George Vella

Principles and Methods for the Analysis and Purification of Synthetic Deoxyribonucleotides by High-Performance Liquid Chromatography

The need for high-purity oligodeoxyribonucleotides for various applications has resulted in the development of novel synthesis, purification, and analytical techniques, A diversity of methods, including polyacrylamide slab gel electrophoresis, capillary gel electrophoresis, as well as high-performance liquid chromatography (HPLC), have been successfully used to aid in the characterization and isolation of these synthetic compounds. The information contained in this review article primarily details both the theoretical and practical aspects related to the use of HPLC for the analysis and purfication of synthetic DNA. In addition, a variety of postsynthesis sample preparation protocols, commonly employed prior to and after HPLC, are described.

Multidimensional chromatography coupled with mass spectrometry for target-based screening

SummaryThe synthesis of structural analogs and the process of drug discovery have evolved dramatically through recent advances in solid-phase synthesis reagents and automated screening systems. As molecular diversity strategies emerge, the need for automated target-based selection of lead candidates becomes equally important. Multidimensional automated chromatographic techniques coupled to electrospray ionization mass spectrometry facilitate the selection process and provide maximum characterization information in a single screening run. The capture of tightly bound affinity leads by target biomolecules, followed by subsequent release and high-resolution separation with sensitive detection, significantly reduces the time required to identify and characterize lead compounds. This automated multidimensional chromatographic approach coupled with mass spectrometry, Selectronics™, was used with several organic and natural libraries to demonstrate an automated target-based screening technique to select for high-affinity binders as potential lead compounds.

Automated proteolytic mapping of proteins

Abstract A new, rapid, automated method for peptide mapping has been developed that requires less than two hours to complete. This method first (i) reduces the protein with dithiothreitol (DTT) or s-mercaptoethanol at 50°C, (ii) then alkylates it with an alkylating agent selected from iodoacetamide, iodoacetic acid, or vinylpyridine, (iii) digests the protein completely with immobilized, TPCK treated trypsin, and (iv) finally analyzes the tryptic fragments by high resolution, reversed-phase liquid chromatography (RPLC) in less than two hours. Reduction and alkylation are achieved in the autosampler of the instrument where the sample, reagents, and reaction protocol are specified by the operator in the system computer. Proteins with up to seven disulfide bridges were quantitatively reduced and alkylated by the system. Immobilized enzyme columns coupled in tandem with an RPLC column were shown to generate protein digests and reproducibly separate the fragments for many cycles of analysis. Based on the fact that any one of several alkylating agents could be used in the mapping process, it was demonstrated that a campaign of experiments could be executed automatically in a search for the optimum alkylating agent. The mapping technique was applied to five different proteins.

Rapid purification and monitoring of immunoglobulin M from ascites by perfusion ion-exchange chromatography

A purification and on-line monitoring procedure for IgM was developed. Perfusion ion-exchange chromatography was used for rapid purification of IgM from ascites fluid and hybridoma supernatant. Crude ascites was directly loaded onto an ion exchanger. Due to the complexity of IgM, a two-step ion-exchange procedure had to be developed. This procedure involved a rapid cation-exchange chromatography capture step followed by further purification using anion-exchange chromatography. High linear velocities, in excess of 3500 cm/h, enabled separations to be performed under 5 min. Purity of the final product by SDS-PAGE was shown to be greater than 95%. Furthermore, the antibodies retained biological activity as measured by indirect immunofluorescence (IIF) and ELISA. The IgM peak was also monitored on-line using a novel peak tracking approach. This involved placing an antibody column (specific to the IgM) prior to the ion-exchange column and operating the ion-exchange column with and without the antibody column in-line. The missing peak that is identified by comparing the two chromatograms indicates where the IgM elutes.

Systematic development and validation of sanitization protocols for a chromatographic system designed for biotherapeutics purification

Production of protein therapeutics through the application of genetic engineering and biotechnology techniques requires comprehensive attention to good manufacturing practice and good laboratory practice (GMP/GLP) guidelines for product recovery and purification. Validated clean-in-place procedures are part of the master method and require analysis of microbial bioburden to assess the efficacy of cleaning protocols. This article describes the extensive microbial challenge of a chromatography system, the use of membrane filtration methods for high sensitivity microbial contamination measurement, and the effectiveness of sodium hydroxide and ethanol solutions in achieving multilog reduction of microbial contamination.

Purification of proteins on an epoxy-activated support by high-performance affinity chromatography

Abstract The use of a rigid silica-based packing material with large particle and pore size, 37–55 μm and 500 A pore, for affinity chromatography makes it possible to combine high selectivity with short analysis times. Both large and small molecules have been covalently bonded to the Protein-Pak TM Affinity Epoxy-Activated bulk packing for purification of glycoproteins, immunoglobulins, enzymes, lectins and other proteins. Recombinant protein A, GammaBind TM G, heparin, Cibacron Blue F3G-A, sulfanilamide, N-acetyl- D -glucosamine, concanavalin A and aminophenylboronic acid were covalently attached to the affinity packing for selective purification of proteins.

New family of high-resolution ion exchangers for protein and nucleic acid purifications from laboratory to process scales

A new family of polymer-based ion exchangers was tested for the purification of acidic and basic proteins on both the analytical and preparative scales. Protein-Pak HR series packings are available as strong cation (SP) and weak anion (DEAE) exchangers, allowing the development of a purification method regardless of the isoelectric point of protein. Three particle sizes, 8, 15 and 40 microns, are offered in scalable Advanced Purification (AP) glass columns or as bulk packings. The lower back pressures of the 15- and 40-microns packings compared to that of the 8-microns material allow rapid throughput of large volumes without exceeding the pressure limitations of the resin or the column. The capacity of the AP1 (100 mm x 10 mm) glass columns, containing these ion-exchange packings, is comparable to other ion-exchange columns. The resolution of mouse serum, plasmids, and a standard protein mixture was demonstrated and compared with the results obtained with other resin-based ion exchangers of similar particle size. Proteins were purified without significant loss of biological activity or mass.