A critical review of analytical ultracentrifugation and field flow fractionation methods for measuring protein aggregation

Analytical ultracentrifugation (AUC) and field flow fractionation (FFF) are 2 important biophysical methods for measuring protein aggregates. Both methods can separate protein monomer from its aggregate forms under a broad range of solution conditions. Recent advances in instrumentation and data analysis, particularly in the field of analytical ultracentrifugation technology, have significantly improved the capability and sensitivity of these biophysical methods for detecting protein aggregates. These advances have resulted in an increased use of these methods in the biopharmaceutical industry for characterization of therapeutic proteins. However, despite their many advantages over conventional methods, the difficulty in the use of the instrumentation and the complexity of data analysis process, have often hampered the widespread use and proper interpretation of data. This article reviews the recent progress in both technologies, and a few case studies are also presented to discuss their advantages and limitations.     

Characterization of chitosan and its derivatives using asymmetrical flow field-flow-fractionation: a comparison with traditional methods

Chitosan and its derivatives are an important group of polymers used extensively in pharmaceutics. Thus, their physicochemical properties are of considerable interest and need to be characterized carefully. The most important feature to determine is their molecular weight and molecular weight distribution while the molecular weight of polymers plays an important role as pharmaceutical excipients. In this study, the feasibility of using asymmetrical flow field-flow-fractionation (AF4) connected online to a multi-angle light scattering (MALS) detector to measure the molecular weight of chitosans, trimethyl chitosans were studied and compared with the results from some traditional measurement methods. It was found that the influence of trimethyl chitosan synthesis process on the resulting molecular weight decrease depends on the initial molecular weight of chitosan. Significant molecular weight decrease was observed when chitosan molecular weight was larger than 100 kDa. In contrast, the influence was marginal when the molecular weight of chitosan was less than 50 kDa. The AF4-MALS was found to be a suitable method for the characterization of pure chitosans and trimethyl chitosans.     

Synthesis and characterization of 99mTc‐labelled biologically active molecules using borohydride exchange resin as a reducing agent for the preparation of radiopharmaceuticals

A novel and efficient method for preparing ^99mTc‐complexes of radiopharmaceuticals has been developed by reacting [^99mTc]pertechnetate with a ligand in the presence of borohydride exchange resin (BER) as a reducing agent. The latter is stable over a wide range of pH (2–11) and thus can be used with biologically active compounds without the formation of insoluble ^99mTcO₂ or SnO₂ colloids. Since the radiolabelled complexes are produced with high radiochemical purity and labelling efficiency under milder conditions than those required for the conventional reducing agents, the latter can be replaced. The method is expected to be applicable to the preparation of ^99mTc‐radiopharmaceuticals. Copyright © 2004 John Wiley & Sons, Ltd.     

Development and validation of HPLC methods for the determination of potential extractables from elastomeric stoppers in the presence of a complex surfactant vehicle used in the preparation of parenteral drug products

HPLC methods were developed and validated for potential extractables [zinc dithiocarbamate, 2,6-di-tert-butyl-para-cresol (BHT), octylated diphenylamine antioxidant, sulfur, pentylphenol, and tetrakis(methylene(3,5-di-tert-butyl-4-hydroxyhydro cinnamate))methane] from commercial elastomeric stoppers in a complex surfactant matrix. These stoppers were proposed to be part of the container-closure system for experimental formulations containing the surfactant, polyoxyethylated Castor oil (USP/NF) (POE Castor oil) and ethanol. The presence of POE Castor oil in the formulation posed unique challenges to the development and validation of the HPLC methods. POE Castor oil, also known as Cremophor, is a viscous and complex solubilizing agent with a number of uncharacterized fractions. Hence the goal was to identify HPLC conditions that would be suitable for the separation, detection, and quantitation of the potential stopper extractables in the presence of such a complex drug product matrix. A number of experiments were performed to evaluate the effects of different columns, mobile phase composition, injection volume, and gradient profile on the separation and detection of the potential stopper extractables. The quantitation limits of these stopper extractables are between 1 and 10 ppm. The methods demonstrate good linearity, acceptable accuracy, and precision.     

The development and validation of methods for evaluating the immune system in preweaning piglets

The preweaning piglet has been found to be a valuable research model for testing ingredients used in infant formula. As part of the safety assessment, the neonates' immune system is an important component that has to be evaluated. In this study three concurrent strategies were developed to assess immune system status. The methods included (1) immunophenotying to assess circulating innate immune cell populations, (2) monitoring of circulating cytokines, particularly in response to a positive control agent, and (3) monitoring of localized gastrointestinal tissue cytokines using immunohistochemistry (IHC), particularly in response to a positive control agent. All assays were validated using white papers and regulatory guidance within a GLP environment. To validate the assays precision, accuracy and sample stability were evaluated as needed using a fit for purpose approach. In addition animals were treated with proinflammtory substances to detect a positive versus negative signal. In conclusion, these three methods were confirmed to be robust assays to evaluate the immune system and GIT-specific immune responses of preweaning piglets.     

Application and potential of capillary electroseparation methods to determine antioxidant phenolic compounds from plant food material

Antioxidants are one of the most common active ingredients of nutritionally functional foods which can play an important role in the prevention of oxidation and cellular damage inhibiting or delaying the oxidative processes. In recent years there has been an increased interest in the application of antioxidants to medical treatment as information is constantly gathered linking the development of human diseases to oxidative stress.     

Contribution of alpha4beta1 integrin to the antiallergic effect of levocabastine

Levocabastine is an antiallergic drug acting as a histamine H1-receptor antagonist. In allergic conjunctivitis (AC), it may also antagonize up-regulation of the intercellular adhesion molecule-1 (ICAM-1) expressed on epithelial conjunctival cells. However, little is known about its effects on eosinophils, important effector cells in AC. The adhesion molecule integrin alpha(4)beta(1) is expressed in eosinophils; it interacts with the vascular cell adhesion molecule-1 (VCAM-1) and fibronectin (FN) in vascular endothelial cells and contributes to eosinophil activation and infiltration in AC. This study provides evidence that in a scintillation proximity assay levocabastine (IC(50) 406 microM), but not the first-generation antihistamine chlorpheniramine, displaced (125)I-FN binding to human integrin alpha(4)beta(1) and, in flow cytometry analysis, levocabastine antagonized the binding of a primary antibody to integrin alpha(4) expressed on the Jurkat cell surface. Levocabastine, but not chlorpheniramine, binds the alpha(4)beta(1) integrin and prevents eosinophil adhesion to VCAM-1, FN or human umbilical vascular endothelial cells (HUVEC) in vitro. Similarly, levocabastine affects alpha(L)beta(2)/ICAM-1-mediated adhesion of Jurkat cells. In a model of AC levocabastine eye drops reduced the clinical aspects of the late-phase reaction and the conjunctival expression of alpha(4)beta(1) integrin by reducing infiltrated eosinophils. We propose that blockade of integrin-mediated cell adhesion might be a target of the antiallergic action of levocabastine and may play a role in preventing eosinophil adhesion and infiltration in AC.