Method Comparison Study-One Step Forward toward Robust Silicone Layer Thickness Measurements.

In the past decades, the silicone layer thickness and its distribution on the inner glass barrels of prefilled syringes have been characterized in several studies. However, the limited number of adequate methods to characterize thin baked-on silicone layers and the destructive nature of some analytical techniques suggest challenges to the inter-lab reproducibility of some methods. In this study, the measured silicone layer thickness of baked-on siliconized syringes was compared between two laboratories, both equipped with white light reflectometry coupled to laser interferometry instrumentation (Bouncer, LE UT 1.0, LE UT 2.0). The quantity of silicone oil of a subset of those syringes was measured by Fourier transform infrared spectroscopy. Glide force tests were realized as complementary measurements on both syringes analyzed by white light reflectometry coupled to laser interferometry instrumentation and on non-analyzed identical syringes from the same lot. Silicone profiles of all prefilled syringes including the limit of detection results replaced with 20 nm were comparable, but values were slightly lower when measured with the Bouncer instrument. An increase of the layer thickness from the finger flange to the needle side was found for all syringes with all instruments (20 nm to 130-140 nm). Glide force results were similar except for a difference in peak width in the break loose region between the laboratories. The mean quantities of silicone oil found by both laboratories were similar (64 µg/syringe and 69 µg/syringe). Overall, comparable results between laboratories suggest a good reproducibility of the thickness measurement method as a result of thorough method understanding and defining key method parameters. Hence this study presents a robust inter-lab comparison between silicone layer thickness measurements that has been a lack in the literature up to now.

Methods To Determine the Silicone Oil Layer Thickness in Sprayed-On Siliconized Syringes.

The silicone lubricant layer in prefilled syringes has been investigated with regards to siliconization process performance, prefilled syringe functionality, and drug product attributes, such as subvisible particle levels, in several studies in the past. However, adequate methods to characterize the silicone oil layer thickness and distribution are limited, and systematic evaluation is missing. In this study, white light interferometry was evaluated to close this gap in method understanding. White light interferometry demonstrated a good accuracy of 93-99% for MgF2 coated, curved standards covering a thickness range of 115-473 nm. Thickness measurements for sprayed-on siliconized prefilled syringes with different representative silicone oil distribution patterns (homogeneous, pronounced siliconization at flange or needle side, respectively) showed high instrument (0.5%) and analyst precision (4.1%). Different white light interferometry instrument parameters (autofocus, protective shield, syringe barrel dimensions input, type of non-siliconized syringe used as base reference) had no significant impact on the measured average layer thickness. The obtained values from white light interferometry applying a fully developed method (12 radial lines, 50 mm measurement distance, 50 measurements points) were in agreement with orthogonal results from combined white and laser interferometry and 3D-laser scanning microscopy. The investigated syringe batches (lot A and B) exhibited comparable longitudinal silicone oil layer thicknesses ranging from 170-190 nm to 90-100 nm from flange to tip and homogeneously distributed silicone layers over the syringe barrel circumference (110- 135 nm). Empty break-loose (4-4.5 N) and gliding forces (2-2.5 N) were comparably low for both analyzed syringe lots. A silicone oil layer thickness of 100-200 nm was thus sufficient for adequate functionality in this particular study. Filling the syringe with a surrogate solution including short-term exposure and emptying did not significantly influence the silicone oil layer at the investigated silicone level. It thus appears reasonable to use this approach to characterize silicone oil layers in filled syringes over time. The developed method characterizes non-destructively the layer thickness and distribution of silicone oil in empty syringes and provides fast access to reliable results. The gained information can be further used to support optimization of siliconization processes and increase the understanding of syringe functionality.LAY ABSTRACT: Silicone oil layers as lubricant are required to ensure functionality of prefilled syringes. Methods evaluating these layers are limited, and systematic evaluation is missing. The aim of this study was to develop and assess white light interferometry as an analytical method to characterize sprayed-on silicone oil layers in 1 mL prefilled syringes. White light interferometry showed a good accuracy (93-99%) as well as instrument and analyst precision (0.5% and 4.1%, respectively). Different applied instrument parameters had no significant impact on the measured layer thickness. The obtained values from white light interferometry applying a fully developed method concurred with orthogonal results from 3D-laser scanning microscopy and combined white light and laser interferometry. The average layer thicknesses in two investigated syringe lots gradually decreased from 170-190 nm at the flange to 100-90 nm at the needle side. The silicone layers were homogeneously distributed over the syringe barrel circumference (110-135 nm) for both lots. Empty break-loose (4-4.5 N) and gliding forces (2-2.5 N) were comparably low for both analyzed syringe lots. Syringe filling with a surrogate solution, including short-term exposure and emptying, did not significantly affect the silicone oil layer. The developed, non-destructive method provided reliable results to characterize the silicone oil layer thickness and distribution in empty siliconized syringes. This information can be further used to support optimization of siliconization processes and increase understanding of syringe functionality.

Influence of particle shedding from silicone tubing on antibody stability.

OBJECTIVES: Peristaltic pumps are increasingly employed during fill & finish operations of a biopharmaceutical drug, due to sensitivity of many biological products to rotary piston pump-related stresses. Yet, possibly also unit operations using peristaltic pumps may shed particulates into the final product due to abrasion from the employed tubing. It was the aim of this study to elucidate the potential influence of particles shed from peristaltic pump tubing on the stability of a drug product. METHODS: Spiking solutions containing shed silicone particles were prepared via peristaltic pumping of placebo under recirculating conditions and subsequently characterized. Two formulated antibodies were spiked with two realistic, but worst-case levels of particles and a 6-month accelerated stability study with storage at 2-8, 25 and 40°C were conducted. KEY FINDINGS: Regarding the formation of aggregates and fragments, both mAbs degraded at their typically expected rates and no additional impact of spiked particles was observed. No changes were discerned however in turbidity, subvisible and visible particle assessments. Flow imaging data for one of the mAb formulations with spiked particles suggested limited colloidal stability of shed particles as indicated by a similar increase in spiked placebo. CONCLUSIONS: Shed silicone particles from peristaltic pump tubing are assumed to not impair drug product stability.

Physico-chemical Stability of MabThera Drug-product Solution for Subcutaneous Injection under in-use Conditions with Different Administration Materials.

MabThera is an essential component of the standard-of-care regimens in the treatment of non-Hodgkin lymphoma and Chronic Lymphatic Leukemia. MabThera for subcutaneous injection is a novel line extension that has been approved by the European Medicines Agency for the treatment of patients with follicular lymphoma and diffuse large B-cell lymphoma. This study aimed to evaluate in-use stability data of MabThera subcutaneous drug-product solution in single-use syringes for subcutaneous administration according to the European Medicines Agency guideline. The drug-product solution was exposed to material contact surfaces of five different administration setups commonly used in subcutaneous drug delivery. MabThera subcutaneous was transferred under aseptic conditions into polypropylene and polycarbonate syringes and stored for 1, 2, and 4 weeks at 2°C to 8°C followed by 24 hours at 30°C. After storage, subcutaneous administration was simulated and MabThera subcutaneous drug-product solution quality attributes were evaluated by using compendial physico-chemical tests, as well as suitable and validated molecule- and formulation-specific analytical methods. MabThera subcutaneous vials were treated and analyzed in parallel. The physico-chemical results of MabThera subcutaneous in the different setups were comparable to the control for all timepoints. No change in drug-product quality after storage and simulated administration was found compared to the control. However, since single-dose products do not contain preservatives, microbial contamination and growth needs to be avoided and product sterility needs to be ensured. The results showed that MabThera subcutaneous remains compatible and stable, from a physico-chemical perspective, for up to 4 weeks at 2°C to 8°C followed by 24 hours at 30°C with the contact materials tested in this study. In order to avoid and minimize microbial growth, MabThera subcutaneous should be used immediately after removal from the original packaging container and strict aseptic handling conditions need to be followed.

Kinetic Modeling of Methionine Oxidation in Monoclonal Antibodies from Hydrogen Peroxide Spiking Studies.

UNLABELLED: When isolator technology is applied to biotechnology drug product fill-finish process, hydrogen peroxide (H2O2) spiking studies for the determination of the sensitivity of protein to residual peroxide in the isolator can be useful for assessing a maximum vapor phase hydrogen peroxide (VPHP) level. When monoclonal antibody (mAb) drug products were spiked with H2O2, an increase in methionine (Met 252 and Met 428) oxidation in the Fc region of the mAbs with a decrease in H2O2 concentration was observed for various levels of spiked-in peroxide. The reaction between Fc-Met and H2O2 was stoichiometric (i.e., 1:1 molar ratio), and the reaction rate was dependent on the concentrations of mAb and H2O2. The consumption of H2O2 by Fc-Met oxidation in the mAb followed pseudo first-order kinetics, and the rate was proportional to mAb concentration. The extent of Met 428 oxidation was half of that of Met 252, supporting that Met 252 is twice as reactive as Met 428. Similar results were observed for free L-methionine when spiked with H2O2. However, mAb formulation excipients may affect the rate of H2O2 consumption. mAb formulations containing trehalose or sucrose had faster H2O2 consumption rates than formulations without the sugars, which could be the result of impurities (e.g., metal ions) present in the excipients that may act as catalysts. Based on the H2O2 spiking study results, we can predict the amount Fc-Met oxidation for a given protein concentration and H2O2 level. Our kinetic modeling of the reaction between Fc-Met oxidation and H2O2 provides an outline to design a H2O2 spiking study to support the use of VPHP isolator for antibody drug product manufacture. LAY ABSTRACT: Isolator technology is increasing used in drug product manufacturing of biotherapeutics. In order to understand the impact of residual vapor phase hydrogen peroxide (VPHP) levels on protein product quality, hydrogen peroxide (H2O2) spiking studies may be performed to determine the sensitivity of monoclonal antibody (mAb) drug products to residual peroxide in the isolator. In this study, mAbs were spiked with H2O2; an increase in methionine (Met) oxidation of the mAbs with a decrease in H2O2 concentration was observed for various levels of spiked-in peroxide. The reaction between Met and H2O2 was 1:1, and its rate was dependent on mAb and H2O2 concentrations. Consumption of H2O2 by Met followed pseudo first-order kinetics; the rate was proportional to mAb concentration. Formulations containing trehalose or sucrose had faster consumption rates than formulations without the sugars, which could be due to excipient impurities. Based on H2O2 spiking study results, we can predict the amount of Met oxidation for a given mAb concentration and H2O2 level. Our modeling of the reaction between Fc-Met oxidation and H2O2 provides an outline to design a H2O2 spiking study that supports using VPHP isolators during manufacture of mAb products.

Particle shedding from peristaltic pump tubing in biopharmaceutical drug product manufacturing.

In a typical manufacturing setup for biopharmaceutical drug products, the fill and dosing pump is placed after the final sterile filtration unit in order to ensure adequate dispensing accuracy and avoid backpressure peaks. Given the sensitivity of protein molecules, peristaltic pumps are often preferred over piston pumps. However, particles may be shed from the silicone tubing employed. In this study, particle shedding and a potential turbidity increase during peristaltic pumping of water and buffer were investigated using three types of commercially available silicone tubing. In the recirculates, mainly particles of around 200 nm next to a very small fraction of particles in the lower micrometer range were found. Using 3D laser scanning microscopy, surface roughness of the inner tubing surface was found to be a determining factor for particle shedding from silicone tubing. As the propensity toward particle shedding varied between tubing types and also cannot be concluded from manufacturer's specifications, individual testing with the presented methods is recommended during tubing qualification. Choosing low abrasive tubing can help to further minimize the very low particle counts to be expected in pharmaceutical drug products.

Assessment of chemical modifications of sites in the CDRs of recombinant antibodies: Susceptibility vs. functionality of critical quality attributes.

Modifications like asparagine deamidation, aspartate isomerization, methionine oxidation, and lysine glycation are typical degradations for recombinant antibodies. For the identification and functional evaluation of antibody critical quality attributes (CQAs) derived from chemical modifications in the complementary-determining regions (CDRs) and the conserved regions, an approach employing specific stress conditions, elevated temperatures, pH, oxidizing agents, and forced glycation with glucose incubation, was applied. The application of the specific stress conditions combined with ion exchange chromatography, proteolytic peptide mapping, quantitative liquid chromatography mass spectrometry, and functional evaluation by surface plasmon resonance analysis was adequate to identify and functionally assess chemical modification sites in the CDRs of a recombinant IgG1. LC-Met-4, LC-Asn-30/31, LC-Asn-92, HC-Met-100c, and HC Lys-33 were identified as potential CQAs. However, none of the assessed degradation products led to a complete loss of functionality if only one light or heavy chain of the native antibody was affected.

Analytical protein a chromatography as a quantitative tool for the screening of methionine oxidation in monoclonal antibodies.

The presence of oxidized methionine residues in therapeutic monoclonal antibodies can potentially impact drug efficacy, safety, as well as antibody half-life in vivo. Therefore, methionine oxidation of antibodies is a strong focus during pharmaceutical development and a well-known degradation pathway. The monitoring of methionine oxidation is currently routinely performed by peptide mapping/liquid chromatography-mass spectrometry techniques, which are laborious and time consuming. We have established analytical protein A chromatography as a method of choice for fast and quantitative screening of total Fc methionine oxidation during formulation and process development. The principle of this method relies on the lower binding affinity of protein A for immunoglobulin G-Fc domains containing oxidized methionines, compared with nonoxidized Fc domains. Our data reveal that highly conserved Fc methionines situated close to the binding site to protein A can serve as marker for the oxidation of other surface-exposed methionine residues. In case of poor separation of oxidized species by protein A chromatography, analytical protein G chromatography is proposed as alternative. We demonstrate that analytical protein A chromatography, and alternatively protein G chromatography, is a valuable tool for the screening of methionine oxidation in therapeutic antibodies during formulation and process development.

The degradation of polysorbates 20 and 80 and its potential impact on the stability of biotherapeutics.

PURPOSE: To study the potential impact of the degradation of Polysorbates (PS) 20 and 80 on the stability of therapeutic proteins in parenteral formulations. METHOD: First, degradation products of PS20 and 80 were identified. Subsequently, the effect of degraded polysorbate on physical characteristics and long-term stability of protein formulations was assessed. Further, the impact of polysorbate degradation on protein stability was evaluated via shaking stress studies on formulations spiked with artificially degraded polysorbate or degradants like fatty acids. Additionally, aged formulations with reduced polysorbate content were shaken. RESULTS: The degradation of polysorbate leads to a buildup of various molecules, some of which are poorly soluble, including fatty acids and polyoxyethylene (POE) esters of fatty acids. Spiking studies showed that the insoluble degradants could potentially impact protein stability and that the presence of sufficient intact polysorbate was crucial to prevent this. End-of-shelf-life shaking of protein formulations showed that the stability of various monoclonal antibodies was, however, not affected. CONCLUSIONS: Although some degradants can potentially influence the stability of the protein (as discerned from spiking studies), degradation of polysorbates did not impact the stability of the different proteins tested in pharmaceutically relevant temperature and storage conditions.

Efficient IgM assembly and secretion require the plasma cell induced endoplasmic reticulum protein pERp1.

Plasma cells daily secrete their own mass in antibodies, which fold and assemble in the endoplasmic reticulum (ER). To reach these levels, cells require pERp1, a novel lymphocyte-specific small ER-resident protein, which attains expression levels as high as BiP when B cells differentiate into plasma cells. Although pERp1 has no homology with known ER proteins, it does contain a CXXC motif typical for oxidoreductases. In steady state, the CXXC cysteines are locked by two parallel disulfide bonds with a downstream C(X)(6)C motif, and pERp1 displays only modest oxidoreductase activity. pERp1 emerged as a dedicated folding factor for IgM, associating with both heavy and light chains and promoting assembly and secretion of mature IgM.

The structure of a cytolytic alpha-helical toxin pore reveals its assembly mechanism.

Pore-forming toxins (PFTs) are a class of potent virulence factors that convert from a soluble form to a membrane-integrated pore. They exhibit their toxic effect either by destruction of the membrane permeability barrier or by delivery of toxic components through the pores. Among the group of bacterial PFTs are some of the most dangerous toxins, such as diphtheria and anthrax toxin. Examples of eukaryotic PFTs are perforin and the membrane-attack complex, proteins of the immune system. PFTs can be subdivided into two classes, alpha-PFTs and beta-PFTs, depending on the suspected mode of membrane integration, either by alpha-helical or beta-sheet elements. The only high-resolution structure of a transmembrane PFT pore is available for a beta-PFT--alpha-haemolysin from Staphylococcus aureus. Cytolysin A (ClyA, also known as HlyE), an alpha-PFT, is a cytolytic -helical toxin responsible for the haemolytic phenotype of several Escherichia coli and Salmonella enterica strains. ClyA is cytotoxic towards cultured mammalian cells, induces apoptosis of macrophages and promotes tissue pervasion. Electron microscopic reconstructions demonstrated that the soluble monomer of ClyA must undergo large conformational changes to form the transmembrane pore. Here we report the 3.3 A crystal structure of the 400 kDa dodecameric transmembrane pore formed by ClyA. The tertiary structure of ClyA protomers in the pore is substantially different from that in the soluble monomer. The conversion involves more than half of all residues. It results in large rearrangements, up to 140 A, of parts of the monomer, reorganization of the hydrophobic core, and transitions of -sheets and loop regions to -helices. The large extent of interdependent conformational changes indicates a sequential mechanism for membrane insertion and pore formation.

Protein aggregation: pathways, induction factors and analysis.

Control and analysis of protein aggregation is an increasing challenge to pharmaceutical research and development. Due to the nature of protein interactions, protein aggregation may occur at various points throughout the lifetime of a protein and may be of different quantity and quality such as size, shape, morphology. It is therefore important to understand the interactions, causes and analyses of such aggregates in order to control protein aggregation to enable successful products. This review gives a short outline of currently discussed pathways and induction methods for protein aggregation and describes currently employed set of analytical techniques and emerging technologies for aggregate detection, characterization and quantification. A major challenge for the analysis of protein aggregates is that no single analytical method exists to cover the entire size range or type of aggregates which may appear. Each analytical method not only shows its specific advantages but also has its limitations. The limits of detection and the possibility of creating artifacts through sample preparation by inducing or destroying aggregates need to be considered with each method used. Therefore, it may also be advisable to carefully compare analytical results of orthogonal methods for similar size ranges to evaluate method performance.

Cytotoxin ClyA from Escherichia coli assembles to a 13-meric pore independent of its redox-state.

ClyA is a pore-forming toxin from virulent Escherichia coli and Salmonella enterica strains. Here, we show that the intrinsic hemolytic activity of ClyA is independent of its redox state, and that the assembly of both reduced and oxidized ClyA to the ring-shaped oligomer is triggered by contact with lipid or detergent. A rate-limiting conformational transition in membrane-bound ClyA monomers precedes their assembly to the functional pore. We obtained a three-dimensional model of the detergent-induced oligomeric complex at 12 A resolution by combining cryo- and negative stain electron microscopy with mass measurements by scanning transmission electron microscopy. The model reveals that 13 ClyA monomers assemble into a cylinder with a hydrophobic cap region, which may be critical for membrane insertion.

High-resolution structures of Escherichia coli cDsbD in different redox states: A combined crystallographic, biochemical and computational study.

Escherichia coli DsbD transports electrons from cytoplasmic thioredoxin to periplasmic target proteins. DsbD is composed of an N-terminal (nDsbD) and a C-terminal (cDsbD) periplasmic domain, connected by a central transmembrane domain. Each domain possesses two cysteine residues essential for electron transport. The transport proceeds via disulfide exchange reactions from cytoplasmic thioredoxin to the central transmembrane domain and via cDsbD to nDsbD, which then reduces the periplasmic target proteins. We determined four high-resolution structures of cDsbD: oxidized (1.65 A resolution), chemically reduced (1.3 A), photo-reduced (1.1 A) and chemically reduced at pH increased from 4.6 to 7. The latter structure was refined at 0.99 A resolution, the highest achieved so far for a thioredoxin superfamily member. The data reveal unprecedented structural details of cDsbD, demonstrating that the domain is very rigid and undergoes hardly any conformational change upon disulfide reduction or interaction with nDsbD. In full agreement with the crystallographic results, guanidinium chloride-induced unfolding and refolding experiments indicate that oxidized and reduced cDsbD are equally stable. We confirmed the structural rigidity of cDsbD by molecular dynamics simulations. A remarkable feature of cDsbD is the pKa of 9.3 for the active site Cys461: this value, determined using two different experimental methods, surprisingly was around 2.5 units higher than expected on the basis of the redox potential. Additionally, taking advantage of the very high quality of the cDsbD structures, we carried out pKa calculations, which gave results in agreement with the experimental findings. In conclusion, our wide-scope analysis of cDsbD, encompassing atomic-resolution crystallography, computational chemistry and biophysical measurements, highlighted two so far unrecognized key aspects of this domain: its unusual redox properties and extreme rigidity. Both are likely to be correlated to the role of cDsbD as a covalently linked electron shuttle between the membrane domain and the N-terminal periplasmic domain of DsbD.

Staphylococcus aureus DsbA is a membrane-bound lipoprotein with thiol-disulfide oxidoreductase activity.

DsbA proteins, the primary catalysts of protein disulfide bond formation, are known to affect virulence and penicillin resistance in Gram-negative bacteria. We identified a putative DsbA homologue in the Gram-positive pathogen Staphylococcus aureus that was able to restore the motility phenotype of an Escherichia coli dsbA mutant and thus demonstrated a functional thiol oxidoreductase activity. The staphylococcal DsbA (SaDsbA) had a strong oxidative redox potential of -131 mV. The persistence of the protein throughout the growth cycle despite its predominant transcription during exponential growth phase suggested a rather long half-life for the SaDsbA. SaDsbA was found to be a membrane localised lipoprotein, supporting a role in disulfide bond formation. But so far, neither in vitro nor in vivo phenotype could be identified in a staphylococcal dsbA mutant, leaving its physiological role unknown. The inability of SaDsbA to interact with the E. coli DsbB and the lack of an apparent staphylococcal DsbB homologue suggest an alternative re-oxidation pathway for the SaDsbA.

Structural basis and kinetics of DsbD-dependent cytochrome c maturation.

DsbD from Escherichia coli transports two electrons from cytoplasmic thioredoxin to the periplasmic substrate proteins DsbC, DsbG and CcmG. DsbD consists of an N-terminal periplasmic domain (nDsbD), a C-terminal periplasmic domain, and a central transmembrane domain. Each domain possesses two cysteines required for electron transport. Herein, we demonstrate fast (3.9 x 10(5) M(-1)s(-1)) and direct disulfide exchange between nDsbD and CcmG, a highly specific disulfide reductase essential for cytochrome c maturation. We determined the crystal structure of the disulfide-linked complex between nDsbD and the soluble part of CcmG at 1.94 A resolution. In contrast to the other two known complexes of nDsbD with target proteins, the N-terminal segment of nDsbD contributes to specific recognition of CcmG. This and other features, like the possibility of using an additional interaction surface, constitute the structural basis for the adaptability of nDsbD to different protein substrates.

Pilus chaperones represent a new type of protein-folding catalyst.

Adhesive type 1 pili from uropathogenic Escherichia coli strains have a crucial role during infection by mediating the attachment to and potentially the invasion of host tissue. These filamentous, highly oligomeric protein complexes are assembled by the 'chaperone-usher' pathway, in which the individual pilus subunits fold in the bacterial periplasm and form stoichiometric complexes with a periplasmic chaperone molecule that is essential for pilus assembly. The chaperone subsequently delivers the subunits to an assembly platform (usher) in the outer membrane, which mediates subunit assembly and translocation to the cell surface. Here we show that the periplasmic type 1 pilus chaperone FimC binds non-native pilus subunits and accelerates folding of the subunit FimG by 100-fold. Moreover, we find that the FimC-FimG complex is formed quantitatively and very rapidly when folding of FimG is initiated in the presence of both FimC and the assembly-competent subunit FimF, even though the FimC-FimG complex is thermodynamically less stable than the FimF-FimG complex. FimC thus represents a previously unknown type of protein-folding catalyst, and simultaneously acts as a kinetic trap preventing spontaneous subunit assembly in the periplasm.

Structural basis and kinetics of inter- and intramolecular disulfide exchange in the redox catalyst DsbD.

DsbD from Escherichia coli catalyzes the transport of electrons from cytoplasmic thioredoxin to the periplasmic disulfide isomerase DsbC. DsbD contains two periplasmically oriented domains at the N- and C-terminus (nDsbD and cDsbD) that are connected by a central transmembrane (TM) domain. Each domain contains a pair of cysteines that are essential for catalysis. Here, we show that Cys109 and Cys461 form a transient interdomain disulfide bond between nDsbD and cDsbD in the reaction cycle of DsbD. We solved the crystal structure of this catalytic intermediate at 2.85 A resolution, which revealed large relative domain movements in DsbD as a consequence of a strong overlap between the surface areas of nDsbD that interact with DsbC and cDsbD. In addition, we have measured the kinetics of all functional and nonfunctional disulfide exchange reactions between redox-active, periplasmic proteins and protein domains from the oxidative DsbA/B and the reductive DsbC/D pathway. We show that both pathways are separated by large kinetic barriers for nonfunctional disulfide exchange between components from different pathways.

Mechanism of the electron transfer catalyst DsbB from Escherichia coli.

The membrane protein DsbB from Escherichia coli is essential for disulfide bond formation and catalyses the oxidation of the periplasmic dithiol oxidase DsbA by ubiquinone. DsbB contains two catalytic disulfide bonds, Cys41-Cys44 and Cys104-Cys130. We show that DsbB directly oxidizes one molar equivalent of DsbA in the absence of ubiquinone via disulfide exchange with the 104-130 disulfide bond, with a rate constant of 2.7 x 10 M(-1) x s(-1). This reaction occurs although the 104-130 disulfide is less oxidizing than the catalytic disulfide bond of DsbA (E(o)' = -186 and -122 mV, respectively). This is because the 41-44 disulfide, which is only accessible to ubiquinone but not to DsbA, is the most oxidizing disulfide bond in a protein described so far, with a redox potential of -69 mV. Rapid intramolecular disulfide exchange in partially reduced DsbB converts the enzyme into a state in which Cys41 and Cys44 are reduced and thus accessible for reoxidation by ubiquinone. This demonstrates that the high catalytic efficiency of DsbB results from the extreme intrinsic oxidative force of the enzyme.