Semiautomated pH gradient ion-exchange chromatography of monoclonal antibody charge variants.

A new approach using a chromatography system equipped with isocratic pumps and an electrolytic eluent generator (EG) is introduced, replacing external pH gradient delivery using conventional gradient systems, in which bottled buffers with preadjusted pH are mixed using a gradient pump. The EG is capable of generating high purity base or acid required for online preparation of the buffer at the point of use, utilizing deionized water as the only carrier stream. Typically, the buffer was generated from online titration of a reagent composed of low molecular weight amines.

Charge heterogeneity profiling of monoclonal antibodies using low ionic strength ion-exchange chromatography and well-controlled pH gradients on monolithic columns.

In this work, the suitability of employing shallow pH gradients generated using single component buffer systems as eluents through cation-exchange (CEX) monolithic columns is demonstrated for the high-resolution separation of monoclonal antibody (mAb) charge variants in three different biopharmaceuticals. A useful selection of small molecule buffer species is described that can be used within very narrow pH ranges (typically 1 pH unit) defined by their buffer capacity for producing controlled and smooth pH profiles when used together with porous polymer monoliths. Using very low ionic strength eluents also enabled direct coupling with electrospray ionisation mass spectrometry.

Poly(ethylene glycol)-based monolithic capillary columns for hydrophobic interaction chromatography of immunoglobulin G subclasses and variants.

Polymer monoliths were prepared in 150 μm id capillaries by thermally initiated polymerization of PEG diacrylate for rapid hydrophobic interaction chromatography of immunoglobulin G (IgG) subclasses and related variants. Using only one monomer in the polymerization mixture allowed ease of optimization and synthesis of the monolith. The performance of the monolith was demonstrated by baseline resolution of IgG subclasses and variants, including mixtures of the κ variants of IgG1, IgG2, and IgG3 as well as the κ and λ variants associated with IgG1 and IgG2.

Hybrid capillary/microfluidic system for comprehensive online liquid chromatography-capillary electrophoresis-electrospray ionization-mass spectrometry.

A hybrid multidimensional separation system was made by coupling capillary liquid chromatography (LC) to a microfluidic device. The microfluidic device integrated flow splitting, capillary electrophoresis (CE), electroosmotic pumping, and electrospray ionization (ESI) emitter functional elements. The system was used with a time-of-flight mass spectrometer for comprehensive online LC-CE-MS of proteolytic digests.

Comparison of ZIC-HILIC and graphitized carbon-based analytical approaches combined with exoglycosidase digestions for analysis of glycans from monoclonal antibodies.

Two LC approaches for analysis of therapeutic monoclonal antibodies (MAbs) are presented and compared. In the first approach, zwitterionic-type hydrophilic interaction chromatography (ZIC-HILIC) of 2-aminobenzamide-labelled glycans was coupled with fluorescence or electrospray ionisation mass spectrometric (ESI-MS) detection. The ZIC-HILIC method enabled relative quantification and identification of major glycan species.

Epoxy-based monoliths for capillary liquid chromatography of small and large molecules.

A versatile epoxy-based monolith was synthesised by polycondensation polymerisation of glycidyl ether 100 with ethylenediamine using a porogenic system consisting of polyethylene glycol, M(w) = 1000, and 1-decanol. Polymerisation was performed at 80 °C for 22 h. A simple acid hydrolysis of residual epoxides resulted in a mixed diol-amino chemistry.

Zwitterionic-type hydrophilic interaction nano-liquid chromatography of complex and high mannose glycans coupled with electrospray ionisation high resolution time of flight mass spectrometry.

In this study we describe a new method for rapid and sensitive analysis of reduced high mannose and complex glycans using zwitterionic-type hydrophilic interaction nano-liquid chromatography (nano ZIC-HILIC, 75 μm I.D.×150 mm) coupled with high resolution nanoelectrospray ionisation time of flight mass spectrometry (nano ESI-TOF-MS).

Glycan profiling of monoclonal antibodies using zwitterionic-type hydrophilic interaction chromatography coupled with electrospray ionization mass spectrometry detection.

We present a new method for the analysis of glycans enzymatically released from monoclonal antibodies (MAbs) employing a zwitterionic-type hydrophilic interaction chromatography (ZIC-HILIC) column coupled with electrospray ionization mass spectrometry (ESI-MS). Both native and reduced glycans were analyzed, and the developed procedure was compared with a standard HILIC procedure used in the pharmaceutical industry whereby fluorescent-labeled glycans are analyzed using a TSK Amide-80 column coupled with fluorescence detection. The separation of isobaric alditol oligosaccharides present in monoclonal antibodies and ribonuclease B is demonstrated, and ZIC-HILIC is shown to have good capability for structural recognition.

Control of selectivity via nanochemistry: monolithic capillary column containing hydroxyapatite nanoparticles for separation of proteins and enrichment of phosphopeptides.

New monolithic capillary columns with embedded commercial hydroxyapatite nanoparticles have been developed and used for protein separation and selective enrichment of phosphopeptides. The rod-shaped hydroxyapatite nanoparticles were incorporated into the poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) monolith by simply admixing them in the polymerization mixture followed by in situ polymerization. The effect of percentages of monomers and hydroxyapatite nanoparticles in the polymerization mixture on the performance of the monolithic column was explored in detail.

Rapid and refined separation of human IgG2 disulfide isomers using superficially porous particles.

A rapid reversed-phase HPLC separation of recombinant human immunoglobulin gamma 2 (IgG2) disulfide isomers using columns packed with superficially porous particles is reported. Under optimal conditions, a separation of monoclonal IgG2 disulfide isomers was achieved in 10 min using a Poroshell™ 300SB-C8 column via a combination of high column temperature (85°C), mobile phases with high eluotropic strength (e.g.

Capillary gel electrophoresis with laser-induced fluorescence of plasmid DNA in untreated capillary.

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.

Analysis of identity, charge variants, and disulfide isomers of monoclonal antibodies with capillary zone electrophoresis in an uncoated capillary column.

A set of related capillary zone electrophoresis (CZE) methods have been developed for the analysis of identity, charge variants, and disulfide isoforms of IgG monoclonal antibodies (mAbs). These methods utilize an uncoated capillary column. The combined use of concentrated zwitterionic (e-amino-caproic acid) buffer and acid flushing was effective in minimizing the adsorption of protein to the inner wall of a bare capillary.

Development of a capillary gel electrophoresis method for monitoring disulfide isomer heterogeneity in IgG2 antibodies.

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.

Development, validation, and implementation of capillary gel electrophoresis as a replacement for SDS-PAGE for purity analysis of IgG2 mAbs.

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.

High binding capacity surface grafted monolithic columns for cation exchange chromatography of proteins and peptides.

Poly(glycidyl methacrylate-co-ethylene methacrylate) monoliths have been prepared in 100 microm i.d. capillaries and their epoxy groups hydrolyzed to obtain poly(2,3-dihydroxypropyl methacrylate-co-ethylene methacrylate) matrix.

Multidimensional system enabling deglycosylation of proteins using a capillary reactor with peptide-N-glycosidase F immobilized on a porous polymer monolith and hydrophilic interaction liquid chromatography-mass spectrometry of glycans.

A reactor with immobilized peptide-N-glycosidase F on a monolithic polymer support in a capillary has been developed that allows fast and efficient release of N-linked glycans from immunoglobulin G molecules. Two different monolithic scaffolds based on poly(glycidyl methacrylate-co-ethylene dimethacrylate) and poly(butyl methacrylate-co-ethylene dimethacrylate) were prepared. A multistep photografting process was used to reduce non-specific adsorption of proteins and to obtain support containing reactive azlactone functionalities enabling the preparation of highly active immobilized peptide-N-glycosidase F.

Highly efficient enzyme reactors containing trypsin and endoproteinase LysC immobilized on porous polymer monolith coupled to MS suitable for analysis of antibodies.

Capillary enzymatic microreactors containing trypsin and endoproteinase LysC immobilized on a porous polymer monolith have been prepared and used for the characterization and identification of proteins such as cytochrome c, bovine serum albumin, and high-molecular weight human immunoglobulin G. The hydrophilicity of diol functionalities originating from the hydrolyzed poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith was not sufficient to avoid adsorption of hydrophobic albumin in a highly aqueous mobile phase. Therefore, this monolith was first hydrophilized via photografting of poly(ethylene glycol) methacrylate followed by photografting of a 4-vinyl-2,2-dimethylazlactone to provide the pore surface with reactive functionalities required for immobilization.

Microchip capillary electrophoresis: application to peptide analysis.

The development of analytical methodologies to elucidate mechanisms of peptide transport and metabolism is important for the understanding of disease states and the design of effective drug therapies. Interest in the use of microchip capillary electrophoresis (CE) devices for peptide analysis stems from the ability to perform fast, highly efficient separations combined with small sample volume requirements. Many of the separation modes developed on conventional systems, including electrochromatography, isoelectric focusing, and electrophoretic bioaffinity assays, have been demonstrated on microchip devices.

Development of a microfabricated palladium decoupler/electrochemical detector for microchip capillary electrophoresis using a hybrid glass/poly(dimethylsiloxane) device.

The fabrication and evaluation of a palladium decoupler and working electrode for microchip capillary electrophoresis (CE) with electrochemical detection is described. The use of the Pd decoupler allows the working electrode to be placed directly in the separation channel and eliminates the band-broadening characteristic of the end-channel configuration. The method used for fabrication of the decoupler and working electrode was based on thin-layer deposition of titanium followed by palladium onto a glass substrate.

Comparison of the performance characteristics of poly(dimethylsiloxane) and Pyrex microchip electrophoresis devices for peptide separations.

A comparative study of electrophoretic separations of fluorescently labeled peptides and amino acids on poly(dimethylsiloxane) (PDMS) and Pyrex microchips is presented. The separation parameters for each microchip substrate were compared, including electroosmotic flow, plate numbers, resolution, and limits of detection. The effect of buffer composition on the separation was also investigated.

Separation and detection of angiotensin peptides by Cu(II) complexation and capillary electrophoresis with UV and electrochemical detection.

The use of capillary electrophoresis (CE) with on-capillary Cu(II) complexation for the determination of angiotensin and its metabolites is described. The resulting copper-peptide complexes can be detected using either UV or electrochemical (EC) detection. Optimal reaction and separation conditions for the angiotensin peptides were first determined using CE with UV detection.

Detection of homocysteine by conventional and microchip capillary electrophoresis/electrochemistry.

A method based on capillary electrophoresis (CE) with electrochemical (EC) detection for the determination of both total homocysteine (tHcy) and protein-bound homocysteine (pbHcy) in plasma is described. Both end-column and off-column amperometric detection were investigated. Off-column detection resulted in a more sensitive assay for the determination of homocysteine (Hcy).