Christopher J. Siebert

Rapid protein profiling with a novel anion-exchange material

A new anion-exchange material has been developed which allows very rapid resolution of protein mixtures. The Microanalyzer MA7P matrix consists of small (7 micron), spherical, non-porous, polymethacrylate beads with polyethyleneimine (PEI) covalently coupled to the surface. When packed 30 X 4.6 mm I.D. columns, this matrix is particularly well suited for applications in which 1-300 micrograms of a protein mixture must be resolved in a minimum of time. Recoveries of injected proteins are usually quantitative, even when the amounts of individual proteins are in the submicrogram range. Chromatography on Microanalyzer MA7P columns is characterized by very narrow bandwidths, even at relatively high flow-rates. This is due to the combined effects of short column length, high selectivity, and the lack of velocity-dependent bandbroadening attributable to diffusion into and out of pores. These columns have no discernable gel filtration effects in the molecular weight range from 10(3) to 10(6) daltons. Columns are very rapidly equilibrated with new solvents, further reducing cycle-to-cycle times.

Efficient endotoxin removal with a new sanitizable affinity column: affi-prep polymyxin

A new affinity column packing for removal of endotoxins has been prepared by coupling USP drug-quality polymyxin B to Affi-Prep, a novel synthetic macroporous polymer. Affi-Prep Polymyxin binds endotoxins from a number of different strains of gram-negative bacteria. Endotoxin binding is not significantly affected by 10 mg/ml of bovine serum albumin or human immunoglobulin G, by 1 mg/ml sodium dodecyl sulphate, or by 1 mg/ml deoxycholate. Affi-Prep Polymyxin is stable to treatment with 1.0 M sodium hydroxide, an important property for sanitizing the resin. The resin shows a high ligand stability, since no leakage of polymyxin B from the packing could be detected. Affi-Prep Polymyxin exhibited the highest endotoxin binding efficiency when compared with several commercial agarose affinity packings.

Performance evaluation of non-porous versus porous ion-exchange packings in the separation of proteins by high-performance liquid chromatography

The performance of a non-porous, anion-exchange packing was evaluated and compared with a number of similar porous high-performance liquid chromatography packings. The non-porous columns were found to be equally efficient for proteins spanning a wide range of molecular weights, while the porous columns exhibited decreasing efficiency as the proteins became larger. The porous materials also exhibited size exclusion effects that were not seen with the non-porous materials, which partially accounts for the loss of efficiency with large proteins. When increasingly steep gradients were employed, the loss of resolution was less with the non-porous materials.

Optimization of oxidation of glycoproteins: An assay for predicting coupling to hydrazide chromatographic supports

A rapid, simple assay for aldehydes generated by oxidation of saccharide units in glycoproteins, using dyes containing hydrazide functionalities, is described. The assay is used, in conjunction with tests of biological activity, to predict oxidation conditions that will result in a maximum of active protein coupled to a hydrazide chromatographic support. Glycoproteins are labeled with Lucifer Yellow CH or Texas Red Hydrazide, and the extent of labeling is determined. Using the assay, it is shown that the efficiency of coupling to Affi-Prep Hydrazide is proportional to oxidation.

Oligonucleotide analysis by capillary polymer sieving electrophoresis using acryloylaminoethoxyethanol-coated capillaries

Capillary polymer sieving electrophoresis using dynamic sieving polymer solutions provides size-based separations of oligonucleotides. The polymer sieving system described here resolves single-stranded oligonucleotides with single-base resolution up to lengths of 30 bases within 10 min. The effect of temperature on the separation of synthetic oligonucleotide standards was examined between 20 degrees C and 40 degrees C, with optimal performance at 35-40 degrees C. By adding urea to the sieving buffer single-base resolution could be extended to 60 bases in about 40 min. Best performance was achieved with capillaries coated with a new monomer, acryloylaminoethoxyethanol. This coating provides the necessary stability to ensure long lifetimes.

Covalent immobilization of proteins to N-hydroxysuccinimide ester derivatives of agarose: Effect of protein charge on immobilization

An uncharged N-hydroxysuccinimide ester derivative of agarose, Affi-Gel 10, exhibited excellent capacity for immobilization, at pH 7.5, of proteins having isoelectric points of 6.5--11.0. Under identical conditions, acidic proteins with isoelectric points of 3.3--5.9 did not couple well to this activated gel. Immobilization of acidic proteins increased in the presence of 80 mM CaCl2, or at a pH equal to or less than the isoelectric point. Affi-Gel 15, a new N-hydroxysuccinimide ester derivative of agarose containing a tertiary amine in the spacer arm, coupled acidic proteins efficiently at pH 7.5 but basic proteins coupled poorly. The immobilization of basic proteins to Affi-Gel 15 was increased to useful levels by increasing the ionic strength, or the pH, of the reaction medium. The lectin concanavalin A was efficiently immobilized using either activated gel, and the concanavalin A-agarose derivatives bound 3.9--4.1 mg ovalbumin/ml gel. These studies demonstrate that the charge of the protein relative to the charge of the gel is an important factor affecting the level of protein immobilization to active ester gels.

Characterization of synthetic macroporous ion-exchange resins in low-pressure cartridges and columns: Evaluation of the performance of Macro-Prep 50 S resin in the purification of anti-Klenow antibodies from goat serum

Three ion-exchange materials and one hydrophobic-interaction chromatography packing, based on a rigid macroporous polymer with large, relatively uniform pores, have been evaluated for low-pressure liquid chromatography of antibodies. These sorbents have high capacities for both small and large proteins and are mechanically, chemically, and thermally stable. Macro-Prep 50 S. CM and Q ion-exchange materials are strongly acidic, weakly acidic, and strongly basic, respectively. Protein binding and recovery, pressure-flow properties, and chemical and thermal stability were determined for each sorbent. A rapid, two-step method for the purification of anti-Klenow antibodies from goat serum was developed, based on the Macro-Prep 50 S strong-acid cation-exchange material and the Econo-Pac HIC prepacked hydrophobic-interaction cartridge.

Characterization of synthetic macroporous packing materials in low-pressure cartridges and columns

Abstract Three ion-exchange packing materials, Macro-Prep 50 S, Macro-Prep 50 CM and Macro-Prep 50 Q (strong cation, weak cation and strong anion, respectively) were characterized with respect to dynamic protein-binding capacities and chemical stability and resolution following exposure to 0.1 and 1.0 M NaOH. The pore-size distribution in the hydrated state was determined by size-exclusion chromatography on a Macro-Prep t-Butyl hydrophobic interaction (HIC) sorbent. Isolation of anti-Klenow antibodies from goat serum was performed on a 1-1 Macro-Prep 50 S column. Specific anti-Klenow antibodies were affinity purified on a semi-preparative scale using Klenow coupled to an Affi-Prep 10 (NHS activated) sorbent. Recombinant Klenow polymerase from Escherichia coli was purified in one chromatographic step on an Econo-Pac heparin 5-ml cartridge.