ß-FeIII(His)2-cySOx]{PhNHOH} was obtained. Our results reveal, for the first time, the X-ray structural determination of a ß-hemichrome in a human Hb derivative. Our UV-vis and X-ray crystal structural result reveal that although Hb(PhNO) and Hb(RNO) complexes may form as primary products, attempted isolation of these products by crystallization may result in the structural determination of their secondary products which may contain ß-hemichromes en route to further protein degradation.

A Liquid Chromatography High-Resolution Tandem Mass Spectrometry Method to Quantify QS-21 Adjuvant and Its Degradation Products in Liposomal Drug Formulations.

Identification and quantification of an active adjuvant and its degradation product/s in drug formulations are important to ensure drug product safety and efficacy. QS-21 is a potent adjuvant that is currently involved in several clinical vaccine trials and a constituent of licensed vaccines against malaria and shingles. In an aqueous milieu, QS-21 undergoes pH- and temperature-dependent hydrolytic degradation to form a QS-21 HP derivative that may occur during manufacturing and/or long-term storage. Intact QS-21 and deacylated QS-21 HP elicit different immune response profiles; thus, it is imperative to monitor QS-21 degradation in vaccine adjuvant formulation. To date, a suitable quantitative analytical method for QS-21 and its degradation product in drug formulations is not available in the literature. In view of this, a new liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and qualified to accurately quantify the active adjuvant QS-21 and its degradation product (QS-21 HP) in liposomal drug formulations. The method was qualified according to the FDA Guidance for Industry: Q2(R1). Study results showed that the described method presents good specificity for QS-21 and QS-21 HP detection in a liposomal matrix, good sensitivity characterized by the limit of detection (LOD)/limit of quantitation (LOQ) in the nanomolar range, linear regressions with correlation coefficients, R2 > 0.999, recoveries in the range of 80-120%, and precise detection and quantification with % relative standard deviation (RSD) < 6% for QS-21 and < 9% for the QS-21 HP impurity assay. The described method was successfully used to accurately evaluate in-process and product release samples of the Army Liposome Formulation containing QS-21 (ALFQ).

A Rapid Method for Direct Quantification of Antibody Binding-Site Concentration in Serum.

The quantitation of the available antibody binding-site concentration of polyclonal antibodies in serum is critical in defining the efficacy of vaccines against substances of abuse. We have conceptualized an equilibrium dialysis (ED)-based approach coupled with fluorimetry (ED-fluorimetry) to measure the antibody binding-site concentration to the ligand in an aqueous environment. The measured binding-site concentrations in monoclonal antibody (mAb) and sera samples from TT-6-AmHap-immunized rats by ED-fluorimetry are in agreement with those determined by a more established equilibrium dialysis coupled with ultraperformance liquid chromatography tandem mass spectrometry (ED-UPLC-MS/MS). Importantly, we have shown that the measured antibody binding-site concentrations to the ligand by ED-fluorimetry were not influenced by the sample serum matrix; thus, this method is valid for determining the binding-site concentration of polyclonal antibodies in sera samples. Further, we have demonstrated that under appropriate analytical conditions, this method resolved the total binding-site concentrations on a nanomolar scale with good accuracy and repeatability within the microliter sample volumes. This simple, rapid, and sample preparation-free approach has the potential to reliably perform quantitative antibody binding-site screening in serum and other more complex biological fluids.

Insights into the Observed trans-Bond Length Variations upon NO Binding to Ferric and Ferrous Porphyrins with Neutral Axial Ligands.

NO is well-known for its trans effect. NO binding to ferrous hemes of the form (por)Fe(L) (L = neutral N-based ligand) to give the {FeNO}7 (por)Fe(NO)(L) product results in a lengthening of the axial trans Fe-L bond. In contrast, NO binding to the ferric center in [(por)Fe(L)]+ to give the {FeNO}6 [(por)Fe(NO)(L)]+ product results in a shortening of the trans Fe-L bond. NO binding to both ferrous and ferric centers involves the lowering of their spin states. Density functional theory (DFT) calculations were used to probe the experimentally observed trans-bond shortening in some NO adducts of ferric porphyrins. We show that the strong σ antibonding interaction of d z 2 and the axial (L) ligand p orbitals present in the Fe(II) systems is absent in the Fe(III) systems, as it is now in an unoccupied orbital. This feature, combined with a lowering of spin state upon NO binding, provides a rationale for the observed net trans-bond shortening in the {FeNO}6 but not the {FeNO}7 derivatives.

Insight into the preferential N-binding versus O-binding of nitrosoarenes to ferrous and ferric heme centers.

Nitrosoarenes (ArNOs) are toxic metabolic intermediates that bind to heme proteins to inhibit their functions. Although much of their biological functions involve coordination to the Fe centers of hemes, the factors that determine N-binding or O-binding of these ArNOs have not been determined. We utilize X-ray crystallography and density functional theory (DFT) analyses of new representative ferrous and ferric ArNO compounds to provide the first theoretical insight into preferential N-binding versus O-binding of ArNOs to hemes. Our X-ray structural results favored N-binding of ArNO to ferrous heme centers, and O-binding to ferric hemes. Results of the DFT calculations rationalize this preferential binding on the basis of the energies of associated spin-states, and reveal that the dominant stabilization forces in the observed ferrous N-coordination and ferric O-coordination are dπ-pπ* and dσ-pπ*, respectively. Our results provide, for the first time, an explanation why in situ oxidation of the ferrous-ArNO compound to its ferric state results in the observed subsequent dissociation of the ligand.

Not Limited to Iron: A Cobalt Heme-NO Model Facilitates N-N Coupling with External NO in the Presence of a Lewis Acid to Generate N2 O.

Some bacterial heme proteins catalyze the coupling of two NO molecules to generate N2 O. We previously reported that a heme Fe-NO model engages in this N-N bond-forming reaction with NO. We now demonstrate that (OEP)CoII (NO) similarly reacts with 1 equiv of NO in the presence of the Lewis acids BX3 (X=F, C6 F5 ) to generate N2 O. DFT calculations support retention of the CoII oxidation state for the experimentally observed adduct (OEP)CoII (NO⋅BF3 ), the presumed hyponitrite intermediate (P.+ )CoII (ONNO⋅BF3 ), and the porphyrin π-radical cation by-product of this reaction, and that the π-radical cation formation likely occurs at the hyponitrite stage. In contrast, the Fe analogue undergoes a ferrous-to-ferric oxidation state conversion during this reaction. Our work shows that cobalt hemes are chemically competent to engage in the NO-to-N2 O conversion reaction.

Lewis Acid Activation of the Ferrous Heme-NO Fragment toward the N-N Coupling Reaction with NO To Generate N2O.

Bacterial NO reductase (bacNOR) enzymes utilize a heme/non-heme active site to couple two NO molecules to N2O. We show that BF3 coordination to the nitrosyl O-atom in (OEP)Fe(NO) activates it toward N-N bond formation with NO to generate N2O. 15N-isotopic labeling reveals a reversible nitrosyl exchange reaction and follow-up N-O bond cleavage in the N2O formation step. Other Lewis acids (B(C6F5)3 and K+) also promote the NO coupling reaction with (OEP)Fe(NO). These results, complemented by DFT calculations, provide experimental support for the cis: b3 pathway in bacNOR.

Carbon-Nitrogen and Nitrogen-Nitrogen Bond Formation from Nucleophilic Attack at Coordinated Nitrosyls in Fe and Ru Heme Models.

The conversion of inorganic NOx species to organo-N compounds is an important component of the global N-cycle. Reaction of a C-based nucleophile, namely the phenyl anion, with the ferric heme nitrosyl [(OEP)Fe(NO)(5-MeIm)]+ generates a mixture of the C-nitroso derivative (OEP)Fe(PhNO)(5-MeIm) and (OEP)Fe(Ph). The related reaction with [(OEP)Ru(NO)(5-MeIm)]+ generates the (OEP)Ru(PhNO)(5-MeIm) product. Reactions with the N-based nucleophile diethylamide results in the formation of free diethylnitrosamine, whereas the reaction with azide results in N2O formation; these products derive from attack of the nucleophiles on the bound NO groups. These results provide the first demonstrations of C-N and N-N bond formation from attack of C-based and N-based nucleophiles on synthetic ferric-NO hemes.

Hydride Attack on a Coordinated Ferric Nitrosyl: Experimental and DFT Evidence for the Formation of a Heme Model-HNO Derivative.

Heme-HNO species are crucial intermediates in several biological processes. To date, no well-defined Fe heme-HNO model compounds have been reported. Hydride attack on the cationic ferric [(OEP)Fe(NO)(5-MeIm)]OTf (OEP = octaethylporphyrinato dianion) generates an Fe-HNO product that has been characterized by IR and (1)H NMR spectroscopy. Results of DFT calculations reveal a direct attack of the hydride on the N atom of the coordinated ferric nitrosyl.

A bridged di-iron porphyrin hyponitrite complex as a model for biological N2O production from hyponitrite.

Heme-hyponitrites are intermediates that form at the bimetallic active sites of bacterial nitric oxide reductases. To probe a possible effect of the Fe-Fe distance on hyponitrite stability, we prepared a bridged bis-porphyrin Fe-hyponitrite compound, namely [(OEP-CH2)Fe]2(µ2,η(1),η(1)-ONNO). Its υNO of 992 cm(-1) (υ15NO of 976 cm(-1)) is close to the υNO of 983 cm(-1) reported previously by us for the crystallographically characterized [(OEP)Fe]2(µ2,η(1),η(1)-ONNO) compound. The bridged bis-porphyrin Fe-hyponitrite complex is unstable with respect to N2O production, supporting the role of the bis-Fe porphyrin system in hyponitrite conversion to N2O. The preparation and crystallographic determination of the bridging sulfato derivative is also reported.

Six-coordinate ferric porphyrins containing bidentate N-t-butyl-N-nitrosohydroxylaminato ligands: structure, magnetism, IR spectroelectrochemisty, and reactivity.

NONOates (diazeniumdiolates) containing the [X{N2O2}](-) functional group are frequently employed as nitric oxide (NO) donors in biology, and some NONOates have been shown to bind to metalloenzymes. We report the preparation, crystal structures, detailed magnetic behavior, redox properties, and reactivities of the first isolable alkyl C-NONOate complexes of heme models, namely (OEP)Fe(η(2)-ON(t-Bu)NO) (1) and (TPP)Fe(η(2)-ON(t-Bu)NO) (2) (OEP = octaethylporphyrinato dianion, TPP = tetraphenylporphyrinato dianion). The compounds display the unusual NONOate O,O-bidentate binding mode for porphyrins, resulting in significant apical Fe displacements (+0.60 Å for 1, and +0.69 Å for 2) towards the axial ligands. Magnetic susceptibility and magnetization measurements made from 1.8-300 K at magnetic fields from 0.02 to 5 T, yielded magnetic moments of 5.976 and 5.974 Bohr magnetons for 1 and 2, respectively, clearly identifying them as high-spin (S = 5/2) ferric compounds. Variable-frequency (9.4 GHz and 34.5 GHz) EPR measurements, coupled with computer simulations, confirmed the magnetization results and yielded more precise values for the spin Hamiltonian parameters: g(avg) = 2.00 ± 0.03, |D| = 3.89 ± 0.09 cm(-1), and E/D = 0.07 ± 0.01 for both compounds, where D and E are the axial and rhombic zero-field splittings. IR spectroelectrochemistry studies reveal that the first oxidations of these compounds occur at the porphyrin macrocycles and not at the Fe-NONOate moieties. Reactions of 1 and 2 with a histidine mimic (1-methylimidazole) generate RNO and NO, both of which may bind to the metal center if sterics allow, as shown by a comparative study with the Cupferron complex (T(p-OMe)PP)Fe(η(2)-ON(Ph)NO). Protonation of 1 and 2 yields N2O as a gaseous product, presumably from the initial generation of HNO that dimerizes to the observed N2O product.

(1-Methyl-1H-imidazole-κN (3))(1-methyl-2-nitroso-benzene-κN)(5,10,15,20-tetra-phenyl-porphyrinato-κ(4) N)iron(II) di-chloro-methane monosolvate.

The solvated title compound, [Fe(C44H28N4)(C4H6N2)(C7H7NO)]·CH2Cl2, is a porphyrin complex containing an octahedrally coordinated Fe(II) atom with 1-methylimidazole [Fe-N = 2.0651 (17) Å] and o-nitro-sotoluene ligands at the axial positions. The o-nitro-sotoluene ligand is N-bound to iron(II) [Fe-N = 1.8406 (18)Å and Fe-N-O = 122.54 (14)°]. The axial N-Fe-N linkage is almost linear, with a bond angle of 177.15 (7)°. One phenyl group of the porphyrin ligand is disordered over two orientations in a 0.710 (3):0.290 (3) ratio. The di-chloro-methane solvent mol-ecule was severely disordered and its contribution to the scattering was removed with the SQUEEZE routine [van der Sluis & Spek (1990 ▶). Acta Cryst. A46, 194-201].