Avinash L. Lagu

Capillary electrophoretic protein separations in polyacrylamide-coated silica capillaries and buffers containing ionic surfactants

Abstract Capillary electrophoretic protein separations of high efficiency and resolution were obtained using polyacrylamide-coated silica capillaries and buffers containing ionic surfactants. The presence of micellar concentrations of sodium dodecyl sulfate or cetyltrimethyl- ammonium chloride minimized protein-capillary wall interactions, and facilitated concurrent separations of a mixture of both acidic and basic proteins, while the polyacrylamide coating provided increased resolution and migration time reproducibility via a reduction in electroosmotic flow. Attempts to obtain size-based protein separations via sieving through buffers containing the hydrophilic polymers methylcellulose and polyethylene glycol were unsuccessful.

Review applications of capillary electrophoresis to the analysis of biotechnology-derived therapeutic proteins.

The number of proteins produced by recombinant DNA technology continues to grow at a rapid pace. In this review, the emphasis is on proteins that are of therapeutic interest. Aspects of protein analysis, such as glycoform separation of proteins produced in mammalian cells and the separation of oligosaccharides for structure elucidation, are covered. The use of antibodies as therapeutic proteins is growing and currently antibodies are the largest class of proteins produced by biotechnology. This has merited a separate section on analysis of antibodies by capillary electrophoresis (CE). Applications of mass spectrometry as an ancillary technique, used in conjunction with CE, are also covered briefly. This review covers the literature since 1999.

Studies of migration time reproducibility of capillary electrophoretic protein separations

Abstract Studies of the migration time reproducibility of trypsinogen were performed using a capillary electrophoresis system designed for high resolution protein separations, where analyte adsorpt...

Applications of capillary electrophoresis in biotechnology

Capillary electrophoresis (CE)‐related techniques are increasingly being used as a matter of routine practice in the biotechnology discipline. Since recombinant DNA‐derived proteins and the antisense oligonucleotides constitute a large portion of the applications of these techniques, they have been emphasized in this review. Analyses by CE of Escherichia coli‐derived proteins and glycosylated proteins derived from mammalian cell cultures are summarized, as well as those of the carbohydrate chains that have been enzymatically removed from the protein. Applications of CE in the analysis of the antisense oligonucleotides for the determination of purity and the analytical studies on the metabolism of these modified oligonucleotides, by CE are reviewed. The literature mainly covers the period from 1996.

Capillary electrophoresis of proteins and peptides

Surfactant-Based Methods for Prevention of Protein Adsorption in Capillary Electrophoresis Charles A. Lucy, Nicole E. Baryla, and Ken K.-C. Yeung Capillary Coating for Protein Separation Based on Si-O and Si-C Covalent Bond Formation for Capillary Electrophoresis With Laser-Induced Fluorescence Detection Hossein Ahmadzadeh, Norman J. Dovichi, and Sergey Krylov On-Column Labeling Reaction for Analysis of Protein Contents of a Single Cell Using Capillary Electrophoresis With Laser-Induced Fluorescence Detection Hossein Ahmadzadeh and Sergey Krylov Covalent and Noncovalent Labeling Schemes for Near-Infrared Dyes in Capillary Electrophoresis Protein Applications John Sowell, Jozef Salon, Lucjan Strekowski, and Gabor Patonay Capillary Electrophoresis in the Analysis and Monitoring of Biotechnological Processes Vadim Klyushnichenko Capillary Electrophoresis of Proteins in a Quality Control Environment David L. Good, Stacey Cummins-Bitz, Raeann M. Fields, and Brian K. Nunnally Analysis of Neutral N-Linked Oligosaccharides From Antibodies Using Free-Solution Capillary Electrophoresis in Bare Fused-Silica Capillaries Jeffrey S. Patrick, Brenda P. Rener, Gregory S. Clanton, and Avinash L. Lagu Affinity Capillary Electrophoresis to Examine Receptor-Ligand Interactions Maryam Azad, John Kaddis, Valerie Villareal, Lili Hernandez, Catherine Silverio, and Frank A. Gomez Screening Major Binding Sites on Human Serum Albumin by Affinity Capillary Electrophoresis Hee Seung Kim, John Austin, and David S. Hage Using Charge Ladders and Capillary Electrophoresis to Measure the Charge, Size, and Electrostatic Interactions of Proteins Upma Sharma and Jeffrey D. Carbeck Frontal Analysis Continuous Capillary Electrophoresis for Protein-Polyelectrolyte Binding Studies Emek Seyrek, Toshiaki Hattori, and Paul L. Dubin Analysis of Proteins by CE, CIEF, and Microfluidic Devices With Whole-Column-Imaging Detection Jiaqi Wu, Xing-Zheng Wu, Tiemin Huang, and Janusz Pawliszyn Capillary Electrophoresis-Electrospray Ionization Mass Spectrometry of Amino Acids, Peptides, and Proteins Mehdi Moini Capillary Isoelectric Focusing-Mass Spectrometry of Proteins and Protein Complexes Suzana Martinovic, Ljiljana Pasa-Tolic, and Richard D. Smith Integrated System for Rapid Proteomics Analyses Using Microfluidic Devices Coupled to Nanoelectrospray Mass Spectrometry Jianjun Li, Tammy-Lynn Tremblay, Jed Harrison, and Pierre Thibault Index

Micellar electrokinetic chromatography of proteins

Abstract Micellar electrokinetic capillary chromatography (MECC) of proteins is a high resolution capillary electrophoretic (CE) analysis method that utilizes the hydrophobic and electrostatic interaction of protein analytes with surfactant micelles present in the buffer medium to facilitate separation. Through the manipulation of the protein-micelle interaction by the adjustment of variables such as surfactant concentration, solution pH, ionic strength, the presence of an organic modifier and the use of coated capillaries, MECC analyses of a wide variety of proteins have been optimized. MECC has been demonstrated to provide resolution of mixtures consisting of proteins with minor structural variations and also has provided the successful quantitative analysis of protein present in complex matrices. The adoption of protein MECC as a routine analytical technique may be dependent upon the successful interface of MECC with detection methodology, such as mass spectrometry, which can provide analyte characterization information.

Capillary electrophoresis as a tool for the analysis of protein folding

Abstract The utility of capillary electrophoresis (CE) was demonstrated for the analysis of a model protein, bovine trypsinogen, as it underwent oxidation from a fully reduced molecule through a distribution of intermediate species until the disulfide bond conformation corresponding to that of the globular native structure was reached. Through the use of CE, completely refolded (native) trypsinogen was resolved from both the reduced protein and intermediate refolded conformations. In addition, the presence of polyethylene glycol in the separation buffer was determined to provide size-based protein separations of significantly higher resolution than those obtained in phosphate buffer alone. Quantitation of native trypsinogen in refolded material revealed an excellent correlation between CE determinations and analyses by established techniques such as biological activity assay and high-performance size-exclusion and cation-exchange chromatography. These results, together with a comparative consistency of CE separations with those obtained via non-denaturing slab gel electrophoresis and isoelectric focusing, suggest that CE can be an effective technique useful for the analysis of protein refolding.

Analysis of recombinant human growth hormone in Escherichia coli fermentation broth by micellar high-performance liquid chromatography

A method for the reversed-phase high-performance liquid chromatographic (HPLC) determination of recombinant methionylaspartyl-human growth hormone (MD-HGH) in Escherichia coli fermentation broth is described. The technique utilizes mobile phases containing n-propanol and the anionic surfactant sodium dodecyl sulfate (SDS) under micellar conditions at pH 6.4. The methodology is directly applicable to the analysis of samples solubilized via sulfitolysis in the presence of SDS, and offers superior resolution in comparison with chromatography in the absence of the surfactant. Using this method, acceptable precision (day-to-day R.S.D. = 4.9%), accuracy, selectivity, range, linearity and ruggedness were achieved.

Anion-exchange chromatography of DNA restriction fragments

The abilities of several high-performance liquid chromatography (HPLC) anion-exchange packings to separate DNA restriction fragments, ranging in size from 50 to 23,000 base pairs, were studied. The ion exchangers investigated include the porous packings Protein-Pak DEAE-5PW, Nucleogen-DEAE 4000-7, Poros-Q and BakerBond WP-PEI, and the non-porous packings TSK Gel DEAE-NPR, Gen-Pak FAX, and ProPac PA1. The results indicated that the non-porous packings could separate all 18 fragments (less than 600 base pairs) in a pBR322 DNA-HaeIII digest, while of the porous packings, only Nucleogen-DEAE 4000-7 could resolve DNA fragments in this size range. Only Gen-Pak FAX and TSK Gel DEAE-NPR could significantly resolve the very large DNA fragments (125-23,000 base pairs) of a lambda DNA-HindIII digest. The chromatographic parameters governing this separation by Gen-Pak FAX were optimized so that six of eight fragments were resolved. Split-peak phenomena were observed at low flow-rates when employing non-poros packings, but were eliminated by the incorporation of organic modifiers or surfactants, suggesting that, under certain conditions, hydrophobicity may play a significant role in separations on this packing. Gen-Pak FAX also separated 21 of 23 fragments in a 1000-base pair DNA ladder, a performance which, in addition to the quantitative capabilities of HPLC, makes anion-exchange chromatography a powerful method complementary to slab-gel electrophoresis, and perhaps preferable over agarose gel electrophoresis for applications such as the confirmation of plasmid integrity.

Analysis of Neutral N-Linked Oligosaccharides From Antibodies Using Free-Solution Capillary Electrophoresis in Bare Fused-Silica Capillaries

Conditions for the enzymatic release, chemical derivatization, and analysis of oligosaccharides from the consensus glycosylation sites on antibodies are described. Release of the oligosaccharides is from the native protein. The APTS derivatives of the released oligosaccharides are then analyzed by capillary electrophoresis (CE) using a free solution separation in a bare fused silica capillary. An example of the application of the method to the analysis of the oligosaccharide population from antibodies obtained from different cell lines is provided. The separation conditions provide for resolution of various galactose positional isomers, including those derived from different linkage configurations.