A simple micro-volume ultrafiltration method for protein concentration, desalting, buffer exchang... more A simple micro-volume ultrafiltration method for protein concentration, desalting, buffer exchange and purification.
Encyclopedia of Membrane Science and Technology, 2013
Membranes are widely used in biotechnology with the main applications being focused around biopro... more Membranes are widely used in biotechnology with the main applications being focused around bioproduct purification. Membranes are also used for laboratory filtration and analysis as components of membrane bioreactors, for biomedical applications such as dialysis and blood oxygenation, and for drug delivery. This article reviews the application of membranes in biological separation processes. The membranes techniques commonly used in these applications are microfiltration, ultrafiltration, and membrane chromatography. This article first reviews the common features and then discusses them individually, highlighting working principles and specific applications. Keywords: membrane; purification; biotechnology; ultrafiltration; microfiltration; membrane chromatography
N-terminus-specific PEGylation was used to produce mono-PEGylated lysozyme. However, some di- and... more N-terminus-specific PEGylation was used to produce mono-PEGylated lysozyme. However, some di- and tri-PEGylated proteins were also produced due to side chain reaction. The reaction products were characterized by chromatographic and electrophoretic methods. Commercial cation exchange membrane Sartobind S was used for chromatographic purification of PEGylated lysozyme, the basis of separation being the shielding of protein charge by PEG. Using the membrane chromatographic method, lysozyme and mono-, di-, and tri-PEGylated lysozyme could be resolved into separate peaks. Increasing the superficial velocity during chromatographic separation from 24 cm/h to 240 cm/h increased both protein binding capacity and resolution due to enhancement of protein mass transfer coefficient.
ABSTRACT Protein-A affinity chromatography is the standard capture technique used for purificatio... more ABSTRACT Protein-A affinity chromatography is the standard capture technique used for purification of monoclonal antibodies (mAbs). A major problem with this technique is its inability to remove monoclonal antibody aggregates from the monomeric form of the antibody. Moreover, the elution of mAbs from a protein-A column is carried out using acidic conditions which in turn could accelerate the formation of antibody aggregates and causes leaching of protein-A from its supporting media. These impurities have to be removed using appropriate separation methods before the mAb can be used for therapeutic applications. There is currently a limited repertoire of polishing techniques which simultaneously remove both mAb aggregates and protein-A. In this paper, we describe a polishing method based on hydrophobic interaction membrane chromatography for simultaneously removing these impurities. The flow-through mode was used whereby monomeric mAb flowed through a membrane stack while impurities were retained by reversible adsorption. These impurities were subsequently eluted by lowering the salt concentration and the membrane stack was thus regenerated. The operating conditions for this method were systematically optimized using pure mAb, aggregates, protein-A, and mAb/protein-A complexes. The mechanisms by which aggregates and leached protein-A were simultaneously removed are hypothesized.
ABSTRACT The current standard technique for evaluating the macromolecule sieving behavior for the... more ABSTRACT The current standard technique for evaluating the macromolecule sieving behavior for the ultrafiltration (UF) of dextran solutes relies on steady-state operation, at constant trans-membrane pressure and it provides no information on membrane fouling or concentration polarization. This paper describes the use of a pulsed sample injection technique (PSIT), a technique that obviates these limitations, to characterize UF of dextran solutes. Using neutral dextran solutions and a well characterized thin-film composite (TFC) membrane, PSIT yields apparent sieving coefficients (Sapp) that accurately follow trends from previous studies where the standard technique was used. To demonstrate the versatility of PSIT as a characterization method, a series of experiments were done with charged dextran solutes. As compared to neutral dextran, the Sapp value for polycationic diethylaminoethyl (DEAE)-dextran and polyanionic dextran sulfate sodium salt (DSSS) were lower than neutral dextran due to the combined steric and electrostatic repulsion effects. The degree of solute–membrane interaction was assessed using solute-transport times and indicated significant interactions between DEAE-dextran and the membrane but minimal interactions between DSSS and the membrane. Permeate refractive index profiles were used to estimate the amount of solute bound to the membrane and good correlation was found between the amount of DEAE-dextran bound and the change in membrane permeability. The charged dextran sieving behavior was explained according to solute–solute and solute–membrane interactions using the exclusive information on solute transport kinetics and fouling behavior that is available from the PSIT. Overall, the PSIT is shown to be an excellent characterization technique for dextran UF with TFC membranes.
ABSTRACT This paper discusses a novel approach for predicting permeate flux decline in constant p... more ABSTRACT This paper discusses a novel approach for predicting permeate flux decline in constant pressure ultrafiltration of protein solutions. A constant pressure process is assumed to be made up of a large number of small, sequential, constant flux ultrafiltration steps: the flux decreasing due to fouling and other related factors at the end of each step. The advantage of this approach is that constant flux ultrafiltration is easier to study, characterize, and model than constant pressure ultrafiltration. Consequently model parameters can be obtained in reliable and reproducible manner. Constant pressure ultrafiltration is dynamic in nature since both the magnitude of osmotic back-pressure and the extent of membrane fouling decrease as the permeate flux decreases with time. The proposed model takes into consideration the interplay between permeate flux, concentration polarization, and membrane fouling. The model demonstrates that the initial rapid flux decline is due to a combination of concentration polarization and membrane fouling while during the remaining part of the process, the effect of concentration polarization becomes negligible. The model also shows that concentration polarization affects the initial flux decline only at higher transmembrane pressures. This model which was validated using experimental data is conceptually simpler than other available models and easy to use. In addition to its value as a predictive tool it would particularly be useful for deciding appropriate start-up conditions in ultrafiltration processes.
Transgenic plants offer a promising system for large-scale production of therapeutic proteins suc... more Transgenic plants offer a promising system for large-scale production of therapeutic proteins such as monoclonal antibodies (mAbs). This paper describes a membrane-based process suitable for purification of a humanized mAb expressed in tobacco. Most monoclonal ...
This paper demonstrates the feasibility of using cascade ultrafiltration systems run at constant ... more This paper demonstrates the feasibility of using cascade ultrafiltration systems run at constant flux for efficient purification of lysozyme from chicken egg white. Integration of high-resolution purification with concentration is a key requirement in the purification of lysozyme, which is present at very low purity and concentration in egg white. A three-stage cascade ultrafiltration system, which focused on purification alone,
Gas sparging is recognized as an effective way to increase permeate flux in ultrafiltration proce... more Gas sparging is recognized as an effective way to increase permeate flux in ultrafiltration processes. This paper discusses the mechanism of flux enhancement in ultrafiltration processes by gas sparging, in the special case of upward slug flow in tubular membrane module. An ...
Membrane chromatography can overcome some of the problems associated with packed bed chromatograp... more 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.
Journal of Biomaterials Science, Polymer Edition, 2008
Biocompatible polymer-based materials whose properties respond to environmental stimuli are of gr... more Biocompatible polymer-based materials whose properties respond to environmental stimuli are of great value in biomedical and biotechnological applications (e.g., controlled release of drugs and sensitive reactants, bioseparations, spontaneous adjusting of flow of liquids including flow control in microfluidic devices). This study describes preparation and properties of biocompatible poly(N-vinyllactam)-based environment responsive composite membranes. The non-charged network N-vinyl lactam polymers form hydrogels that can exhibit swelling and de-swelling behaviour and, thus, regulate porosity of the membrane. The first part of the study has involved investigation of bulk polymerization reactivity of N-vinylcaprolactam (VCL)-based systems. VCL has been combined with up to 20 mol% of N-vinylpyrrolidone (N-vinylbutyrolactam). These synthetic processes have been investigated as thermal polymerizations (with and without a free-radical initiator) and photopolymerizations. Subsequently, the most reactive systems have been used to prepare composite materials by polymerization-modifying borosilicate microfibre membranes. The modified membranes have been characterized by the net mass gain, infrared spectra, and change in permeability in response to changes in ionic strength of the aqueous media. The permeability experiments have been carried out at constant fluxes and the resultant changes in trans-membrane pressure observed. Those membranes that are ionic-strength responsive could potentially be used for bio-separations and other types of biomedical and biotechnological applications.
Journal of Biomaterials Science, Polymer Edition, 2009
Novel concepts are proposed for interpenetrating polymer networks (IPNs) that can be used in biom... more Novel concepts are proposed for interpenetrating polymer networks (IPNs) that can be used in biomaterials responsive to multiple types of environmental stimuli. The IPNs can be a route to the tunable biomaterials whose chemical structure and/or morphology can be adjusted to the type of task to be performed. The significance of utilizing new types of processes in polymer chemistry such as the Suzuki polycondensation and living/controlled radical polymerizations to the possibility of extending/modifying component networks in the IPNs and fine-tuning their properties is emphasized. Incorporation of nanofillers into IPNs and application of monomers other than traditional alkylacrylamides as a way to enhance versatility of IPNs are also considered.
A simple micro-volume ultrafiltration method for protein concentration, desalting, buffer exchang... more A simple micro-volume ultrafiltration method for protein concentration, desalting, buffer exchange and purification.
Encyclopedia of Membrane Science and Technology, 2013
Membranes are widely used in biotechnology with the main applications being focused around biopro... more Membranes are widely used in biotechnology with the main applications being focused around bioproduct purification. Membranes are also used for laboratory filtration and analysis as components of membrane bioreactors, for biomedical applications such as dialysis and blood oxygenation, and for drug delivery. This article reviews the application of membranes in biological separation processes. The membranes techniques commonly used in these applications are microfiltration, ultrafiltration, and membrane chromatography. This article first reviews the common features and then discusses them individually, highlighting working principles and specific applications. Keywords: membrane; purification; biotechnology; ultrafiltration; microfiltration; membrane chromatography
N-terminus-specific PEGylation was used to produce mono-PEGylated lysozyme. However, some di- and... more N-terminus-specific PEGylation was used to produce mono-PEGylated lysozyme. However, some di- and tri-PEGylated proteins were also produced due to side chain reaction. The reaction products were characterized by chromatographic and electrophoretic methods. Commercial cation exchange membrane Sartobind S was used for chromatographic purification of PEGylated lysozyme, the basis of separation being the shielding of protein charge by PEG. Using the membrane chromatographic method, lysozyme and mono-, di-, and tri-PEGylated lysozyme could be resolved into separate peaks. Increasing the superficial velocity during chromatographic separation from 24 cm/h to 240 cm/h increased both protein binding capacity and resolution due to enhancement of protein mass transfer coefficient.
ABSTRACT Protein-A affinity chromatography is the standard capture technique used for purificatio... more ABSTRACT Protein-A affinity chromatography is the standard capture technique used for purification of monoclonal antibodies (mAbs). A major problem with this technique is its inability to remove monoclonal antibody aggregates from the monomeric form of the antibody. Moreover, the elution of mAbs from a protein-A column is carried out using acidic conditions which in turn could accelerate the formation of antibody aggregates and causes leaching of protein-A from its supporting media. These impurities have to be removed using appropriate separation methods before the mAb can be used for therapeutic applications. There is currently a limited repertoire of polishing techniques which simultaneously remove both mAb aggregates and protein-A. In this paper, we describe a polishing method based on hydrophobic interaction membrane chromatography for simultaneously removing these impurities. The flow-through mode was used whereby monomeric mAb flowed through a membrane stack while impurities were retained by reversible adsorption. These impurities were subsequently eluted by lowering the salt concentration and the membrane stack was thus regenerated. The operating conditions for this method were systematically optimized using pure mAb, aggregates, protein-A, and mAb/protein-A complexes. The mechanisms by which aggregates and leached protein-A were simultaneously removed are hypothesized.
ABSTRACT The current standard technique for evaluating the macromolecule sieving behavior for the... more ABSTRACT The current standard technique for evaluating the macromolecule sieving behavior for the ultrafiltration (UF) of dextran solutes relies on steady-state operation, at constant trans-membrane pressure and it provides no information on membrane fouling or concentration polarization. This paper describes the use of a pulsed sample injection technique (PSIT), a technique that obviates these limitations, to characterize UF of dextran solutes. Using neutral dextran solutions and a well characterized thin-film composite (TFC) membrane, PSIT yields apparent sieving coefficients (Sapp) that accurately follow trends from previous studies where the standard technique was used. To demonstrate the versatility of PSIT as a characterization method, a series of experiments were done with charged dextran solutes. As compared to neutral dextran, the Sapp value for polycationic diethylaminoethyl (DEAE)-dextran and polyanionic dextran sulfate sodium salt (DSSS) were lower than neutral dextran due to the combined steric and electrostatic repulsion effects. The degree of solute–membrane interaction was assessed using solute-transport times and indicated significant interactions between DEAE-dextran and the membrane but minimal interactions between DSSS and the membrane. Permeate refractive index profiles were used to estimate the amount of solute bound to the membrane and good correlation was found between the amount of DEAE-dextran bound and the change in membrane permeability. The charged dextran sieving behavior was explained according to solute–solute and solute–membrane interactions using the exclusive information on solute transport kinetics and fouling behavior that is available from the PSIT. Overall, the PSIT is shown to be an excellent characterization technique for dextran UF with TFC membranes.
ABSTRACT This paper discusses a novel approach for predicting permeate flux decline in constant p... more ABSTRACT This paper discusses a novel approach for predicting permeate flux decline in constant pressure ultrafiltration of protein solutions. A constant pressure process is assumed to be made up of a large number of small, sequential, constant flux ultrafiltration steps: the flux decreasing due to fouling and other related factors at the end of each step. The advantage of this approach is that constant flux ultrafiltration is easier to study, characterize, and model than constant pressure ultrafiltration. Consequently model parameters can be obtained in reliable and reproducible manner. Constant pressure ultrafiltration is dynamic in nature since both the magnitude of osmotic back-pressure and the extent of membrane fouling decrease as the permeate flux decreases with time. The proposed model takes into consideration the interplay between permeate flux, concentration polarization, and membrane fouling. The model demonstrates that the initial rapid flux decline is due to a combination of concentration polarization and membrane fouling while during the remaining part of the process, the effect of concentration polarization becomes negligible. The model also shows that concentration polarization affects the initial flux decline only at higher transmembrane pressures. This model which was validated using experimental data is conceptually simpler than other available models and easy to use. In addition to its value as a predictive tool it would particularly be useful for deciding appropriate start-up conditions in ultrafiltration processes.
Transgenic plants offer a promising system for large-scale production of therapeutic proteins suc... more Transgenic plants offer a promising system for large-scale production of therapeutic proteins such as monoclonal antibodies (mAbs). This paper describes a membrane-based process suitable for purification of a humanized mAb expressed in tobacco. Most monoclonal ...
This paper demonstrates the feasibility of using cascade ultrafiltration systems run at constant ... more This paper demonstrates the feasibility of using cascade ultrafiltration systems run at constant flux for efficient purification of lysozyme from chicken egg white. Integration of high-resolution purification with concentration is a key requirement in the purification of lysozyme, which is present at very low purity and concentration in egg white. A three-stage cascade ultrafiltration system, which focused on purification alone,
Gas sparging is recognized as an effective way to increase permeate flux in ultrafiltration proce... more Gas sparging is recognized as an effective way to increase permeate flux in ultrafiltration processes. This paper discusses the mechanism of flux enhancement in ultrafiltration processes by gas sparging, in the special case of upward slug flow in tubular membrane module. An ...
Membrane chromatography can overcome some of the problems associated with packed bed chromatograp... more 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.
Journal of Biomaterials Science, Polymer Edition, 2008
Biocompatible polymer-based materials whose properties respond to environmental stimuli are of gr... more Biocompatible polymer-based materials whose properties respond to environmental stimuli are of great value in biomedical and biotechnological applications (e.g., controlled release of drugs and sensitive reactants, bioseparations, spontaneous adjusting of flow of liquids including flow control in microfluidic devices). This study describes preparation and properties of biocompatible poly(N-vinyllactam)-based environment responsive composite membranes. The non-charged network N-vinyl lactam polymers form hydrogels that can exhibit swelling and de-swelling behaviour and, thus, regulate porosity of the membrane. The first part of the study has involved investigation of bulk polymerization reactivity of N-vinylcaprolactam (VCL)-based systems. VCL has been combined with up to 20 mol% of N-vinylpyrrolidone (N-vinylbutyrolactam). These synthetic processes have been investigated as thermal polymerizations (with and without a free-radical initiator) and photopolymerizations. Subsequently, the most reactive systems have been used to prepare composite materials by polymerization-modifying borosilicate microfibre membranes. The modified membranes have been characterized by the net mass gain, infrared spectra, and change in permeability in response to changes in ionic strength of the aqueous media. The permeability experiments have been carried out at constant fluxes and the resultant changes in trans-membrane pressure observed. Those membranes that are ionic-strength responsive could potentially be used for bio-separations and other types of biomedical and biotechnological applications.
Journal of Biomaterials Science, Polymer Edition, 2009
Novel concepts are proposed for interpenetrating polymer networks (IPNs) that can be used in biom... more Novel concepts are proposed for interpenetrating polymer networks (IPNs) that can be used in biomaterials responsive to multiple types of environmental stimuli. The IPNs can be a route to the tunable biomaterials whose chemical structure and/or morphology can be adjusted to the type of task to be performed. The significance of utilizing new types of processes in polymer chemistry such as the Suzuki polycondensation and living/controlled radical polymerizations to the possibility of extending/modifying component networks in the IPNs and fine-tuning their properties is emphasized. Incorporation of nanofillers into IPNs and application of monomers other than traditional alkylacrylamides as a way to enhance versatility of IPNs are also considered.
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Papers by Raja Ghosh