Purification of equine IgG using membrane based enhanced hybrid bioseparation technique: a potential method for manufacturing hyperimmune antibody

Hyperimmune equine IgG is widely used as antivenom and anti-rabies agents. This article discusses a membrane based enhanced hybrid bioseparation technique for efficient and scalable purification of equine immunoglobulin G (IgG) from horse serum. This technique is an improved version of a standard hybrid bioseparation technique developed within our group earlier for fractionation of human plasma proteins (Ghosh. 2004. J Membr Sci 237: 109-117). In the presence of a high antichaotropic salt concentration, equine IgG is selectively and reversibly captured within a stirred cell membrane module from horse serum, partly due to precipitation and microfiltration, and partly due to hydrophobic interaction based membrane adsorption, while the impurities are washed out from the device. The reversibly sequestered IgG is then released by lowering the salt concentration which favor both dissolution of the precipitated IgG and desorption of the membrane bound IgG. The enhanced hybrid bioseparation technique improves the IgG recovery from the membrane module by switching from a stirring to non-stirring mode during the IgG release phase. It also reduces membrane fouling by an appropriate pH switch. The effects of operating conditions on equine IgG capture were first systematically studied. The enhanced hybrid bioseparation technique was followed by an ultrafiltration step to remove ammonium sulfate and low molecular weight impurities. The equine IgG purity obtained under optimized conditions was 88% and its recovery was over 90%, both being significantly higher than corresponding values obtained using currently used purification techniques.     

Optimized recovery of monoclonal antibodies from transgenic goat milk by microfiltration

The Predictive Aggregate Transport Model for microfiltration is used in combination with optimum fluid mechanics and electrostatics to maximize recovery of a heterologous immunoglobulin (IgG) from transgenic goat milk. The optimization algorithm involved varying pH (6.8-9), transmembrane pressure (2-4.5 psi), milk feed concentration (1-2X), membrane module type (linear vs. helical design), and axial velocity (Reynolds number: 830-1170). Operation in the pressure-dependent regime at low uniform transmembrane pressures (approximately 2 psi) using permeate circulation in co-flow, at the pI of the protein (9 in this case) was used to increase IgG recovery from less than 1% to over 95%. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and attenuated total reflection Fourier transform infrared spectroscopy of the microfiltration permeate samples confirmed that all the fat globules and most of the casein micelles were retained in the MF membrane whereas a large amount of the target IgG was transported through the membrane. Transmembrane pressure and hence permeation flux was kept low (approximately 15 lmh) to maximize IgG membrane transport and thus recovery, due to a sparse deposit on the membrane which facilitated high solute transport. Next, an analytical method was used to optimize the diafiltration process using the aggregate transport model, experimental target protein sieving coefficients and permeation flux (Baruah and Belfort, 2003). The methodology reported here should be generalizable to the recovery of target proteins found in other complex suspensions of biological origin using the microfiltration process.     

Effects of intermolecular thiol-disulfide interchange reactions on bsa fouling during microfiltration

Several studies have shown that one of the critical factors governing protein fouling of microfiltration membranes is the presence of denaturedand/or aggregated protein in the bulk solutions. Experiments were performed to evaluate the role of intermolecular disulfide interchange reactionson protein aggregation and membrane fouling during stirred cell microfiltration of bovine serum albumin (BSA). The flux decline during BSA filtration was quite dramatic due to the formation of a protein deposit thatfully covered the membrane pores. This flux decline could be completely eliminated by capping the free sulfhydryl group present on the BSA with eithera carboxymethyl or cysteinyl group, demonstrating the critical importance of this free thiol in the intermolecular aggregation reactions and, in turn, protein fouling. BSA aggregation during storage could be reduced by the addition of metal chelators (EDTA and citrate) or dithiothreitol, orby storage at lower pH (7.0) these solutions all had a significantly lower rate of fouling upon subsequent filtration. This behavior is completely consistent with the known chemistry of the thiol-disulfide interchange reaction, demonstrating that an understanding of these intermolecular (aggregation) reactions can provide a rational framework for the analysis and control of protein fouling in these membrane systems. (c) 1994 John Wiley & Sons, Inc.     

Optimized removal of soluble host cell proteins for the recovery of met-human growth hormone inclusion bodies from Escherichia coli cell lysate using crossflow microfiltration

Cross-flow membrane microfiltration was used under optimal conditions to recover met-growth hormone inclusion bodies (IBs) from Escherichia coli cell lysate by removal of the host-cell (bacterial) proteins (HCP) under minimal fouling conditions. This is the first step of a two-step process in which the goal was to isolate IBs at high yield from the HCP. These undesired soluble HCP were removed by passing them through the membrane while retaining the insolubles, including the aggregated IBs. Experiments were conducted at constant permeate flux with flat-sheet membranes of different pore sizes and chemistry, with feeds of varying pH and ionic strengths to determine the optimum combination for HCP removal. Diafiltration, the washing away of impurities with protein-free buffer, was then employed to ensure removal of the host cell proteins at the optimum conditions. About 90% removal of the HCP was obtained in about 5 diavolumes, maintaining high protein transmission and low membrane fouling.     

Membrane fouling by cell-protein mixtures: in situ characterisation using multi-photon microscopy

Fouling of the membrane by cell and protein mixtures can result in severe flux declines, leading to the eventual need to clean or replace the membrane. In this study multi-photon microscopy, a fluorescence-based technique is used to 3-D image in situ the fouling of microfiltration membranes by suspensions containing combinations of washed yeast, bovine serum albumin (BSA) and ovalbumin. Appropriate fluorescent labelling allows the three foulant species to be clearly identified. Images correlate well with filtration data and clearly show the cake of yeast cells capturing protein aggregates. The proteins exhibited very different filtration behaviour. When filtering washed yeast together with ovalbumin and/or a 50:50 mixture by mass of BSA and ovalbumin, the ovalbumin fouling dominates the system. Capture of aggregates by the cake did not reduce fouling of the membrane by the protein and increased the resistance of the cake. For mixtures of BSA and washed yeast, the presence of a cake of yeast cells did reduce fouling of the membrane by the protein, however, the extra resistance due to the cake resulted in a flux lower than that when filtering BSA alone.     

Purification of monoclonal antibodies derived from transgenic goat milk by ultrafiltration

With the goal of recovering heterologous immunoglobulin (IgG), which comprises 10-15% of the total proteins, from transgenic goat milk at 80% yield and 80% purity, we have developed and tested a two-step membrane isolation and purification process. In the first step, reported earlier by Baruah and Belfort, microfiltration was used to fractionate the milk proteins and recover > 90% of the original IgG at a purity of about 15-20% in the permeate stream. Here, we focus on ultrafiltration (UF) to increase the purity of the target protein to 80%, while maintaining a relatively high IgG yield (80%). Tangential flow UF experiments in diafiltration mode were conducted with 100 kDa cellulosic membranes to evaluate the optimal pH, ionic strength, and uniform transmembrane pressure (TMP). The TMP was kept uniform by permeate circulation in co-flow mode. The traditional approach of conducting the UF process close to the pI of the predominant whey proteins (15-40 kDa, pI 5.2), to transmit these proteins while retaining heterologous IgG (155 kDa), could not be applied here because of precipitation of residual casein at pH values lower than 8.5. Instead, the packing characteristics of the cake layer on the membrane wall, as elucidated in the Aggregate Transport Model presented by Baruah et al. was utilized to achieve a selectivity of > 15, which was sufficient to meet the stated goals of purity and yield for this difficult separation. This combined process is expected to reduce the load on subsequent purification and polishing steps for eventual therapeutic use.     

A new integrated membrane filtration and chromatographic device

To improve protein separation, a novel integrated device combining membrane filtration and chromatography has been developed. The device basically consists of a hollow fiber filtration module whose shell side is filled with chromatographic resin beads. However, there is an essentially impermeable coated zone near the hollow fiber module outlet. The integrated device enjoys the advantages of both membrane filtration and chromatography; it also allows one to load the chromatographic media directly from the fermentation broth or lysate and separate the adsorbed proteins through the subsequent elution step in a cyclic process. Interfacial polymerization was carried out to coat the bottom section of the hollow fiber membrane; the rest of the hollow fiber membrane remained unaffected. Myoglobin (Mb) and alpha-lactalbumin (alpha-LA) were primarily used as model proteins in a binary mixture; binary mixtures of Mb and bovine serum albumin (BSA) were also investigated. Separation behaviors of binary protein mixtures were studied in devices having either an ultrafiltration (UF) or a microfiltration (MF) membrane. Experimental results show that the breakthrough time and the protein loading capacities were dramatically improved after introducing the impermeable coating in both UF and MF modules. For a synthetic yeast fermentation broth feed, four loading-washing-elution-reequilibration-based cyclic runs for separation of Mb and alpha-LA were performed in the device using a MF membrane with a coated zone without cleaning in between. The Mb and alpha-LA elution profiles for the four consecutive runs were almost superimposable. Due to lower transmembrane flux in this device plus the periodical washing-elution during the chromatographic separation, fouling was not a problem, unlike in conventional microfiltration.     

An integrated process for mammalian cell perfusion cultivation and product purification using a dynamic filter

In the present work, a dynamic filter was employed to develop an integrated perfusion/purification process. A recombinant CHO cell line producing a human anti-HIV IgG was employed in the experiments. In the first part of this work, the dynamic filter was fitted with conventional microfiltration membranes and tested as a new external cell retention device for perfusion cultivations. The filter was connected to a running perfusion bioreactor and operated for approximately 400 h at an average cell concentration of 10 million cells mL(-)(1), whereby cell viability remained above 90% and no problems of sterility were experienced. In the second part of this work, the dynamic filter was employed to simultaneously carry out cell separation and product purification, using membrane adsorbers containing Protein A affinity ligands. An automated system was built, which integrated the features of an automated perfusion bioreactor and of a liquid chromatography system. The IgG was continuously adsorbed onto the affinity membranes and was periodically recovered through elution cycles. After connection of the filter, the system was operated for approximately 300 h, whereby three elution cycles were carried out. No progressive increase in transmembrane pressure was observed, indicating no membrane fouling problems, and the IgG was recovered practically free of contaminants in a 14-fold concentrated form, indicating that the integrated, one-step perfusion/purification process developed during this work is a promising alternative for the production of biologicals derived from mammalian cells.     

Integration of a perfusion reactor and continuous precipitation in an entirely membrane‐based process for antibody capture

Continuous precipitation coupled with continuous tangential flow filtration is a cost-effective alternative for the capture of recombinant antibodies from crude cell culture supernatant. The removal of surge tanks between unit operations, by the adoption of tubular reactors, maintains a continuous harvest and mass flow of product with the advantage of a narrow residence time distribution (RTD). We developed a continuous process implementing two orthogonal precipitation methods, CaCl₂ precipitation for removal of host-cell DNA and polyethylene glycol (PEG) for capturing the recombinant antibody, with no influence on the glycosylation profile. Our lab-scale prototype consisting of two tubular reactors and two stages of tangential flow microfiltration was continuously operated for up to 8 days in a truly continuous fashion and without any product flow interruption, both as a stand-alone capture and as an integrated perfusion-capture. Furthermore, we explored the use of a negatively charged membrane adsorber for flow-through anion exchange as first polishing step. We obtained a product recovery of approximately 80% and constant product quality, with more than two logarithmic reduction values (LRVs) for both host-cell proteins and host-cell DNA by the combination of the precipitation-based capture and the first polishing step.     

Dean vortex membrane microfiltration and diafiltration of rBDNF E. coli inclusion bodies

Cross-flow microfiltration (CMF) and diafiltration were used to concentrate and purify recombinant Brain-Derived Neutrophic Factor (rBDNF) inclusion bodies from an E. coli cell suspension and a homogenized E. coli cell suspension (homogenate/lysate). Although these processes have been tested industrially in pilot scale with conventional linear membrane microfiltration modules, their performances were severely limited due to membrane fouling. The purpose of this work was to determine whether Dean vortex microfiltration with controlled centrifugal instabilities (Dean vortices produced in helical flow) could be used to improve filtration performance over that observed with conventional linear cross-flow microfiltration (CMF). For the microfiltration experiments with the feeds containing cell and homogenate suspensions, improvements in flux of about 50 and 70%, respectively, were obtained with the helical module as compared with that obtained with the linear module. For diafiltration with the homogenate suspension as feed, solute transport (as measured by mass) was from 100 to 40% higher after 40 and 100 min, respectively, with the helical module as compared with that obtained with the linear module. In the presence of the neutral surfactant, Tween 20, solute transport for diafiltration was at least 25 times higher during the first 10 min of operation and 100% higher after 300 min with the helical module as compared with that obtained with the linear module. Clearly, improved filtration performance, a purer and more concentrated product, and substantial savings can be expected with the new Dean vortex filters.     

Separation of growth-stimulating activity of BSA fraction V from the bulk of albumin using Heparin Sepharose Chromatography

Bovine serum albumin (BSA) is a potential source of biological contamination in cell culture medium. The aim of this work was to attempt to replace BSA in low serum and serum-free medium (SFM). BSA fraction V was subjected to a variety of processes in order to determine if the growth promoting activity observed for NRK cells could be extracted from the BSA molecule. These included solvent extractions, diafiltration, reverse phase HPLC and affinity chromatography using heparin sepharose. Solvent extraction and diafiltration failed to remove the activity from the BSA. Affinity chromatography using heparin sepharose indicated that all of the activity observed with BSA was retained in the 0.5 M NaCl fraction and was associated with less than 3% of the original protein. The major protein band in the 0.5 M NaCl fraction had the same apparent molecular weight as albumin (as seen by SDS-PAGE and analytical reverse phase HPLC). Unlike the untreated BSA, the 0.5 M NaCl fraction was partially susceptible to proteolytic digestion and to variations in pH.Abbreviations HS heparin sepharose - DHS donor horse serum - SFM serum free-medium     

Hybridoma cultures using porcine serum not containing immunoglobulin and dialysis fractions

The whole swine serum was treated with ammonium sulphate to precipitate immunoglobulins. The remained IgG was removed with the use of protein A-sepharose. The hybridoma cells producing monoclonal antibodies to lambda phage (class IgG) were cultured in Dalbecco's modified Eagle medium with addition of a 5% whole swine serum or of a treated unwhole one (final concentration of the protein being 3 mg/ml). Upon these conditions, hybridoma cells had similar growth rate and population density (1-1.3 X 10(6) cells/ml). Maximal antibody concentration was almost similar (80-90 mcg/ml). Purity of a sample of monoclonal antibodies isolated by the method of chromatography with the use of protein A-sepharose from supernatant containing the unwhole serum was no less than 99%, whereas it was considerably lower (12-15%) in the case of the whole serum.     

Ultrafiltration characteristics of immunobiological preparations

The removal of ammonium sulfate from the bulk product of fermented antitoxic serum by continuous diafiltration was not accompanied by changes in the stability of the solution. To concentrate immunoglobulin, eluted from DEAE cellulose, by diafiltration, the stabilization of the solution by adding sodium chloride at high concentration was necessary. The use of membranes purchased from different manufacturers and having similar selectivity characteristics permitted obtaining transfer factor preparations somewhat differing in their biological activity. The process of ultrafiltration, carried out in the atmosphere of compressed carbon dioxide, made it possible to obtain such preparations from donor blood plasma.     

Continuous capture of recombinant antibodies by ZnCl2 precipitation without polyethylene glycol

The capture of recombinant antibodies from cell culture broth is the first critical step of downstream processing. We were able to develop a precipitation‐based method for the capture and purification of monoclonal antibodies based on divalent cations, namely ZnCl₂. Traditional precipitation processes have to deal with high dilution factors especially for resolubilization and higher viscosity due to the use of PEG as precipitation or co‐precipitation agent. By the use of the crosslinking nature of divalent cations without the use of PEG, we kept viscosity from the supernatant and resolubilization dilution factors very low. This is especially beneficial for the solid–liquid separation for the harvest and wash of the precipitate in continuous mode. For this harvest and wash, we used tangential flow filtration that benefits a lot from low viscosity solutions, which minimizes the membrane fouling. With this precipitation based on ZnCl_2, we were able to implement a very lean and efficient process. We demonstrated precipitation studies with three different antibodies, Adalimumab, Trastuzumab, and Denosumab, and a continuous capture case study using tangential flow filtration for precipitate recovery. In this study, we achieved yields of 70%.     

Flux enhancement for membrane filtration of bacterial suspensions using high-frequency backpulsing

A promising method for reducing membrane fouling during crossflow microfiltration of biological suspensions is backpulsing. Very short backpulses (0.1-1.0 s) have been used to increase the net flux for washed bacterial suspensions and whole bacterial fermentation broths. The net fluxes under optimum backpulsing conditions for the washed bacteria are approximately 10-fold higher than those obtained during normal crossflow microfiltration operation, whereas only a 2-fold improvement in the net flux is achieved for the fermentation broths. A theory is presented that is based on external fouling during forward filtration and nonuniform cleaning of the membrane during reverse filtration. The model contains an adjustable parameter which is a measure of the cleaning efficiency during backpulsing; the cleaning efficiency found by fitting the model to the experiments increases with increasing frequency and duration of the backpulses. The theory predicts an optimum backpulsing frequency, as was observed experimentally. An economic analysis shows that crossflow microfiltration with backpulsing has lower costs than centrifugation, rotary vacuum filtration, and crossflow microfiltration without backpulsing.     

Membrane separation of protein precipitates: Studies with cross flow in hollow fibers

Hollow fiber ultrafiltration and microfiltration membranes are examined for the processing of isoelectric soya protein precipitate suspensions. A model based on the various resistances to permeate flux is used to describe membrane performance. The main resistance to permeate flux is due to the interaction between the active membrane and the soluble and precipitated protein; that is, as compared with resistances due to the active membrane itself or the membrane support structure, or arising from concentrated soluble or precipitated protein layers over the membrane surface. Soluble protein rejection and precipitate mean particle diameter are correlated with observed values of this main resistance.In contract to the ultrafiltration of soluble proteins, the flux rates observed when processing protein precipitate suspensions under a similar range of operating conditions do not approach a limiting value with increased transmembrane pressure. At high protein concentrations, greater flux rates may be achieved for precipitated as compared with soluble proteins. The use of a microfiltration membrane does not give further improvement in flux rate; this may be attributed to problems of pore fouling with precipitate particles.     

Purification and properties of buffalo serum albumin

Based on a detailed study of the solubility of serum albumin, a procedure for its purification by selective ammonium sulphate precipitation has been developed. Using buffalo serum, first extraneous proteins were precipitated by making the serum 2.26 M saturated with ammonium sulphate at pH 7.0 and then albumin was precipitated from the supernatant at 1.9 M ammonium sulphate concentration at pH 4.2. The overall yield of serum albumin thus isolated was about 55% with a purity of 97%. The protein preparation gave a single nearly symmetrical peak on Sephadex G-100 column and virtually a single band on polyacrylamide gel electrophoresis in the presence and absence of SDS. Buffalo serum albumin has a molecular weight of 69,000 Da. The hydrodynamic properties such as Stoke's radius (3.70 nm), diffusion coefficient (6.03 X 10(-7) cm2/s) and frictional ratio (1.36) obtained by analytical gel chromatography, bilirubin binding characteristics and its interaction with anti-bovine serum albumin antiserum suggest that buffalo serum albumin is very similar to BSA in its molecular properties.     

Performance of a new protein A affinity membrane for the primary recovery of antibodies

Recovery of antibodies with Protein A affinity chromatography columns has become the standard for the biotechnology industry. Membrane affinity chromatography has not yet experienced extensive application due to the lower capacity of membrane supports compared to chromatographic beads. In this work, new affinity membranes endowed with an interesting binding capacity for human IgG are studied in view of their application in the capturing step of a monoclonal antibody production process. The membranes have been extensively tested with pure IgG solutions and with a cell culture supernatant containing IgG1. The effects of feed flow rate and IgG concentration on the separation performances have been studied in detail, considering in particular binding capacity, selectivity and recovery. These new high capacity affinity membranes appear good candidates to avoid the throughput limitations and other well-known drawbacks of traditional bead-based chromatographic columns.     

Removal of microbial indicators from municipal wastewater by a membrane bioreactor (MBR)

The impact of removable and irremovable fouling on the retention of viral and bacterial indicators by the submerged microfiltration membrane in an MBR pilot plant was evaluated. Escherichia coli, sulphite-reducing Clostridium spores, somatic coliphages and F-specific RNA bacteriophages were used as indicators. The membrane demonstrated almost complete removal of E. coli and sulphite-reducing Clostridium spores. However, there was no correlation with membrane fouling. The phage removal varied in accordance with the irremovable fouling, rising from 2.6 to 5.6 log₁₀ units as the irremovable fouling increased (measured by the change in the transmembrane pressure). In contrast, removable fouling did not have any effect on the retention of viruses by the membrane. These results indicate that irremovable membrane fouling may affect the removal efficiency of MBRs and, therefore, their capacity to ensure the required microbiological standards for the permeate achieved.     

Selective precipitation and purification of monovalent proteins using oligovalent ligands and ammonium sulfate

This paper describes a method for the selective precipitation and purification of a monovalent protein (carbonic anhydrase is used as a demonstration) from cellular lysate using ammonium sulfate and oligovalent ligands. The oligovalent ligands induce the formation of protein-ligand aggregates, and at an appropriate concentration of dissolved ammonium sulfate, these complexes precipitate. The purification involves three steps: (i) the removal of high-molecular-weight impurities through the addition of ammonium sulfate to the crude cell lysate; (ii) the introduction of an oligovalent ligand and the selective precipitation of the target protein-ligand aggregates from solution; and (iii) the removal of the oligovalent ligand from the precipitate by dialysis to release the target protein. The increase of mass and volume of the proteins upon aggregate formation reduces their solubility, and results in the selective precipitation of these aggregates. We recovered human carbonic anhydrase, from crude cellular lysate, in 82% yield and 95% purity with a trivalent benzene sulfonamide ligand. This method provides a chromatography-free strategy of purifying monovalent proteins--for which appropriate oligovalent ligands can be synthesized--and combines the selectivity of affinity-based purification with the convenience of salt-induced precipitation.