Applying high-throughput methods to develop a purification process for a highly glycosylated protein.

Micro-scale chromatography formats are becoming more routinely used in purification process development because of their ability to rapidly screen large number of process conditions at a time with minimal material. Given the usual constraints that exist on development timelines and resources, these systems can provide a means to maximize process knowledge and process robustness compared to traditional packed column formats. In this work, a high-throughput, 96-well filter plate format was used in the development of the cation exchange and hydrophobic interaction chromatography steps of a purification process designed to alter the glycoform distribution of a small protein. The significant input parameters affecting process performance were rapidly identified for both steps and preliminary operating conditions were identified. These ranges were verified in a packed chromatography column in order to assess the ability of the 96-well plate to predict packed column performance. In both steps, the 96-well plate format consistently led to underestimated glycoform-enrichment levels and to overestimated product recovery rates compared to the column-based approach. These studies demonstrate that the plate format can be used as a screening tool to narrow the operating ranges prior to further optimization on packed chromatography columns.

A novel two-zone protein uptake model for affinity chromatography and its application to the description of elution band profiles of proteins fused to a family 9 cellulose binding module affinity tag.

A novel two-zone model (TZM) is presented to describe the rate of solute uptake by the stationary phase of a sorption-type chromatography column. The TZM divides the porous stationary-phase particle into an inner protein-free core and an outer protein-containing zone where intraparticle transport is limited by pore diffusion and binding follows Langmuir theory. The TZM and the classic pore-diffusion model (PDM) of chromatography are applied to the prediction of stationary-phase uptake and elution bands within a cellulose-based affinity chromatography column designed to selectively purify proteins genetically labelled with a CBM9 (family 9 cellulose binding module) affinity tag. Under both linear and nonlinear loading conditions, the TZM closely matches rates of protein uptake within the stationary phase particles as measured by confocal laser scanning microscopy, while the PDM deviates from experiment in the linear-binding region. As a result, the TZM is shown to provide improved predictions of product breakthrough, including elution behavior from a bacterial lysate feed.

A multiple chemical equilibria approach to modeling and interpreting the separation of amino acid enantiomers by chiral ligand-exchange chromatography.

A model of chiral ligand-exchange chromatography (CLEC) is presented that combines the non-ideal equilibrium-dispersion equation for solute transport with equations describing all chemical equilibria within the column. The model connects elution band profiles to the time and space resolved formation of diastereomeric complexes in both the mobile and stationary phases, thereby providing insights into the overall separation mechanism. The stoichiometries and formation constants for all equilibrium complexes formed in the mobile phase are taken from standard thermodynamic databases and independent potentiometric titration experiments. Formation constants for complexes formed with the stationary phase ligand are determined from potentiometric titration data for a water-soluble analogue of the ligand. Together this set of pure thermodynamic parameters can be used to calculate the equilibrium composition of the system at any operating condition. The model includes a temperature-dependent pure-component parameter, determined by regression to a single elution band for the pure component, that corrects for subtle effects associated with immobilizing the ligand (i.e., the chiral selector) onto the stationary phase. Model performance is assessed through comparison with chromatograms for two hydrophobic amino acid racemates loaded on the Nucleosil Chiral-1 CLEC column. The model is also applied to a restricted optimization of column operating conditions to assess its predictive power. In both cases, model predictions compare well with experiment while also providing a molecular understanding of the separation process and its dependence on column operating conditions.

Interpreting the effects of temperature and solvent composition on separation of amino-acid racemates by chiral ligand-exchange chromatography.

Routinely applied at both preparative and analytical scales, chiral ligand-exchange chromatography (CLEC) separates enantiomers capable of chelating a divalent transition-metal-ion through a pair of coordinating electronegative atoms. CLEC separation efficiencies are strongly dependent on column operating conditions, including temperature and mobile-phase solvent composition. Although previous empirical studies provide some useful guidelines for optimizing column operating conditions, the fundamental mechanisms underlying the unusually high sensitivity of CLEC performance to operating temperature and solvent composition remain poorly understood, limiting efforts to develop a comprehensive model for the technology. To address this problem, we report transport and chemical equilibria data for the separation of alpha-amino acids on a Nucleosil chiral-1 column presenting L-hydroxyproline as the immobilized ligand. Solute transport is found to be limited by pore diffusion at all column operating temperatures and solvent compositions, validating the existence of local equilibria throughout the column. Changes in separation performance are found to correlate with changes in chemical equilibria, emphasizing the need to carefully account for all speciation within the column when modeling CLEC and providing important fundamental data to achieve this goal. Each enantiomer participates in a large number of solution-phase complexes. As a result, the thermodynamic driving force for separation is unusually complex, allowing subtle changes in column operating conditions to mediate significant changes in speciation profiles and separation efficiency. A reaction-equilibria model accounting for all speciation within the CLEC column is proposed and used to estimate enantiomer partition coefficients and retention times.

Specificity of the synergistic anion for iron binding by ferric binding protein from Neisseria gonorrhoeae.

Ferric binding protein in Neisseria gonorrhoeae (nFbpA) transports iron from outer membrane receptors for host proteins across the periplasm to a permease in an alternative pathway to the use of siderophores in some pathogenic bacteria. Phosphate and nitrilotriacetate, both at pH 8, and vanadate at pH 9 are shown to be synergistic in promoting ferric binding to nFbpA, in contrast to carbonate and sulfate. Interestingly, only phosphate produces the fully closed conformation of nFbpA as defined by native electrophoresis. The role of phosphate was probed by constructing three mutants: Q58E, Q58R, and G140H. The anion and iron binding properties of the Q58E mutant are similar to the wild-type protein, implying that one phosphate oxygen is a hydrogen bond donor and may in part define the specificity of nFbpA for phosphate over sulfate. Phosphate is a weakly synergistic anion in the Q58R and G140H mutants, and these mutants do not form completely closed structures. Ferric binding was investigated by both isothermal titration and differential scanning calorimetry. The apparent affinity of nFbpA for iron in a solution of 30 mM citrate is 1 order of magnitude larger in the presence (K(app)= 1.7 x 10(5) M(-1)) of phosphate than in its absence (K(app) = 1.6 x 10(4) M(-1)) at pH 7. Similar results were obtained at pH 8. This increase in affinity with phosphate as well as the formation of closed structure allows nFbpA to compete for free ferric ions in solution and suggests that ferric binding to nFbpA is regulated by the synergistic phosphate anion at sites of iron uptake.

Carbohydrate-binding modules recognize fine substructures of cellulose.

Competition isotherms are used to identify the set of cellulose substructures to which cellulose binding modules (CBMs) from families 2a, 3, 4, 9, and 17 bind. The experiments are based on coupling a unique fluorescent tag to each CBM in a manner that does not alter the natural binding properties of the CBM and therefore allows the surface and solution concentrations of each CBM to be monitored as a function of time and composition. Adsorption and surface exchange of like or competing CBMs are monitored using a range of cellulose preparations varying in both crystallinity and provenance. CBMs from families 2a, 3, 4, 9, and 17 are shown to recognize different physical forms of prepared cellulose. The demonstration of the very fine binding specificity of cellulose-specific CBMs implies that the polysaccharide targets of CBMs extend down to the resolution of cellulose microstructures.