Larry J. Cummings

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.

Protein chromatography on hydroxyapatite columns.

The introduction of spherical forms of hydroxyapatite has enabled protein scientists to separate and purify proteins multiple times with the same packed column. Biopharmaceutical companies have driven single column applications of complex samples to simpler samples obtained from upstream column purification steps on affinity, ion exchange or hydrophobic interaction columns. Multiple column purification permits higher protein loads to spherical forms of hydroxyapatite and improved reduction in host cell protein, aggregates, endotoxin, and DNA from recombinant proteins. Adsorption and desorption mechanisms covering the multimodal properties of hydroxyapatite are discussed. The chemical interactions of hydroxyapatite surface with common ions, metals, and phosphate species affect column lifetimes. Adsorbed hydroxonium ions from low ionic strength buffers are noted by a shift in effluent pH during column equilibration. Hydroxonium ion desorption is observed by acidic shifts in the column effluent with the magnitude and duration surprisingly extreme. Buffering reagents with high buffering capacity reduce both the magnitude and duration of the acidic shift. Column packing methods for the robust spherical particles as well as the microcrystalline hydroxyapatite particles are reviewed. Applications covering extracted proteins and recombinant protein purification, especially monoclonal antibodies, with multiple chromatography media in concert with hydroxyapatite are reviewed.

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.

Monoclonal Antibody Purification by Ceramic Hydroxyapatite Chromatography

Hydroxyapatite chromatography is shown to be an excellent method for chromatographically purifying monoclonal antibodies (Mab). Mab contained in eluates from Protein A columns was partially purified on ceramic hydroxyapatite (CHT™) Type I, 40 μm ceramic hydroxyapatite using two scouting methods which provide milligram amounts of Mab typical at laboratory scale. The result from one of the scouting methods was optimized to obtain a high concentration of purified Mab with acceptable clearance of cell culture impurities. Several techniques (linear phosphate screening, linear alkaline salt screening, and two alkaline salt step gradients) are described for obtaining high concentrations of purified Mab in a lab-scale CHT chromatography column.

Hydroxyapatite Chromatography: Purification Strategies for Recombinant Proteins

The purpose of this section is to provide developmental purification strategies for recombinant proteins on a column of hydroxyapatite and then to optimize them for repeated purification cycles using effect additives. Surface neutralization solutions (SNS) (patent pending) and/or the addition of calcium ions reduce the solubility of hydroxyapatite thus extending the column lifetime. This section leads the user through purification protocol screening followed by SNS--calcium ion supplementation step gradients for purifying a monoclonal antibody from its contaminants. The section provides simple analytical tools using a 160 ml scale column to predict column life at the process scale. However, the development engineer is encouraged to test the model in a minimum-sized process column.

Monitoring ceramic hydroxyapatite media degradation using dynamic image analysis and uniaxial confined bulk compression

Ceramic hydroxyapatite (CHT) is a multimodal chromatographic medium widely used in the pharmaceutical industry for the purification of biomolecules. CHT is a sintered form of hydroxyapatite crystals with moderate stability at acidic conditions. This moderate stability may lead to underperformance of CHT packed bed lifetime, especially under acidic conditions, which should be monitored by diagnostic tools to design optimal buffer systems for the step. This study presents the application of dynamic image analysis (DIA) and uniaxial confined bulk compression (UCBC) to monitor CHT particle degradation as a function of buffer composition. DIA was used to evaluate changes in solidity and morphology, while UCBC was used to evaluate changes in resistance to uniaxial compression. All properties were studied as a function of bed position and operational parameters. Results show that when CHT is exposed to acidic pH, adding phosphate and/or calcium at concentrations of 1 mM minimizes changes in particle solidity and mechanical strength. Changes in CHT morphological properties (i.e., convexity, aspect ratio) are also affected by the presence of calcium and/or phosphate in the inlet buffers. Furthermore, calcium and phosphate have a positive effect on the mechanical behavior of CHT, which is related to changes in the CHT particle solidity.