Characterization of Subvisible Particles in Biotherapeutic Prefilled Syringes: The Role of Polysorbate and Protein on the Formation of Silicone Oil and Protein Subvisible Particles After Drop Shock.

Subvisible particles (SbVPs) are a critical quality attribute for biotherapeutics. Particle content in prefilled syringes (PFSs) of a biotherapeutic can include protein particles and silicone oil particles (SiOP). Here, a real-world protein therapeutic PFS shows that although polysorbate is effective in preventing protein particle formation, it also leads to the formation of SiOP.

Unique Impacts of Methionine Oxidation, Tryptophan Oxidation, and Asparagine Deamidation on Antibody Stability and Aggregation.

Monoclonal antibodies are attractive therapeutic agents because of their impressive biological activities and favorable biophysical properties. Nevertheless, antibodies are susceptible to various types of chemical modifications, and the impact of such modifications on antibody physical stability and aggregation remains understudied. Here, we report a systematic analysis of the impact of methionine oxidation, tryptophan oxidation, and asparagine deamidation on antibody conformational and colloidal stability, hydrophobicity, solubility, and aggregation.

Deamidation Can Compromise Antibody Colloidal Stability and Enhance Aggregation in a pH-Dependent Manner.

Monoclonal antibodies must be both chemically and physically stable to be developed into safe and effective drugs. Although there has been considerable progress in separately understanding the molecular determinants of antibody chemical and physical stability, it remains poorly understood how defects in one property (e.g.

Probing the Tryptophan Environment in Therapeutic Proteins: Implications for Higher Order Structure on Tryptophan Oxidation.

Tryptophan (Trp) oxidation in proteins leads to a number of events, including changes in color, higher order structure (HOS), and biological activity. We describe here a number of new findings through a comprehensive characterization of 6 monoclonal antibodies (mAbs) following selective oxidation of Trp residues by 2,2'-azobis(2-amidinopropane) dihydrochloride. Fluorescence spectroscopy, in combination with second derivative analysis, demonstrates that the loss of Trp fluorescence intensity is a sensitive indicator of Trp oxidation in mAbs.

Enhanced Precision of Circular Dichroism Spectral Measurements Permits Detection of Subtle Higher Order Structural Changes in Therapeutic Proteins.

Protein higher order structure (HOS) is an essential quality attribute to ensure protein stability and proper biological function. Protein HOS characterization is performed during comparability assessments for product consistency as well as during forced degradation studies for structural alteration upon stress. Circular dichroism (CD) spectroscopy is a widely used technique for measuring protein HOS, but it remains difficult to assess HOS with a high degree of accuracy and precision.

Detection and Identification of the Vibrational Markers for the Quantification of Methionine Oxidation in Therapeutic Proteins.

Methionine oxidation is a major degradation pathway in therapeutic proteins which can impact the structure and function of proteins as well as risk to drug product quality. Detecting Met oxidation in proteins by peptide mapping followed by liquid chromatography with mass spectrometry (LC-MS) is the industry standard but is also labor intensive and susceptible to artifacts. In this work, vibrational difference spectroscopy in combination with O isotopic shift enabled us to demonstrate the application of Raman and FTIR techniques for the detection and quantification of Met oxidation in various therapeutic proteins, including mAbs, fusion proteins, and antibody drug conjugate.

Protection of therapeutic antibodies from visible light induced degradation: Use safe light in manufacturing and storage.

As macromolecules, biologics are susceptible to light exposure, which induces oxidation of multiple amino acid residues including tryptophan, tyrosine, phenylalanine, cysteine and methionine. Pertaining to safety, efficacy and potency, light-induced oxidation of biologics has been widely studied and necessary precautions need to be taken during biologics manufacturing process, drug substance and products handling and storage. Proteins will degrade to varying extents depending on the protein properties, degradation pathways, formulation compositions and type of light source.

Submicron Protein Particle Characterization using Resistive Pulse Sensing and Conventional Light Scattering Based Approaches.

Purpose: Characterizing submicron protein particles (approximately 0.1-1μm) is challenging due to a limited number of suitable instruments capable of monitoring a relatively large continuum of particle size and concentration. In this work, we report for the first time the characterization of submicron protein particles using the high size resolution technique of resistive pulse sensing (RPS).

Protein Partial Molar Volumes in Multicomponent Solutions from the Perspective of Inverse Kirkwood-Buff Theory.

Inverse Kirkwood-Buff (KB) solution theory can be used to relate macroscopic quantities with molecular scale interactions and correlation functions, in the form of KB integrals. Protein partial specific volumes ([Formula: see text]) from high-precision density measurements can be used to quantify solvent-solute and solute-solute KB integrals. Currently, general expressions for [Formula: see text] as a function of cosolute concentration (c) have been provided for only binary and ternary solutions.

Interference from Proteins and Surfactants on Particle Size Distributions Measured by Nanoparticle Tracking Analysis (NTA).

Purpose: Characterization of submicron protein particles continues to be challenging despite active developments in the field. NTA is a submicron particle enumeration technique, which optically tracks the light scattering signal from suspended particles undergoing Brownian motion. The submicron particle size range NTA can monitor in common protein formulations is not well established.

Identifying protein aggregation mechanisms and quantifying aggregation rates from combined monomer depletion and continuous scattering.

Parallel temperature initial rates (PTIR) from chromatographic separation of aggregating protein solutions are combined with continuous simultaneous multiple sample light scattering (SMSLS) to make quantitative deductions about protein aggregation kinetics and mechanisms. PTIR determines the rates at which initially monomeric proteins are converted to aggregates over a range of temperatures, under initial-rate conditions. Using SMSLS for the same set of conditions provides time courses of the absolute Rayleigh scattering ratio, IR(t), from which a potentially different measure of aggregation rates can be quantified.

Acetate- and Citrate-Specific Ion Effects on Unfolding and Temperature-Dependent Aggregation Rates of Anti-Streptavidin IgG1.

Controlling and predicting unwanted degradation, such as non-native aggregation, is a long-standing challenge for mAbs and other protein-based products. mAb aggregation rates are typically sensitive to temperature, pH, and the addition of excipients. Quantitatively comparing temperature-dependent aggregation rates across multiple possible formulations is a challenge in product development.

Aggregate structure, morphology and the effect of aggregation mechanisms on viscosity at elevated protein concentrations.

Non-native aggregation is a common issue in a number of degenerative diseases and during manufacturing of protein-based therapeutics. There is a growing interest to monitor protein stability at intermediate to high protein concentrations, which are required for therapeutic dosing of subcutaneous injections. An understanding of the impact of protein structural changes and interactions on the protein aggregation mechanisms and resulting aggregate size and morphology may lead to improved strategies to reduce aggregation and solution viscosity.

Specific-ion effects on the aggregation mechanisms and protein-protein interactions for anti-streptavidin immunoglobulin gamma-1.

Non-native protein aggregation is common in the biopharmaceutical industry and potentially jeopardizes product shelf life, therapeutic efficacy, and patient safety. The present article focuses on the relationship(s) among protein-protein interactions, aggregate growth mechanisms, aggregate morphologies, and specific-ion effects for an anti-streptavidin (AS) immunoglobulin gamma 1 (IgG1). Aggregation mechanisms of AS-IgG1 were determined as a function of pH and NaCl concentration with sodium acetate buffer and compared to previous work with sodium citrate.

Calcitonin gene-related peptide neurons mediate sleep-specific circadian output in Drosophila.

Background: Imbalances in amount and timing of sleep are harmful to physical and mental health. Therefore, the study of the underlying mechanisms is of great biological importance. Proper timing and amount of sleep are regulated by both the circadian clock and homeostatic sleep drive.

Learning and memory performance in a cohort of clinically referred breast cancer survivors: the role of attention versus forgetting in patient-reported memory complaints.

Objective: While forgetfulness is widely reported by breast cancer survivors, studies documenting objective memory performance yield mixed, largely inconsistent, results. Failure to find consistent, objective memory issues may be due to the possibility that cancer survivors misattribute their experience of forgetfulness to primary memory issues rather than to difficulties in attention at the time of learning.

Methods: To clarify potential attention issues, factor scores for Attention Span, Learning Efficiency, Delayed Memory, and Inaccurate Memory were analyzed for the California Verbal Learning Test-Second Edition (CVLT-II) in 64 clinically referred breast cancer survivors with self-reported cognitive complaints; item analysis was conducted to clarify specific contributors to observed effects, and contrasts between learning and recall trials were compared with normative data.

Synthesis of zeolitic imidazolate framework films and membranes with controlled microstructures.

Zeolitic imidazolate frameworks (ZIFs) are hybrid organic-inorganic microporous materials that exhibit zeolite-like structures and can be synthesized with a wide range of pore sizes and chemical functionality. ZIFs as thin films and membranes are of interest for their applications in sensors and gas separation. Here, we report a method for ZIF film and membrane fabrication, based on support surface modification and in situ solvothermal growth, which has potential for general application to other ZIF membranes.

Short-term skeletal and dental effects of the Xbow appliance as measured on lateral cephalograms.

Introduction: Our objective was to determine the short-term skeletal and dental effects of the Xbow appliance when compared with an equivalent untreated control group as measured on lateral cephalograms.

Methods: A prospective sample of 69 consecutive Class II patients treated with only the Xbow appliance was compared with 30 historical Class II untreated controls. Standardized lateral cephalograms were used.

Immediate skeletal and dentoalveolar effects of the crown- or banded type Herbst appliance on Class II division 1 malocclusion.

Objective: To evaluate the relative skeletal and dental changes produced by the crown- or banded-type Herbst appliance in growing Class II division 1 malocclusion cases.

Materials And Methods: Several electronic databases were searched with the help of a health sciences librarian, without language limitation. Abstracts that appeared to fulfill the initial selection criteria (Herbst use and clinical trial) were selected by consensus, and their original articles were then retrieved.