Correlated x-ray fluorescence and ptychographic nano-tomography on Rembrandt's The Night Watch reveals unknown lead "layer".

The Night Watch, one of the most famous masterpieces by Rembrandt, is the subject of a large research and conservation project. For the conservation treatment, it is of great importance to understand its current condition. Correlated nano-tomography using x-ray fluorescence and ptychography revealed a-so far unknown-lead-containing "layer", which likely acts as a protective impregnation layer applied on the canvas before the quartz-clay ground was applied. This layer might explain the presence of lead soap protrusions in areas where no other lead components are present. In addition to the three-dimensional elemental mapping, ptychography visualizes and quantifies components not detectable by hard x-ray fluorescence such as the organic fraction and quartz. The first-time use of this combination of synchrotron-based techniques on a historic paint micro-sample shows it to be an important tool to better interpret the results of noninvasive imaging techniques operating on the macroscale.

The Metabolism of Lufotrelvir, a Prodrug Investigated for the Treatment of SARS-COV2 in Humans Following Intravenous Administration.

The metabolism of lufotrelvir, a novel phosphate prodrug of PF-00835231 for the treatment of COVID-19, was evaluated in healthy human volunteers and clinical trial participants with COVID-19 following intravenous infusion. The prodrug was completely converted to PF-00835231 that was subsequently cleared by hydrolysis, hydroxylation, ketoreduction, epimerization, renal clearance, and secretion into the feces. The main circulating metabolite was a hydrolysis product (M7) that was present at concentrations greater than PF-00835231, and this was consistent between healthy volunteers and participants with COVID-19. On administration of [14C]lufotrelvir, only 63% of the dose was obtained in excreta over 10 days and total drug-related material demonstrated a prolonged terminal phase half-life in plasma. A considerable portion of the labeled material was unextractable from fecal homogenate and plasma. The position of the carbon-14 atom in the labeled material was at a leucine carbonyl, and pronase digestion of the pellet derived from extraction of the fecal homogenate showed that [14C]leucine was released. SIGNIFICANCE STATEMENT: Lufotrelvir is an experimental phosphate prodrug intravenous therapy investigated for the potential treatment of COVID-19 in a hospital setting. The overall metabolism of lufotrelvir was determined in human healthy volunteers and clinical trial participants with COVID-19. Conversion of the phosphate prodrug to the active drug PF-00835231 was complete and the subsequent metabolic clearance of the active drug was largely via amide bond hydrolysis. Substantial drug-related material was not recovered due to loss of the carbon-14 label to endogenous metabolism.

Microdosing as a Potential Tool to Enhance Clinical Development of Novel Antibiotics: A Tissue and Plasma PK Feasibility Study with Ciprofloxacin.

BACKGROUND AND OBJECTIVE: In microdose studies, drug pharmacokinetics is measured in humans after administration of subtherapeutic doses. While previous microdose studies focused primarily on plasma pharmacokinetics, we set out to evaluate the feasibility of microdosing for a pharmacokinetic assessment in subcutaneous tissue and epithelial lining fluid. METHODS: Healthy subjects received a single intravenous bolus injection of a microdose of [14C]ciprofloxacin (1.1 µg, 7 kBq) with (cohort A, n = 9) or without (cohort B, n = 9) a prior intravenous infusion of a therapeutic dose of unlabeled ciprofloxacin (400 mg). Microdialysis and bronchoalveolar lavage were applied for determination of subcutaneous and intrapulmonary drug concentrations. Microdose [14C]ciprofloxacin was quantified by accelerator mass spectrometry and therapeutic-dose ciprofloxacin by liquid chromatography-tandem mass spectrometry. RESULTS: The pharmacokinetics of therapeutic-dose ciprofloxacin (cohort A) in plasma, subcutaneous tissue, and epithelial lining fluid was in accordance with previous data. In plasma and subcutaneous tissue, the dose-adjusted area under the concentration-time curve of microdose ciprofloxacin was similar in cohorts A and B and within an 0.8-fold to 1.1-fold range of the area under the concentration-time curve of therapeutic-dose ciprofloxacin. Penetration of microdose ciprofloxacin into subcutaneous tissue was similar in cohorts A and B and comparable to that of therapeutic-dose ciprofloxacin with subcutaneous tissue-to-plasma area under the concentration-time curve ratios of 0.44, 0.44, and 0.38, respectively. Penetration of microdose ciprofloxacin into epithelial lining fluid was highly variable and failed to predict the epithelial lining fluid penetration of therapeutic-dose ciprofloxacin. CONCLUSIONS: Our study confirms the feasibility of microdosing for pharmacokinetic measurements in plasma and subcutaneous tissue. Microdosing combined with microdialysis is a potentially useful tool in clinical antimicrobial drug development, but its applicability for the assessment of pulmonary pharmacokinetics with bronchoalveolar lavage requires further studies. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov NCT03177720 (registered 6 June, 2017).

Quantification of azacitidine incorporation into human DNA/RNA by accelerator mass spectrometry as direct measure of target engagement.

We report an accelerator mass spectrometry (AMS) assay to quantify azacitidine (Aza) incorporation into DNA and RNA from human acute myeloid leukemia (AML) cells, mouse bone marrow (BM) and peripheral blood mononuclear cells (PBMCs). Aza, a cytidine nucleoside analogue, is a disease modifying pharmacological agent used for treatment of myelodysplastic syndromes (MDS) and AML. Our assay was able to directly quantify the complex of Aza incorporated into DNA/RNA, via isolation of DNA/RNA from matrix (i.e., cancer cells, BM and PBMC) and subsequent measurement of total radioactivity (i.e., 14C-Aza) by using AMS. The sensitivity of the method was able to quantify as little as a single Aza molecule incorporated into DNA with approximately 2 × 107 nucleotides from PBMCs. An in vivo mouse model was used for establishing the lower limits of quantification (LLOQs) for Aza incorporated into DNA/RNA in mouse PBMCs (∼ 3.7 × 105) and BM (∼27.8 mg) collected 24 h post-dose after total exposure of 18 nCi/mouse (Aza 1 mg/kg). The LLOQs for PBMC analysis were 2.5 picogram equivalents per microgram (pgEq/µg) DNA and 0.22 pgEq/µg RNA, and for BM analysis were 1.7 pgEq/µg DNA and 0.22 pgEq/µg RNA. A linear relationship (i.e., ∼10-fold) was established of radioactive dose from 14C-Aza 17 nCi/mouse to 188 nCi/mouse and AMS response (i.e., 14C/12C ratio ranging from 2.45 × 10-11 to 2.50 × 10-10), as Aza was incorporated into DNA in mouse BM. The current method enables the direct measurement of Aza incorporation into DNA and RNA from patient PBMCs and BM to provide dosing optimization, and to assess target engagement with as little as ∼5 mL whole blood and ∼3 mL of BM from patients.

The Oral Bioavailability and Metabolism of Midazolam in Stable Critically Ill Children: A Pharmacokinetic Microtracing Study.

Midazolam is metabolized by the developmentally regulated intestinal and hepatic drug-metabolizing enzyme cytochrome P450 (CYP) 3A4/5. It is frequently administered orally to children, yet knowledge is lacking on the oral bioavailability in term neonates up until 1 year of age. Furthermore, the dispositions of the major metabolites 1-OH-midazolam (OHM) and 1-OH-midazolam-glucuronide (OHMG) after oral administration are largely unknown for the entire pediatric age span. We aimed to fill these knowledge gaps with a pediatric [14 C]midazolam microtracer population pharmacokinetic study. Forty-six stable, critically ill children (median age 9.8 (range 0.3-276.4) weeks) received a single oral [14 C]midazolam microtracer (58 (40-67) Bq/kg) when they received a therapeutic continuous intravenous midazolam infusion and had an arterial line in place enabling blood sampling. For midazolam, in a one-compartment model, bodyweight was a significant predictor for clearance (0.98 L/hour) and volume of distribution (8.7 L) (values for a typical individual of 5 kg). The typical oral bioavailability in the population was 66% (range 25-85%). The exposures of OHM and OHMG were highest for the youngest age groups and significantly decreased with postnatal age. The oral bioavailability of midazolam, largely reflective of intestinal and hepatic CYP3A activity, was on average lower than the reported 49-92% for preterm neonates, and higher than the reported 21% for children> 1 year of age and 30% for adults. As midazolam oral bioavailability varied widely, systemic exposure of other CYP3A-substrate drugs after oral dosing in this population may also be unpredictable, with risk of therapy failure or toxicity.

Proof of Concept: First Pediatric [14 C]microtracer Study to Create Metabolite Profiles of Midazolam.

Growth and development affect drug-metabolizing enzyme activity thus could alter the metabolic profile of a drug. Traditional studies to create metabolite profiles and study the routes of excretion are unethical in children due to the high radioactive burden. To overcome this challenge, we aimed to show the feasibility of an absorption, distribution, metabolism, and excretion (ADME) study using a [14 C]midazolam microtracer as proof of concept in children. Twelve stable, critically ill children received an oral [14 C]midazolam microtracer (20 ng/kg; 60 Bq/kg) while receiving intravenous therapeutic midazolam. Blood was sampled up to 24 hours after dosing. A time-averaged plasma pool per patient was prepared reflecting the mean area under the curve plasma level, and subsequently one pool for each age group (0-1 month, 1-6 months, 0.5-2 years, and 2-6 years). For each pool [14 C]levels were quantified by accelerator mass spectrometry, and metabolites identified by high resolution mass spectrometry. Urine and feces (n = 4) were collected up to 72 hours. The approach resulted in sufficient sensitivity to quantify individual metabolites in chromatograms. [14 C]1-OH-midazolam-glucuronide was most abundant in all but one age group, followed by unchanged [14 C]midazolam and [14 C]1-OH-midazolam. The small proportion of unspecified metabolites most probably includes [14 C]midazolam-glucuronide and [14 C]4-OH-midazolam. Excretion was mainly in urine; the total recovery in urine and feces was 77-94%. This first pediatric pilot study makes clear that using a [14 C]midazolam microtracer is feasible and safe to generate metabolite profiles and study recovery in children. This approach is promising for first-in-child studies to delineate age-related variation in drug metabolite profiles.

Dose-linearity of the pharmacokinetics of an intravenous [14 C]midazolam microdose in children.

AIMS: Drug disposition in children may vary from adults due to age-related variation in drug metabolism. Microdose studies present an innovation to study pharmacokinetics (PK) in paediatrics; however, they should be used only when the PK is dose linear. We aimed to assess dose linearity of a [14 C]midazolam microdose, by comparing the PK of an intravenous (IV) microtracer (a microdose given simultaneously with a therapeutic midazolam dose), with the PK of a single isolated microdose. METHODS: Preterm to 2-year-old infants admitted to the intensive care unit received [14 C]midazolam IV as a microtracer or microdose, followed by dense blood sampling up to 36 hours. Plasma concentrations of [14 C]midazolam and [14 C]1-hydroxy-midazolam were determined by accelerator mass spectrometry. Noncompartmental PK analysis was performed and a population PK model was developed. RESULTS: Of 15 infants (median gestational age 39.4 [range 23.9-41.4] weeks, postnatal age 11.4 [0.6-49.1] weeks), 6 received a microtracer and 9 a microdose of [14 C]midazolam (111 Bq kg-1 ; 37.6 ng kg-1 ). In a 2-compartment PK model, bodyweight was the most significant covariate for volume of distribution. There was no statistically significant difference in any PK parameter between the microdose and microtracer, nor in the area under curve ratio [14 C]1-OH-midazolam/[14 C]midazolam, showing the PK of midazolam to be linear within the range of the therapeutic and microdoses. CONCLUSION: Our data support the dose linearity of the PK of an IV [14 C]midazolam microdose in children. Hence, a [14 C]midazolam microdosing approach may be used as an alternative to a therapeutic dose of midazolam to study developmental changes in hepatic CYP3A activity in young children.

Assessment of Dermal Absorption of Aluminum from a Representative Antiperspirant Formulation Using a 26 Al Microtracer Approach.

A clinical pharmacokinetic study was performed in 12 healthy women to evaluate systemic exposure to aluminum following topical application of a representative antiperspirant formulation under real-life use conditions. A simple roll-on formulation containing an extremely rare isotope of aluminum (26 Al) chlorohydrate (ACH) was prepared to commercial specifications. A 26 Al radio-microtracer was used to distinguish dosed aluminum from natural background, using accelerated mass spectroscopy. The 26 Al citrate was administered intravenously (i.v.) to estimate fraction absorbed (Fabs ) following topical delivery. In blood samples after i.v. administration, 26 Al was readily detected (mean area under the curve (AUC) = 1,273 ± 466 hours×fg/mL). Conversely, all blood samples following topical application were below the lower limit of quantitation (LLOQ; 0.12 fg/mL), except two samples (0.13 and 0.14 fg/mL); a maximal AUC was based on LLOQs. The aluminum was above the LLOQ (61 ag/mL) in 31% of urine samples. From the urinary excretion data, a conservative estimated range for dermal Fabs of 0.002-0.06% was calculated, with a mean estimate of 0.0094%.

Successful Use of [14C]Paracetamol Microdosing to Elucidate Developmental Changes in Drug Metabolism.

BACKGROUND: We previously showed the practical and ethical feasibility of using [14C]-microdosing for pharmacokinetic studies in children. We now aimed to show that this approach can be used to elucidate developmental changes in drug metabolism, more specifically, glucuronidation and sulfation, using [14C]paracetamol (AAP). METHODS: Infants admitted to the intensive care unit received a single oral [14C]AAP microdose while receiving intravenous therapeutic AAP every 6 h. [14C]AAP pharmacokinetic parameters were estimated. [14C]AAP and metabolites were measured with accelerator mass spectrometry. The plasma area under the concentration-time curve from time zero to infinity and urinary recovery ratios were related to age as surrogate markers of metabolism. RESULTS: Fifty children [median age 6 months (range 3 days-6.9 years)] received a microdose (3.3 [2.0-3.5] ng/kg; 64 [41-71] Bq/kg). Plasma [14C]AAP apparent total clearance was 0.4 (0.1-2.6) L/h/kg, apparent volume of distribution was 1.7 (0.9-8.2) L/kg, and the half-life was 2.8 (1-7) h. With increasing age, plasma and urinary AAP-glu/AAP and AAP-glu/AAP-sul ratios significantly increased by four fold, while the AAP-sul/AAP ratio significantly decreased. CONCLUSION: Using [14C]labeled microdosing, the effect of age on orally administered AAP metabolism was successfully elucidated in both plasma and urine. With minimal burden and risk, microdosing is attractive to study developmental changes in drug disposition in children.

The impact of early human data on clinical development: there is time to win.

Modern accelerator mass spectrometry (AMS) methods enable the routine application of this technology in drug development. By the administration of a (14)C-labelled microdose or microtrace, pharmacokinetic (PK) data, such as mass balance, metabolite profiling, and absolute bioavailability (AB) data, can be generated easier, faster, and at lower costs. Here, we emphasize the advances and impact of this technology for pharmaceutical companies. The availability of accurate intravenous (iv) PK and human absorption, distribution, metabolism, and excretion (ADME) information, even before or during Phase I trials, can improve the clinical development plan. Moreover, applying the microtrace approach during early clinical development might impact the number of clinical pharmacology and preclinical safety pharmacology studies required, and shorten the overall drug discovery program.

Observational infant exploratory [(14)C]-paracetamol pharmacokinetic microdose/therapeutic dose study with accelerator mass spectrometry bioanalysis.

AIMS: The aims of the study were to compare [(14)C]-paracetamol ([(14)C]-PARA) paediatric pharmacokinetics (PK) after administration mixed in a therapeutic dose or an isolated microdose and to develop further and validate accelerator mass spectrometry (AMS) bioanalysis in the 0-2 year old age group. METHODS: [(14)C]-PARA concentrations in 10-15 µl plasma samples were measured after enteral or i.v. administration of a single [(14)C]-PARA microdose or mixed in with therapeutic dose in infants receiving PARA as part of their therapeutic regimen. RESULTS: Thirty-four infants were included in the PARA PK analysis for this study: oral microdose (n = 4), i.v. microdose (n = 6), oral therapeutic (n = 6) and i.v. therapeutic (n = 18). The respective mean clearance (CL) values (SDs in parentheses) for these dosed groups were 1.46 (1.00) l h(-1), 1.76 (1.07) l h(-1), 2.93 (2.08) l h(-1) and 2.72 (3.10) l h(-1), t(1/2) values 2.65 h, 2.55 h, 8.36 h and 7.16 h and dose normalized AUC(0-t) (mg l(-1) h) values were 0.90 (0.43), 0.84 (0.57), 0.7 (0.79) and 0.54 (0.26). CONCLUSIONS: All necessary ethical, scientific, clinical and regulatory procedures were put in place to conduct PK studies using enteral and systemic microdosing in two European centres. The pharmacokinetics of a therapeutic dose (mg kg(-1)) and a microdose (ng kg(-1)) in babies between 35 to 127 weeks post-menstrual age. [(14)C]-PARA pharmacokinetic parameters were within a two-fold range after a therapeutic dose or a microdose. Exploratory studies using doses significantly less than therapeutic doses may offer ethical and safety advantages with increased bionalytical sensitivity in selected exploratory paediatric pharmacokinetic studies.

Pediatric microdose study of [(14)C]paracetamol to study drug metabolism using accelerated mass spectrometry: proof of concept.

BACKGROUND: Pediatric drug development is hampered by practical, ethical, and scientific challenges. Microdosing is a promising new method to obtain pharmacokinetic data in children with minimal burden and minimal risk. The use of a labeled oral microdose offers the added benefit to study intestinal and hepatic drug disposition in children already receiving an intravenous therapeutic drug dose for clinical reasons. OBJECTIVE: The objective of this study was to present pilot data of an oral [(14)C]paracetamol [acetaminophen (AAP)] microdosing study as proof of concept to study developmental pharmacokinetics in children. METHODS: In an open-label microdose pharmacokinetic pilot study, infants (0-6 years of age) received a single oral [(14)C]AAP microdose (3.3 ng/kg, 60 Bq/kg) in addition to intravenous therapeutic doses of AAP (15 mg/kg intravenous every 6 h). Blood samples were taken from an indwelling catheter. AAP blood concentrations were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and [(14)C]AAP and metabolites ([(14)C]AAP-Glu and [(14)C]AAP-4Sul) were measured by accelerator mass spectrometry. RESULTS: Ten infants (aged 0.1-83.1 months) were included; one was excluded as he vomited shortly after administration. In nine patients, [(14)C]AAP and metabolites in blood samples were detectable at expected concentrations: median (range) maximum concentration (C max) [(14)C]AAP 1.68 (0.75-4.76) ng/L, [(14)C]AAP-Glu 0.88 (0.34-1.55) ng/L, and [(14)C]AAP-4Sul 0.81 (0.29-2.10) ng/L. Dose-normalized oral [(14)C]AAP C max approached median intravenous average concentrations (C av): 8.41 mg/L (3.75-23.78 mg/L) and 8.87 mg/L (3.45-12.9 mg/L), respectively. CONCLUSIONS: We demonstrate the feasibility of using a [(14)C]labeled microdose to study AAP pharmacokinetics, including metabolite disposition, in young children.

Automated combustion accelerator mass spectrometry for the analysis of biomedical samples in the low attomole range.

The increasing role of accelerator mass spectrometry (AMS) in biomedical research necessitates modernization of the traditional sample handling process. AMS was originally developed and used for carbon dating, therefore focusing on a very high precision but with a comparably low sample throughput. Here, we describe the combination of automated sample combustion with an elemental analyzer (EA) online coupled to an AMS via a dedicated interface. This setup allows direct radiocarbon measurements for over 70 samples daily by AMS. No sample processing is required apart from the pipetting of the sample into a tin foil cup, which is placed in the carousel of the EA. In our system, up to 200 AMS analyses are performed automatically without the need for manual interventions. We present results on the direct total (14)C count measurements in <2 µL human plasma samples. The method shows linearity over a range of 0.65-821 mBq/mL, with a lower limit of quantification of 0.65 mBq/mL (corresponding to 0.67 amol for acetaminophen). At these extremely low levels of activity, it becomes important to quantify plasma specific carbon percentages. This carbon percentage is automatically generated upon combustion of a sample on the EA. Apparent advantages of the present approach include complete omission of sample preparation (reduced hands-on time) and fully automated sample analysis. These improvements clearly stimulate the standard incorporation of microtracer research in the drug development process. In combination with the particularly low sample volumes required and extreme sensitivity, AMS strongly improves its position as a bioanalysis method.

Substrate occupancy at the onset of oligomeric transitions of DegP.

The protease-chaperone DegP undergoes secondary through quaternary structural changes, regulating function and preventing indiscriminate proteolysis. Several structures of DegP oligomers have been observed, including the resting state 6-mer and the 12-mer and 24-mer active states. However, the precise events of the transition between the resting and active states still need to be elucidated. We used native mass spectrometry to demonstrate that binding of multiple substrate-mimicking peptide ligands to the DegP resting state occurs prior to the transition to an active conformation. This transition occurred at a 6-mer occupancy of 40% for each peptide ligand. We observed ligand-specific 9-mer formation with a maximum load of 9 peptides, whereas other substrates led to 12-mers accommodating 24 peptides. Based on these data, we present a model for the initial steps of substrate-induced transitions from the resting to active states of DegP.

Sizing up large protein complexes by electrospray ionisation-based electrophoretic mobility and native mass spectrometry: morphology selective binding of Fabs to hepatitis B virus capsids.

The capsid of hepatitis B virus (HBV) is a major viral antigen and important diagnostic indicator. HBV capsids have prominent protrusions ('spikes') on their surface and are unique in having either T = 3 or T = 4 icosahedral symmetry. Mouse monoclonal and also human polyclonal antibodies bind either near the spike apices (historically the 'α-determinant') or in the 'floor' regions between them (the 'ß-determinant'). Native mass spectrometry (MS) and gas-phase electrophoretic mobility molecular analysis (GEMMA) were used to monitor the titration of HBV capsids with the antigen-binding domain (Fab) of mAb 3120, which has long defined the ß-determinant. Both methods readily distinguished Fab binding to the two capsid morphologies and could provide accurate masses and dimensions for these large immune complexes, which range up to ~8 MDa. As such, native MS and GEMMA provide valuable alternatives to a more time-consuming cryo-electron microscopy analysis for preliminary characterisation of virus-antibody complexes.

Structure and activity of the RNA-targeting Type III-B CRISPR-Cas complex of Thermus thermophilus.

The CRISPR-Cas system is a prokaryotic host defense system against genetic elements. The Type III-B CRISPR-Cas system of the bacterium Thermus thermophilus, the TtCmr complex, is composed of six different protein subunits (Cmr1-6) and one crRNA with a stoichiometry of Cmr112131445361:crRNA1. The TtCmr complex copurifies with crRNA species of 40 and 46 nt, originating from a distinct subset of CRISPR loci and spacers. The TtCmr complex cleaves the target RNA at multiple sites with 6 nt intervals via a 5' ruler mechanism. Electron microscopy revealed that the structure of TtCmr resembles a "sea worm" and is composed of a Cmr2-3 heterodimer "tail," a helical backbone of Cmr4 subunits capped by Cmr5 subunits, and a curled "head" containing Cmr1 and Cmr6. Despite having a backbone of only four Cmr4 subunits and being both longer and narrower, the overall architecture of TtCmr resembles that of Type I Cascade complexes.

Crystal structure of 3-hydroxybenzoate 6-hydroxylase uncovers lipid-assisted flavoprotein strategy for regioselective aromatic hydroxylation.

3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is a dimeric flavoprotein that catalyzes the NADH- and oxygen-dependent para-hydroxylation of 3-hydroxybenzoate to 2,5-dihydroxybenzoate. In this study, we report the crystal structure of 3HB6H as expressed in Escherichia coli. The overall fold of 3HB6H is similar to that of p-hydroxybenzoate hydroxylase and other flavoprotein aromatic hydroxylases. Unexpectedly, a lipid ligand is bound to each 3HB6H monomer. Mass spectral analysis identified the ligand as a mixture of phosphatidylglycerol and phosphatidylethanolamine. The fatty acid chains occupy hydrophobic channels that deeply penetrate into the interior of the substrate-binding domain of each subunit, whereas the hydrophilic part is exposed on the protein surface, connecting the dimerization domains via a few interactions. Most remarkably, the terminal part of a phospholipid acyl chain is directly involved in the substrate-binding site. Co-crystallized chloride ion and the crystal structure of the H213S variant with bound 3-hydroxybenzoate provide hints about oxygen activation and substrate hydroxylation. Essential roles are played by His-213 in catalysis and Tyr-105 in substrate binding. This phospholipid-assisted strategy to control regioselective aromatic hydroxylation is of relevance for optimization of flavin-dependent biocatalysts.

Epitope-distal effects accompany the binding of two distinct antibodies to hepatitis B virus capsids.

Infection of humans by hepatitis B virus (HBV) induces the copious production of antibodies directed against the capsid protein (Cp). A large variety of anticapsid antibodies have been identified that differ in their epitopes. These data, and the status of the capsid as a major clinical antigen, motivate studies to achieve a more detailed understanding of their interactions. In this study, we focused on the Fab fragments of two monoclonal antibodies, E1 and 3120. E1 has been shown to bind to the side of outward-protruding spikes whereas 3120 binds to the "floor" region of the capsid, between spikes. We used hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to investigate the effects on HBV capsids of binding these antibodies. Conventionally, capsids loaded with saturating amounts of Fabs would be too massive to be readily amenable to HDX-MS. However, by focusing on the Cp protein, we were able to acquire deuterium uptake profiles covering the entire 149-residue sequence and reveal, in localized detail, changes in H/D exchange rates accompanying antibody binding. We find increased protection of the known E1 and 3120 epitopes on the capsid upon binding and show that regions distant from the epitopes are also affected. In particular, the α2a helix (residues 24-34) and the mobile C-terminus (residues 141-149) become substantially less solvent-exposed. Our data indicate that even at substoichiometric antibody binding an overall increase in the rigidity of the capsid is elicited, as well as a general dampening of its breathing motions.

Exploring an orbitrap analyzer for the characterization of intact antibodies by native mass spectrometry.

Antibody profiling: native mass spectrometry analysis of intact antibodies can be achieved with improved speed, sensitivity, and mass resolution by using a modified orbitrap instrument. Complex mixtures of monoclonal antibodies can be resolved and their glycan "fingerprints" can be profiled. Noncovalent interactions are maintained, thus allowing antibody-antigen binding to be measured.

Qualitative and semiquantitative analysis of composite mixtures of antibodies by native mass spectrometry.

Native mass spectrometry was evaluated for the qualitative and semiquantitative analysis of composite mixtures of antibodies representing biopharmaceutical products coexpressed from single cells. We show that by using automated peak fitting of the ion signals in the native mass spectra, we can quantify the relative abundance of each of the antibodies present in mixtures, with an average accuracy of 3%, comparable to a cation exchange chromatography based approach performed in parallel. Moreover, using native mass spectrometry we were able to identify, separate, and quantify 9 antibodies present in a complex mixture of 10 antibodies, whereas this complexity could not be unraveled by cation exchange chromatography. Native mass spectrometry presents a valuable alternative to existing analytical methods for qualitative and semiquantitative profiling of biopharmaceutical products. It provides both the identity of each species in a mixture by mass determination and the relative abundance through comparison of relative ion signal intensities. Native mass spectrometry is a particularly effective tool for characterization of heterogeneous biopharmaceutical products such as bispecific antibodies and antibody mixtures.