Miguel Saggu

• Dr.
• Genentech

Visible particles are a critical quality attribute for parenteral products and must be monitored. A carefully designed, executed, and controlled drug product manufacturing process including a final 100% visual inspection and appropriate end-product controls ensures that visible particles are consistently minimized and demonstrates that the injectab...

Dry powder inhalers, comprising an active pharmaceutical ingredient (API) and carrier excipients, are often used in the delivery of pulmonary drugs. The stability of the API particle size within a formulation blend is a critical attribute for aerodynamic performance but can be challenging to measure. The presence of excipients, typically at concent...

Visible protein-like particle standards may improve visual inspection and/or appearance testing practices used in the biotechnology industry. They may improve assay performance resulting in better alignment and more standardized training among different companies. The National Institute of Standards and Technology (NIST) has conducted an interlabo...

Purpose Polysorbates (PS) contain polyoxyethylene (POE) sorbitan/isosorbide fatty acid esters that can partially hydrolyze over time in liquid drug products to generate degradants and a remaining intact PS fraction with a modified ester distribution. The degradants are composed of free fatty acids (FFAs) –-primarily lauric acid for PS20 and oleic a...

Rapid release of biopharmaceutical products enables a more efficient drug manufacturing process. Multi-attribute methods that target several product quality attributes (PQAs) at one time are an essential pillar of the rapid-release strategy. The novel, high-throughput, and nondestructive multi-attribute Raman spectroscopy (MARS) method combines Ram...

The measurement of polydisperse protein aggregates and particles in biotherapeutics remains a challenge, especially for particles with diameters of ≈ 1 µm and below (sub-micrometer). This paper describes an interlaboratory comparison with the goal of assessing the measurement variability for the characterization of a sub-micrometer polydisperse par...

PurposeTo evaluate a modified high purity polysorbate 20 (RO HP PS20)—with lower levels of stearate, palmitate and myristate esters than the non-modified HP PS20—as a surfactant in biopharmaceutical drug products (DP). RO HP PS20 was designed to provide functional equivalence as a surfactant while delaying the onset of free fatty acid (FFA) particl...

Biologics subjected to interfacial stress can generate a diverse assortment of aggregated species ranging in size from dimers and other soluble aggregates, through subvisible or micrometer-sized particles, to particles in the hundreds of micrometers that are visible to the unaided eye. The quantification and analysis of these aggregates are an impo...

In recent years, there has been increased scrutiny on the presence and formation of product-related particles in biopharmaceutical formulations. These types of particles, originating from the degradation of the active pharmaceutical ingredient or the excipients, can be challenging to identify and characterize due to their fragility. Additionally, t...

Compendial testing methods are not required to be fully validated but their suitability should be verified under actual conditions of use. This requirement is established in 21 CFR 211.194(a)(2) of the current Good Manufacturing Practice regulations in United States. ANVISA (Agéncia Nacional de Vigiláncia Sanitária) also requires that compendial an...

Biopharmaceutical companies are required to control subvisible and visible particles in their products to ensure a consistent manufacturing process, assess product quality, as well as address potential safety concerns. Subvisible particles cover the size range between 1 and 100 μm, while particles >100 μm are generally considered to be visible [1]....

One of the major product quality challenges for injectable biologics is controlling the amount of protein aggregates and particles present in the final drug product. This article focuses on particles in the submicron range (< 2 μm). A cross-industry collaboration was undertaken to address some of the analytical gaps in measuring submicron particles...

The origin of unidirectional electron transfer in photosynthetic reaction centers (RCs) has been widely discussed. Despite the high level of structural similarity between the two branches of pigments that participate in the initial electron transfer steps of photosynthesis, electron transfer only occurs along one branch. One possible explanation fo...

Purpose: To evaluate a random forest model that counts silicone oil droplets and non-silicone oil particles in protein formulations with large class imbalance. Methods: In this work, we present a novel approach for automated image analysis of flow microscopy data based on random forest classification enabling rapid analysis of large data sets. T...

To study composition and heterogeneity of insoluble subvisible particles in Mab formulations resulting from degradation of polysorbate 20 and to develop a better understanding of the mechanisms of polysorbate degradation leading to particle formation. In this study, we exploit the potential of Raman microscopy for chemical identification of particl...

Slow, ∼50 ps, P* → P(+)HA(-) electron transfer is observed in Rhodobacter capsulatus reaction centers (RCs) bearing the native Tyr residue at M208 and the single amino acid change of isoleucine at M204 to glutamic acid. The P* decay kinetics are unusually homogeneous (single exponential) at room temperature. Comparative solid-state NMR of [4'-(13)C...

Guidelines for submitting commentsPolicy: Comments that contribute to the discussion of the article will be posted within approximately three business days. We do not accept anonymous comments. Please include your email address; the address will not be displayed in the posted comment. Cell Press Editors will screen the comments to ensure that they...

We have developed vibrational Stark effect (VSE) spectroscopy to probe electrostatics and dynamics in organized systems, in particular in proteins where they can report on functionally important electric fields. The strategy involves deploying site-specific vibrational probes (-C≡N, -C-D, -C=O and -C-F) whose sensitivity to an electric field is mea...

Hydrogen bonds are ubiquitous in chemistry and biology. The physical forces that govern hydrogen bonding interactions have been heavily debated, with much of the discussion focused on the relative contributions of electrostatic vs. quantum mechanical effects. In principle, the vibrational Stark effect (VSE), the response of a vibrational mode to el...

Hydrogen bonds and aromatic interactions are of widespread importance in chemistry, biology, and materials science. Electrostatics play a fundamental role in these interactions, but the magnitude of the electric fields that support them has not been quantified experimentally. Phenol forms a weak hydrogen bond complex with the π-cloud of benzene, an...

Aldehyde oxidase (AOX) is characterized by a broad substrate specificity, oxidizing aromatic azaheterocycles, such as N¹-methylnicotinamide and N-methylphthalazinium, or aldehydes, such as benzaldehyde, retinal, and vanillin. In the past decade, AOX has been recognized increasingly to play an important role in the metabolism of drugs through its co...

Hydrogenases are essential for H(2) cycling in microbial metabolism and serve as valuable blueprints for H(2)-based biotechnological applications. However, most hydrogenases are extremely oxygen sensitive and prone to inactivation by even traces of O(2). The O(2)-tolerant membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha H16 is one of the few...

Hydrogenasen sind Metalloenzyme und spielen eine entscheidende Rolle im Metabolismus einer Vielzahl von Mikroorganismen. Diese Enzyme katalysieren die reversible Spaltung von molekularem Wasserstoff in zwei Protonen und zwei Elektronen. Allerdings werden sie durch Sauerstoff katalytisch inaktiviert. [FeFe]-Hydrogenasen werden irreversibel inaktivie...

Ein klares Bild liefern EPR- und FTIR-spektroskopische In-situ-Untersuchungen der loslichen NAD+-reduzierenden [NiFe]-Hydrogenase von Ralstonia eutropha: Das katalytische Zentrum liegt in intakten Zellen vornehmlich im intermediaren Nia-C-Zustand vor, der entweder reversibel zu einem „Nir-B-ahnlichen“ Zustand oxidiert oder aber zu mehreren Nia-SR-S...

A clear picture: In situ EPR and FTIR spectroscopic studies on the soluble, NAD+-reducing [NiFe]-hydrogenase of Ralstonia eutropha reveal that the catalytic site resides predominantly in the intermediate Nia-C state within whole cells. This state, can either be reversibly oxidized to a Nir-B-like state or further reduced to various Nia-SR species....

[NiFe] hydrogenases are widespread among microorganisms and catalyze the reversible cleavage of molecular hydrogen. However, only a few bacteria, such as Ralstonia eutropha H16 (Re), synthesize [NiFe] hydrogenases that perform H(2) cycling in the presence of O(2). These enzymes are of special interest for biotechnological applications. To gain furt...

Dark operative protochlorophyllide oxidoreductase (DPOR) catalyzes the light-independent two-electron reduction of protochlorophyllide a to form chlorophyllide a, the last common precursor of chlorophyll a and bacteriochlorophyll a biosynthesis. During ATP-dependent DPOR catalysis the homodimeric ChlL2 subunit carrying a [4Fe-4S] cluster transfers...

The geometric and electronic structures of the active sites in the oxidized Ni(r)-B state of the [NiFe] hydrogenases from Ralstonia eutropha H16 and Desulfovibrio vulgaris Miyazaki F were investigated in pulsed EPR and ENDOR experiments at two different microwave frequencies (X- and Q-band). Two hyperfine-couplings were clearly resolved in the froz...

The bidirectional [NiFe] hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 was purified to apparent homogeneity by a single affinity chromatography step using a Synechocystis mutant with a Strep-tag II fused to the C terminus of HoxF. To increase the yield of purified enzyme and to test its overexpression capacity in Synechocystis the ps...

Three DNA regions carrying genes encoding putative homologs of xanthine dehydrogenases were identified in Escherichia coli, named xdhABC, xdhD, and yagTSRQ. Here, we describe the purification and characterization of gene products of the yagTSRQ operon, a molybdenum-containing iron-sulfur flavoprotein from E. coli, which is located in the periplasm....

This study provides the first spectroscopic characterization of the membrane-bound oxygen-tolerant [NiFe] hydrogenase (MBH) from Ralstonia eutropha H16 in its natural environment, the cytoplasmic membrane. The H2-converting MBH is composed of a large subunit, harboring the [NiFe] active site, and a small subunit, capable in coordinating one [3Fe4S]...

Mouse aldehyde oxidase (mAOX1) forms a homodimer and belongs to the xanthine oxidase family of molybdoenzymes which are characterized by an essential equatorial sulfur ligand coordinated to the molybdenum atom. In general, mammalian AOs are characterized by broad substrate specificity and an yet obscure physiological function. To define the physiol...

Hydrogenases are metalloenzymes and play a pivotal role in the energy metabolism of a variety of microorganisms. They catalyze the reversible cleavage of molecular hydrogen into two protons and two electrons. However, one drawback is their catalytic inactivation upon exposure to oxygen.[FeFe]-hydrogenases are irreversibly inactivated by oxygen, whe...

[NiFe] hydrogenases catalyze the reversible conversion of H2 into protons and electrons. The reaction takes place at the active site, which is composed of a nickel and an iron atom and three diatomic ligands, two cyanides and one carbon monoxide, bound to the iron. The NiFe(CN-)2CO cofactor is synthesized by an intricate posttranslational maturatio...

Metallo-β-lactamases hydrolyze most β-lactam antibiotics. The lack of a successful inhibitor for them is related to the previous failure to characterize a reaction intermediate with a clinically useful substrate. Stopped-flow experiments together with rapid freeze−quench EPR and Raman spectroscopies were used to characterize the reaction of Co(II)−...

Chlorophyll and bacteriochlorophyll biosynthesis requires the two-electron reduction of protochlorophyllide a ringDbya protochlorophyllide oxidoreductase to form chlorophyllide a. A light-dependent (light-dependent Pchlide oxidoreductase (LPOR)) and an unrelated dark operative enzyme (dark operative Pchlide oxidoreductase (DPOR)) are known. DPOR pl...

Rhodobacter capsulatus xanthine dehydrogenase (XDH) is a molybdo-flavoprotein that is highly homologous to the homodimeric mammalian xanthine oxidoreductase. However, the bacterial enzyme has an (alphabeta)(2) heterotetrameric structure, and the cofactors were identified to be located on two different polypeptides. We have analyzed the mechanism of...