ABSTRACT
This work describes the use of a reverse phase (RP) column for size-based separation of hydrophilic polysaccharides. The entire separation window (before and after void volume) is used to provide unique separation of polysaccharides from other components in a glycol-conjugate vaccine or complex media of fermentation broth. The technique has also been applied to the separation of polysaccharides of different sizes. The effect of chromatographic parameters including type of packing material (CN, C8 and C18), pore size (80 and 300Å) of stationary phase, concentration of organic solvent on the separation was investigated. In addition, characterization of size-based separation was evaluated by coupling of RP column with multi-angle laser light scattering (MALLs) detector.
Subject(s)
Chromatography, Gel , Chromatography, Reverse-Phase , Polysaccharides/isolation & purification , Chromatography, Reverse-Phase/instrumentation , Complex Mixtures/chemistry , DNA/chemistry , DNA/isolation & purification , Hydrophobic and Hydrophilic Interactions , Molecular Weight , Particle Size , Polysaccharides/chemistry , Proteins/chemistry , Proteins/isolation & purificationABSTRACT
A technique utilizing CGE-LIF in a bare capillary has been developed and evaluated for the detection of the three different topoisomers (linear, open circle, and supercoiled) of plasmid DNA along with the prospect of the dimer form of the supercoiled isoform. Utilizing the zwitterionic buffer, HEPES with boric acid sufficiently prevented capillary wall interactions and minimized the EOF, enabling a well-resolved separation of different plasmid isoforms. Multiple run conditions including buffer concentration and pH, hydroxypropylmethylcellulose size and amount, injection parameters, and the presence of an intercalating dye were evaluated and optimized. In addition, the feasibility of using this method as a platform for varying sizes of plasmid was investigated.
Subject(s)
DNA/isolation & purification , Electrophoresis, Capillary/methods , Plasmids/isolation & purification , Boric Acids , Fluorescence , HEPES , LasersABSTRACT
A CGE method for monitoring the disulfide isomer distribution characteristic of IgG2 MAbs is presented. Disulfide heterogeneity of MAbs has been studied using various chromatographic and electrophoretic methods. Although CGE operates using a different selectivity mechanism from that of sorption chromatographic techniques, similar trends are present in the data, which allow the CGE method to be used as a complementary method for studying disulfide isomer distribution. This article focuses on the optimization of a capillary-based gel electrophoresis method that can be used to support antibody development including bioprocess optimization, antibody characterization, release, and formulation stability assessment.
Subject(s)
Antibodies, Monoclonal/chemistry , Disulfides/analysis , Electrophoresis, Capillary/methods , Immunoglobulin G/chemistry , Biopharmaceutics/methods , Disulfides/chemistry , IsomerismABSTRACT
Capillary gel electrophoresis (CGE) methods with UV detection were developed for reduced and non-reduced mAb analysis. These methods can be used to evaluate mAb purity, offering more reproducible quantitation compared with that of traditional SDS-PAGE methods. These CGE methods have been utilized as platform technology for bioprocess development, formulation development, mAb characterization, drug substance/drug product release testing as well as a required methodology for stability testing. We have found these CGE methods to be applicable across a platform of mAbs in preclinical and clinical development, with the majority of mAbs requiring no modification to the method conditions. This methodology has been ICH validated and transferred to several supporting organizations. The data presented herein describes the development of CGE methodology, platform application to mAb purity analysis, ICH validation, reliability metrics, and considerations on technology enhancement for improved performance and throughput.
Subject(s)
Antibodies, Monoclonal/analysis , Capillary Electrochromatography/methods , Immunoglobulin G/analysis , Alkylating Agents/chemistry , Antibodies, Monoclonal/chemistry , Buffers , Drug Stability , Electrophoresis, Polyacrylamide Gel , Equipment Failure Analysis , Hydrogen-Ion Concentration , Immunoglobulin G/chemistry , Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase/chemistry , Reducing Agents/chemistry , Reproducibility of Results , Spectrophotometry, Ultraviolet , TemperatureABSTRACT
PURPOSE: This study investigated the penetration of lidocaine around and through a sutured incision following the application of iontophoretic and passive patches in the CD Hairless rat. MATERIALS AND METHODS: Concentrations in localized areas (suture, dermis, subcutaneous, and vascular) were determined using microdialysis sampling followed by analysis using liquid chromatography with UV detection. RESULTS: Iontophoresis significantly enhanced the dermal penetration of lidocaine. In an intact skin model, dermal concentrations were 40 times greater following iontophoretic delivery compared to passive delivery. In a sutured incision model, iontophoresis enhanced localized concentrations in the dermis, suture, and subcutaneous regions by 6-, 15-, and 20-fold, respectively. Iontophoretic delivery to a region containing a sutured incision was focused to the incision resulting in a greater increase in the suture concentration and in the subcutaneous region directly below the incision. CONCLUSIONS: The four microdialysis probe design was successful in the determination of localized drug penetration in a sutured incision model. Iontophoresis enhanced skin penetration and allowed for site specific delivery when applied to a sutured incision.
