Graham E. McCreath

Modification of polystyrenic matrices for the purification of proteins Effect of the adsorption of poly(vinyl alcohol) on the characteristics of poly(styrene-divinylbenzene) beads for use in affinity chromatography

A poly(styrene-divinylbenzene) (PSDVB) chromatography matrix, CG1000-sd (TosoHaas), has been modified using poly(vinyl alcohol) (PVA) to create a matrix suitable for the attachment of functional groups for the selective purification of proteins. The characteristics of the modified matrix have been studied using a BET nitrogen adsorption/desorption technique and it has been found that the adsorption of PVA results in the bead micropores being filled whilst the bead macropores are left essentially unaltered. There was no protein adsorption onto the modified matrices. A dye ligand (Procion Blue MX-R) has been covalently attached to PVA-PSDVB matrix and the lysozyme capacities of the PVA-PSDVB matrix have been determined. The matrix compares well with commercial Blue Sepharose Fast Flow, an affinity matrix on cross-linked agarose. The dye-PVA-PSDVB matrix is stable when subjected to sanitisation with sodium hydroxide.

Novel affinity separations based on perfluorocarbon emulsions. Use of a perfluorocarbon affinity emulsion for the purification of human serum albumin from blood plasma in a fluidised bed.

A perfluorocarbon affinity emulsion has been generated by homogenisation of a saturated perfluorocarbon oil with a polymeric fluorosurfactant based on poly(vinyl alcohol) (relative molecular mass 9000-10,000) previously derivatised with the triazine dye CI Reactive Blue 4. This affinity emulsion has subsequently been cross-linked in situ and used in a fluidised bed for the purification of human serum albumin (HSA) from blood plasma. HSA was quantitatively recovered in a semi-continuous fashion from plasma at an average purity of 90 +/- 3.3%. The albumin binding capacity of the emulsion has been shown to be 0.59 mg/ml by frontal analysis corresponding to a mol/mol ligand usage of 13.5%. In all regards, when used in a fluidised bed, the emulsions have been shown to behave as a normal chromatographic material. They are stable under operational conditions with no coalescence being observed for periods greater than 1 year. These novel liquid affinity supports present an exciting opportunity to develop a range of unit operations for the continuous purification of proteins.

Novel affinity separations based on perfluorocarbon emulsions. Development of a perfluorocarbon emulsion reactor for continuous affinity separations and its application in the purification of human serum albumin from blood plasma.

Perfluorocarbon affinity emulsions are generated by the homogenisation of a perfluorocarbon oil with a polymeric fluorosurfactant previously derivatised with an affinity ligand and subsequently cross-linked in situ. This procedure gives rise to a novel liquid affinity adsorbent that can be used for continuous protein purification. Discrete emulsion droplets were found to be unstable when pumped for prolonged periods; however, when flocculated, the emulsion floccules with diameters of around 125 microns, were very stable and sedimented faster. A four-stage reactor unit (perfluorocarbon emulsion reactor for continuous affinity separations, PERCAS) was designed and constructed to carry out continuous separations, and exploited the unusual properties of the absorbent, i.e. liquid nature and high density. Each of the four stages of PERCAS consisted of a mixing tank, for contacting between emulsion phase and aqueous phase, adjacent to a settling tank for the subsequent separation of emulsion from the aqueous phase. Using PERCAS adsorption, washing, elution and re-equilibration of the emulsion could be carried out continuously with emulsion recycle. Using single-component adsorption of human serum albumin to a perfluorocarbon affinity emulsion derivatised with the triazine dye C.I. Reactive Blue 2, PERCAS was optimised with respect to flow-rates and input concentrations. The work was then extended to the continuous purification of essentially homogeneous human serum albumin from blood plasma.

A new approach to continuous counter-current protein chromatography: Direct purification of malate dehydrogenase from a Saccharomyces cerevisiae homogenate as a model system.

A novel technique for protein chromatography has been developed, which can be used to extract proteins from particulate-containing solutions (such as fermentation broths or preparations of disrupted cells) on a continuous basis, and delivers clarified streams of purified product. Adsorbents deployed in this type of contactor are based on PVA-coated perfluorocarbons derivitized with affinity ligands such as triazine dyes. In this article, we describe the application of this equipment for the continuous purification of malate dehydrogenase from an unclarified homogenate of Saccharomyces cerevisiae, using a Procion Red HE-7B-derivitized adsorbent. Although operating conditions were not optimized to produce a product of maximized purification factor, concentration, and yield, we have shown that MDH can be purified continuously in 78% yield at a rate of 70 U/min, with a purification factor of approximately 10. This corresponds to specific productivity of approximately 0.35 U/min per milliliter of settled adsorbent, a higher specific productivity than was feasible with the same adsorbent using expanded bed adsorption (EBA). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 427-441, 1997.

Applications of Perfluorocarbon Affinity Emulsions for the Rapid Isolation of Staphylococcus aureus

Human immunoglobulin G (IgG) has been immobilized to poly(vinyl alcohol)‐stabilized liquid perfluorocarbon droplets. This affinity emulsion has been shown to adsorb Staphylococcus aureus cells from solutions with adsorption capacities of 32 × 109 and 48 × 109 cells/mL for affinity emulsions with immobilized IgG densities of 1.15 and 2.45 mg/mL, respectively. The kinetics of cell binding from solution were found to be rapid with clearance of S. aureus cells from a suspension (8 × 108 cell/mL) achievable in under 5 min. S. aureus cells (1.3 × 109 cells) could also be rapidly depleted from a suspension of Saccharomyces cerevisiae (3.4 × 1010 cell/mL) in under 18 min with no cross‐reactivity being observed. The affinity emulsion was stable for at least five cycles of operation with little loss in adsorption capacity when removal of adsorbed cells was carried out at pH 2.5.

Affinity adsorption of Saccharomyces cerevisiae on concanavalin a perflurocarbon emulsions

Perfluorocarbon affinity emulsions have been generated by immobilizing concanavalin A onto the surface of triazine‐activated perfluorocarbon droplets. Immobilized concanavalin A densities of 0.1, 0.7 and 2.1 mg/ml were obtained by varying the concentration of cyanuric chloride used for activation. The affinity emulsions were found to adsorb Saccharomyces cerevisiae cells from solution with adsorption capacities of 1 × 109 cells, 4.6 × 109 and 6 × 109 cell/ml, respectively. Optimal conditions for the elution of bound cells were obtained by studying inhibition curves of concanavalin A–mannan precipitation using simple sugars. Methyl α‐D‐mannopyranoside showed the greatest inhibitory power with 50 per cent inhibition displayed at a concentration of 0.05 mM. Experiments carried out examining the concentrations of methyl α‐D‐mannopyranoside necessary to dissociate a concanavalin A–mannan precipitate demonstrated that at least a seven‐fold higher concentration of methyl α‐D‐mannopyranoside was required for dissociation than that required for inhibition of its formation. The efficiency of elution of bound yeast cells was found to be dependent on the concentration of methyl α‐D‐mannopyranoside used, the time of elution and the immobilized ligand density. Thus, 100 per cent elution was obtained with a concanavalin A affinity emulsion (0.1 mg/ml) by incubation with 500 mM methyl α‐D‐mannopyranoside for 1 h.

Purification of G6PDH from Unclarified Yeast Cell Homogenate Using Expanded Bed Adsorption (EBA) with STREAMLINETM Red H-E7B

The use of expanded beds of STREAMLINETM Red H-E7B in a STREAMLINE 50 EBA apparatus for the direct extraction of glucose-6-phosphate dehydrogenase (G6PDH) from unclarified preparations of disrupted bakers’ yeast has been investigated It has been demonstrated that such crude feedstocks can be applied to the bed without prior clarification steps (centrifugation and microfiltration). The ability of combining clarification, capture, and purification in a single step will greatly simplify downstream processing flowsheets and reduce the costs of protein purification. The purification of G6PDH from unclarified yeast homogenate was chosen as a model system outlining the typical features of direct broth extraction. Procion Red H-E7B STREAMLINETM adsorbent has been assessed for its performance in expanded bed procedures. The absorbent was shown to be successful in achieving isolation of G6PDH from unclarified homogenate with a purification factor of 12 and yield of 86% in a single step process. STREAMLINETM Red H-E7B could be cleaned using simple clean-in-place procedures without affecting either adsorption or the bed expansion properties of the adsorbent. The results clearly demonstrate the potential of expanded bed technology in achieving a significant reduction in the number of processing steps required to purify proteins from crude feedstocks. In addition, the use of biomimetic dyes as affinity ligands seems to be an ideal compromise between selectivity and robustness.

APPLICATION OF PERFLUOROCARBONS IN NOVEL CONTINUOUS COUNTER-CURRENT PROTEIN CHROMATOGRAPHY

In this work the development of a process capable of extracting proteins from particulate‐containing solutions (such as fermentation broths) on a continuous basis, and in which adsorbent and process streams are contacted counter‐currently is described. The process consists of four stages, required for the loading, washing, elution and regeneration of the adsorbent. Because of its counter‐current nature, it has imporved performance over existing (though not yet commercialized) continuous processes, which have been based on CSTR‐type contractors e.g. PERCAS (McCreath et al., 1993). The improved efficiency has been shown by carrying out extraction of lysozyme from a single component feed stream. The adsorbent used in this work is a Procion Red HE‐7B‐derivatized perfluorocarbon support, which has shown particular suitability for continuous processes due to its inherently high mechanical strength and high density. Results illustrating yields obtained using this equipment are presented and discussed.