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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

In vitro folding, functional characterization, and disulfide pattern of the extracellular domain of human GLP-1 receptor.

The N-terminal, extracellular domain of the receptor for glucagon-like peptide 1 (GLP-1 receptor) was expressed at a high level in E. coli and isolated as inclusion bodies. Renaturation with concomitant disulfide bond formation was achieved from guanidinium-solubilized material.