Raja Ghosh

Protein separation using membrane chromatography: opportunities and challenges

Some of the problems associated with packed bed chromatography can be overcome by using synthetic macroporous and microporous membranes as chromatographic media. This paper reviews the current state of development in the area of membrane chromatographic separation of proteins. The transport phenomenon of membrane chromatography is briefly discussed and work done in this area is reviewed. The various separation chemistries which have been utilised for protein separation, along with different applications, are also reviewed. The technical challenges facing membrane chromatography are highlighted and the scope for future work is discussed.

Fractionation of BSA and lysozyme using ultrafiltration: effect of pH and membrane pretreatment

Selective transmission of a solute through membranes proves to be a challenge in ultrafiltration processes. This is because the transport of a solute through an ultrafiltration membrane does not depend on size alone, but on several other factors such as solute-solute and solute-membrane interactions. By manipulating physicochemical parameters and process variables (eg. pH, ionic strength, concentration of solute, etc.) and by membrane modification, it is possible to enhance the transmission of a particular solute and thus enhance fractionation of solutes. In this paper, the effect of pH on fractionation of BSA and lysozyme by ultrafiltration through 50 kDa MWCO (molecular weight cut off) polysulfone membrane has been examined. It was found that the selectivity of solute separation for dilute mixtures of BSA and lysozyme was very much pH dependent and varied from 3.3 at pH 5.2 to 220.0 at pH 8.8. However, at a higher feed concentration, the transmission of lysozyme through polysulfone membrane decreases quite dramatically resulting in lower throughput of product. An attempt has been made to enhance the transmission of lysozyme through the polysulfone ultrafiltration membrane by pretreating the surface of the membrane by adsorption of another protein, myoglobin. An increase in lysozyme transmission of up to 63% with respect to native membrane was observed. The stability of this pretreatment and its effect on permeate flux have been examined. The pretreated membrane was used to fractionate BSA/lysozyme mixtures. Even at higher feed concentration, enhanced fractionation with respect to native membrane was observed due to highly enhanced transmission of lysozyme through the pretreated membrane.

Lysozyme separation by hollow-fibre ultrafiltration

This paper discusses the purification of lysozyme from chicken egg white using hollow-fibre ultrafiltration (30kDa MWCO, polysulphone membrane). Lysozyme is preferentially transmitted through the membrane while the membrane largely retains other egg white proteins. Improvement in system hydrodynamics resulted in an increase in permeate flux while lysozyme transmission remained unaffected, leading to higher productivity. The percentage purity of lysozyme obtained was generally insensitive to system hydrodynamics. The permeate flux and productivity increased with increase in transmembrane pressure (TMP) before levelling off around 0.7bar. However, the TMP did not have any pronounced effect on the transmission and the purity of lysozyme. Experiments carried out in the diafiltration mode showed that moderately pure lysozyme (80-90%) could be obtained in an extended operation.

Separation of proteins using hydrophobic interaction membrane chromatography

Membrane chromatography can overcome some of the problems associated with packed bed chromatography. In most membrane chromatographic studies reported so far, ion-exchange and affinity interactions have been utilised. In this paper the use of hydrophobic interactions for chromatographic separation is described. A polyvinylidene fluoride membrane was identified which could bind specific proteins in the presence of high ammonium sulphate concentration. The separation of CAMPATH-IG monoclonal antibody and bovine serum albumin using this membrane is discussed.

Purification of lysozyme using ultrafiltration.

This article examines the separation of lysozyme from chicken egg white by ultrafiltration with 25 kDa and 50 kDa MWCO polysulfone membranes. The effects of pH, system hydrodynamics, feed concentration, and transmembrane pressure on permeate flux, lysozyme transmission, purification factor, and productivity have been discussed. With both types of membranes, higher permeate flux and lysozyme transmission were observed at higher pH. Higher lysozyme purity was generally obtained with the 25 kDa MWCO membrane. Purity of lysozyme decreased when the feed concentration was increased. With the 50 kDa MWCO membrane permeate flux, productivity and the purity of lysozyme were found to increase with increase in transmembrane pressure. The possibility of using a two-step ultrafiltration process for achieving high productivity along with high purity of lysozyme was also investigated.

Enhancement of ultrafiltration by gas sparging with flat sheet membrane modules

Gas sparged ultrafiltration has been applied to a flat sheet membrane module and the enhancing effect from the injected bubbles is examined experimentally. Two membranes, polysulphone (PS) and polyethersulfone (PES), were used in the experiments, and four proteins, human serum albumin (HSA), human immunoglobulin G (IgG), bovine serum albumin (BSA) and lysozyme (Lys), were chosen as the test media. The effects of gas sparging on permeate flux, single protein transmission and protein fractionation have been investigated. Experimental results show that gas sparging can increase permeate flux and improve the efficiency of protein fractionation.

High-resolution plasma protein fractionation using ultrafiltration☆

The effects of pH and ionic strength (salt concentration) on transmission of single protein human serum albumin (HSA) and human immunoglobulins (HIgG) through a 100 kDa molecular weight cut-off (MWCO) polyethersulfone membrane have been studied using a pulsed sample injection technique. Experimental results obtained clearly demonstrate the effectiveness of changing solution pH and ionic strength optimisation for selective transmission of HSA or HIgG. These also demonstrate the usefulness of pulsed injection experimental technique for the optimization of ultrafiltration processes. A pulse input carrier phase ultrafiltration (CPUF) was employed to fractionate a binary protein mixture of HSA and HIgG at low NaCl concentration, showing that it is feasible to separate the binary protein at optimised conditions.

Analysis of protein transport and polarization through membranes using pulsed sample injection technique

Abstract Protein fractionation using ultrafiltration is feasible after extensive process optimization. However, conventional process optimisation methods are time consuming and require large amounts of pure proteins, which are sometimes very expensive. Pulsed sample injection ultrafiltration allows rapid determination of sieving coefficient data and may prove to be an efficient process optimisation technique. Very small amounts of pure protein are required for these experiments. A ‘quasi-steady state’ approximation is proposed for calculating the actual bulk and membrane surface concentrations from permeate concentration data. The sieving coefficient data obtained from the pulsed sample injection technique are in good agreement with data obtained from ‘steady-state’ ultrafiltration experiments. Experimental data clearly suggest that for a given permeate flux, the sieving coefficients are independent of the value of the feed concentration. The permeate concentration–time curves show that at higher permeate flux, lower time duration is required for the development of concentration polarization.

Purification of a human immunoglobulin G1 monoclonal antibody from transgenic tobacco using membrane chromatographic processes.

Efficient purification of protein biopharmaceuticals from transgenic plants is a major challenge, primarily due to low target protein expression levels, and high impurity content in the feed streams. These challenges may be addressed by using membrane chromatography. This paper discusses the use of cation-exchange and Protein A affinity-based membrane chromatographic techniques, singly and in combination for the purification of an anti-Pseudomonas aerugenosa O6ad human IgG1 monoclonal antibody from transgenic tobacco. Protein A membrane chromatography on its own was unable to provide a pure product, mainly due to extensive non-specific binding of impurities. Moreover, the Protein A membrane showed severe fouling tendency and generated high back-pressure. With cation-exchange membrane chromatography, minimal membrane fouling and high permeability were observed but high purity could not be achieved using one-step. Therefore, by using a combination of the cation-exchange and Protein A membrane chromatography, in that order, both high purity and recovery were achieved with high permeability. The antibody purification method was first systematically optimized using a simulated feed solution. Anti-P. aeruginosa human IgG1 type monoclonal antibody was then purified from transgenic tobacco juice using this optimized method.

Purification of lysozyme by microporous PVDF membrane-based chromatographic process

Abstract Microporous and macroporous membranes can be used to carry out chromatographic separation processes, thereby overcoming some of the limitations associated with packed bed columns. A commercially available polyvinylidine fluoride (PVDF) membrane was identified which could selectively bind lysozyme by a cation exchange mechanism. The PVDF membrane used was reported to be resistant to a wide variety of organic solvents, acids and bases, and could be sterilised by heat. The purification of lysozyme from chicken egg white (CEW) was carried out using this membrane. Based on this, a robust and reproducible process for manufacturing lysozyme could potentially be developed.