Simon C. Burton

Hydrophobic charge induction chromatography: salt independent protein adsorption and facile elution with aqueous buffers

A new form of protein chromatography, hydrophobic charge induction, is described. Matrices prepared by attachment of weak acid and base ligands were uncharged at absorption pH. At low ligand densities, protein adsorption was typically promoted with lyotropic salts. At higher ligand densities, chymosin, chymotrypsinogen and lysozyme were adsorbed independently of ionic strength. A pH change released the electrostatic potential of the matrix and weakened hydrophobic interactions, inducing elution. Matrix hydrophobicity and titration range could be matched to protein requirements by ligand choice and density. Both adsorption and elution could be carried out within the pH 5-9 range.

One step purification of chymosin by mixed mode chromatography.

Mixed mode Sepharose and Perloza bead cellulose matrices were prepared using various chemistries. These matrices contained hydrophobic (aliphatic and/or aromatic) and ionic (carboxylate or alkylamine) groups. Hydrophobic amine ligands were attached to epichlorohydrin activated Sepharose (mixed mode amine matrices). Hexylamine, aminophenylpropanediol and phenylethylamine were the preferred ligands, on the basis of cost and performance. Other mixed mode matrices were produced by incomplete attachment (0-80%) of the same amine ligands to carboxylate matrices. The best results were obtained using unmodified or partially ligand-modified aminocaproic acid Sepharose and Perloza. High ligand densities were used, resulting in high capacity. Furthermore, chymosin was adsorbed at high and low ionic strengths, which reduced sample preparation requirements. Chymosin, essentially homogeneous by electrophoresis, was recovered by a small pH change. The methods described were simple, efficient, inexpensive and provided very good resolution of chymosin from a crude recombinant source. The carboxylate matrices had the best combination of capacity and regeneration properties. The performance of Sepharose and Perloza carboxylate matrices was similar, but higher capacities were found for the latter. Because it is cheaper and can be used at higher flow rates, Perloza should be better suited to large scale application. High capacity chymosin adsorption was found with carboxymethyl ion exchange matrices, but low ionic strength was essential for adsorption and the purity was inferior to that of the mixed mode matrices.

Salt-independent adsorption chromatography: new broad-spectrum affinity methods for protein capture

The role of chromatography in capture is reviewed in terms of the special requirements imposed by the processing of very crude feedstocks. Adsorption methods which are not significantly affected by variations of feedstock ionic strength are highlighted. Methods are compared in terms of simplicity, robustness, selectivity and ease of elution. The application of such methods to enzyme and antibody purifications is summarised. Particular emphasis is placed on high ligand density methods, which have potential for broad-spectrum application.

Development of a mixed mode adsorption process for the direct product sequestration of an extracellular protease from microbial batch cultures

Direct product sequestration of extracellular proteins from microbial batch cultures can be achieved by continuous or intermittent broth recycle through an external extractive loop. Here, we describe the development of a fluidisable, mixed mode adsorbent, designed to tolerate increasing ionic strength (synonymous with extended productive batch cultures). This facilitated operations for the integrated recovery of an extracellular acid protease from cultures of Yarrowia lipolytica. Mixed mode adsorbents were prepared using chemistries containing hydrophobic and ionic groups. Matrix hydrophobicity and titration ranges were matched to the requirements of integrated protease adsorption. A single expanded bed was able to service the productive phase of growth without recourse to the pH adjustment of the broth previously required for ion exchange adsorption. This resulted in increased yields of product, accompanied by further increases in enzyme specific activity. A step change from pH 4.5 to 2.6, across the isoelectric point of the protease, enabled high resolution fixed bed elution induced by electrostatic repulsion. The generic application of mixed mode chemistries, which combine the physical robustness of ion-exchange ligands in sanitisation and sterilisation procedures with a selectivity, which approaches that of affinity interactions, is discussed.

High-density ligand attachment to brominated allyl matrices and application to mixed mode chromatography of chymosin

Abstract Allylated cellulose, agarose and methacrylate matrices, activated with allyl bromide or allyl glycidyl ether, were modified by aqueous bromination, preferably with N-bromosuccinimide. Amine, thiol and sulphite ligands were attached efficiently and in high densities (1–1.5 mmol/g dry) to the brominated matrices, using concentrated reaction mixtures. Organic solvents were not required. Bromohydroxypropyl matrices and amine or carboxylate derivatives could thus be used to produce matrices for all major forms of adsorption chromatography. This chemistry was used to prepare high-density mercaptoalkyl acid matrices, which were successfully applied to mixed mode purification of crude chymosin and compared with matrices used previously.

Impact of the physical and topographical characteristics of adsorbent solid-phases upon the fluidised bed recovery of plasmid DNA from Escherichia coli lysates.

A comparison is made of the performance of two types of adsorbent solid phases (commercially sourced Streamline composites and custom-assembled Zirblast pelliculates), derivatised with similar anion exchange chemistries and applied to the recovery of plasmid DNA from Escherichia coli extracts prepared by chemical lysis and coarse filtration. Streamline and Zirblast adsorbents were characterised by average particle diameters of 200 and 95 microm, densities of 1.16 and 3.85 g/m2, and small ion capacities of 170 and 8 micromol/ml settled adsorbent, respectively. Detailed analysis of products and impurities in a full operational cycle of adsorption, washing, pre-elution, elution and regeneration processes was enabled by the harnessing of a battery of analyses for nucleic acid and organic solute content of feedstocks and bed effluents exploiting ultra-violet spectrophotometry, agarose gel electrophoresis and specific reactions with the fluorescent probe PicoGreen. In comparative tests operated under near identical conditions, Streamline and Zirblast adsorbents exhibited plasmid recoveries of 76 and 90% of bound product characterised by purity ratios (relative PicoGreen and A254 estimates of mass) of 9 and 32, respectively. Conclusions are drawn regarding the specific impact of the physical and topographical characteristics of solid-phase geometry upon product throughput, achievable product purity, process time-scales and operational economics for the manufacture of plasmid DNA.

Preparation of chromatographic matrices by free radical addition ligand attachment to allyl groups

Chromatographic matrices were produced by free radical addition reactions between immobilised allyl groups and suitable ligands. Efficient addition and high ligand densities were readily obtained with mercaptoacids, glutathione, mercaptoethanol and sodium bisulphite, using aqueous solvation. Addition of nitrogen containing thiol ligands other than glutathione was also demonstrated, although heat or radiation catalysis was required. Addition of cysteamine or mercaptoacids resulted in spacer arm derivatives, suitable for further ligand attachment chemistry. Mercaptoacetic acid and bisulphite attachment was used to prepare cation-exchange matrices. Glutathione derivatives were compared with matrices prepared conventionally, for affinity chromatography of glutathione transferase.

Bifunctional etherification of a bead cellulose for ligand attachment with allyl bromide and allyl glycidyl ether

Abstract Efficient carboxymethylation of a bead cellulose was achieved with dimethyl sulphoxide–water mixtures but low activation levels were found with epoxidation reagents. The highest activation levels were obtained with allyl bromide and allyl glycidyl ether. This was attributed to the relative inertness of the allyl group towards water and cellulose, limiting hydrolysis and crosslinking. Advantages over epoxidation chemistry for bead cellulose were efficient reagent use, reproducible activation up to high levels and specificity of matrix modification. Predictable activation was obtained and facile titration methods were developed. Efficient activations of agarose and methacrylate matrices were also obtained with the allyl reagents.

Design and assembly of solid-phases for the effective recovery of nanoparticulate bioproducts in fluidised bed contactors

Practical recovery of nanoparticulate bioproducts from suspension feedstocks has been studied in batch, fixed bed and fluidised bed adsorptive contactors. The performance of five discrete configurations of adsorbent solid phase has been critically evaluated in the anion exchange recovery of mg quantities of BSA nanoparticles. These have served as surrogate size mimics of less easily sourced viral and plasmid gene therapy vectors, characterised by high value and a shortage of supply in quantities sufficient for research and development. Performance parameters of binding capacity, efficacy of washing, desorption efficiency and total cycle time were strongly influenced by the external and internal topographies of solid phases, together with the localised concentrations of interacting chemical ligands which modulate adsorption. In respect of a full operational recovery cycle, porous adsorbents developed for refined chromatographic fractionation of macromolecules, appear less suited overall than solid, non-porous particles, or solid particles coated with a shallow pellicle of active adsorbent material. Such findings have been confirmed in a detailed demonstration of the recovery of plasmid DNA (7.8 Kb) from chemical lysates of Escherichia coli.

Efficient substitution of 1,1′ -carbonyldiimidazole activated cellulose and Sepharose matrices with amino acyl spacer arms

Abstract Previous procedures for addition of the spacer arm, 6-aminocaproic acid, to 1,1′-carbonyldiimidazole (CDI) activated matrices used dilute, largely aqueous reaction conditions. Only 20–50% of the activated groups were substituted. Treatment of CDI activated matrices in an organic solvent, with a concentrated aqueous solution of sodium 6-aminocaproate, allowed efficient (up to 95%) substitution of CDI activated groups. The substitution efficiency of aminocaprylic acid was also improved considerably using an organic solvent although the maximum efficiency was lower (66%). The new procedure allows reproducible levels of spacer arm substitution to be obtained. A benzamidine affinity resin prepared by the new method had a 50% higher capacity for trypsin than one prepared by the original method.