Ion-exchange Membrane Chromatography Research Articles

An unconventional strategy for purifying recombinant SARS-CoV-2 spike protein

The soluble domain of the trimeric SARS-CoV-2 spike protein is a promising candidate for a COVID-19 vaccine. Purification of this protein from mammalian cell culture supernatant using conventional resin-based chromatography is challenging as its large size (∼550 kDa) restricts its access and mobility within the pores of the resin particles. This reduces binding capacity and process robustness very significantly as extremely low flow rates need to be used during purification. Convection-based ion-exchange membrane chromatography has been found to be suitable in this respect. However, the high ionic strength of mammalian cell culture supernatant makes it difficult to bind this protein on charged membranes without dilution with a suitable buffer. An unconventional strategy involving size-exclusion chromatography as the first step, followed by cation exchange membrane chromatography as the second step is proposed in this paper. In the size exclusion chromatography step, the spike protein is excluded from the pores and can therefore be isolated in the void volume fraction. This step removes small molecule impurities and also serves as a desalting and buffer exchange step, making the partially purified material suitable for the cation exchange membrane chromatography step. The proposed process is variant-independent, fast and scalable and addresses some of the challenges associated with the currently used purification methods.

Towards next generation high throughput ion exchange membranes for downstream bioprocessing: A review

Membrane chromatography is recognised as a potential solution to streamline downstream processing for protein purification, where ion exchange membrane chromatography (IEMC) as a polishing step to remove impurities has been successfully demonstrated in small scales. Despite limited commercial adoption in large-scale production, the concept of IEMC attracts many interests and tremendous progress is made. To fill the review gap for advancements in the last decade, this article provides a timely analysis on key performance-determining aspects in IEMC systems. Modern laboratory-made membranes with polymeric chains of tuneable surface area and charge allow for high binding capacity (up to 10-fold higher than that of traditional resins) while simultaneously mitigating the loss of permeance due to the introduction of grafted layers up to 40%. Nevertheless, robust evaluation are yet to be conducted. Despite making equal contribution to binding, the review on process-related work was supported by only <1/3 of the cited articles, where a transition of empirical to mechanistic models was identified, enabling rationale system design and upscaling. The use of molecular simulation into binding studies reveals the roles of membrane properties but limited work was found. While highlighting disconnection between academic and commercial efforts, research gaps for future work were identified.

Comparison of the performance of anion exchange membrane materials for adenovirus purification using laterally-fed membrane chromatography

Viruses are an emerging class of biotherapeutics used to treat a variety of diseases. Ion-exchange membrane chromatography is commonly used for virus purification, with the Mustang Q (Pall) and Sartobind Q (Sartorius) being two of the most widely used strong anion-exchange membranes. These membranes are made from different materials and have different pore sizes, and while previous studies have attempted to compare their performance in various virus purification applications, those studies have been hindered by the additional fact that these membranes are sold in different module formats. Thus, in order to establish a direct comparison of the capabilities and purification performance of anion exchange membrane materials, we first fabricated equivalent laterally-fed membrane chromatography (LFMC) devices containing either 1 mL of Mustang Q membrane or 1 mL of Sartobind Q membrane and then compared their performance based on residence times, the elution patterns of pre-purified biomolecules, and the recovery and purity of adenovirus from a cell lysate. Under the same linear gradient elution conditions, purified adenovirus eluted approximately one membrane volume earlier for the Sartobind Q membrane; a similar result was obtained during purification of adenovirus from infected suspension 293 cells. While the total recovery of adenovirus was the same for both membranes (~60%), the Sartobind Q membrane provided a much greater removal of DNA impurities in the virus containing elution fractions.

CDNA microarray assays to evaluate immune responses following intracranial injection of baculoviral vectors in non-human primates.

Recombinant insect baculoviral vectors efficiently transduce several types of cells in the brain and can possibly be used in gene therapy for brain disorders. However, together with contaminating insect cell proteins, they trigger immune responses that might damage host brain cells. To substantially reduce unwanted immune responses due to the insect cell impurities, we purified and concentrated baculoviral vectors by combining an ion-exchange membrane chromatography method with high-speed centrifugation and demonstrated reduced immune responses of the vector preparations in the mouse brain. To verify the suitability of using these viral vectors for gene therapy strategies in the brain, we evaluated immune reactions and vector toxicity upon acute administration of baculoviral vectors into the brains of cynomolgus macaques. We demonstrated that the virus inoculation caused no abnormalities to non-human primates but induced host anti-viral responses in the brain. With global cellular gene expression profiling, using cDNA microarray technology, we detected that the affected genes were mainly associated with innate immunity, involving the genes of RIG-1-like receptor signaling pathway as the major interferon production pathway. These findings in non-human primates, which share close physiological and genomic similarities with man, may better mimic the responses to baculoviral transduction in the human brain and should be useful in guiding rational therapeutic applications of baculoviral vectors to treat brain disorders.

Modeling and simulation of anion-exchange membrane chromatography for purification of Sf9 insect cell-derived virus-like particles

Recombinant protein-based virus-like particles (VLPs) are steadily gaining in importance as innovative vaccines against cancer and infectious diseases. Multiple VLPs are currently evaluated in clinical phases requiring a straightforward and rational process design. To date, there is no generic platform process available for the purification of VLPs. In order to accelerate and simplify VLP downstream processing, there is a demand for novel development approaches, technologies, and purification tools. Membrane adsorbers have been identified as promising stationary phases for the processing of bionanoparticles due to their large pore sizes. In this work, we present the potential of two strategies for designing VLP processes following the basic tenet of ‘quality by design’: High-throughput experimentation and process modeling of an anion-exchange membrane capture step. Automated membrane screenings allowed the identification of optimal VLP binding conditions yielding a dynamic binding capacity of 5.7mg/mL for human B19 parvovirus-like particles derived from Spodoptera frugiperda Sf9 insect cells. A mechanistic approach was implemented for radial ion-exchange membrane chromatography using the lumped-rate model and stoichiometric displacement model for the in silico optimization of a VLP capture step. For the first time, process modeling enabled the in silico design of a selective, robust and scalable process with minimal experimental effort for a complex VLP feedstock. The optimized anion-exchange membrane chromatography process resulted in a protein purity of 81.5%, a DNA clearance of 99.2%, and a VLP recovery of 59%.

Ion-Exchange Membrane Chromatography as an Alternative Method of Separation of Potato y Virus

Abstract Procedures of separation of virus particles from a plant material are multistage. Furthermore often they are difficult in terms of methodology and require use of expensive, highly specialist equipment and yield of separation is often low. The antigen obtained is often degraded and contains admixtures of other proteins. Therefore, generation of high quality and specificity antibodies based on such antigen is very difficult and quality of the antibodies has impact on reliability, sensitivity and unambiguity of results of immunodiagnostic tests (e.g. ELISA) that are currently conventionally used to detect vegetable viruses. In this study three conventionally-performed methods of separation of potato virus Y (PVY) were compared and a method of separation based on membrane chromatography, as an alternative separation technique, has been presented. It has been demonstrated that in proper process conditions good quality virus preparation can be obtained.

Simple method for purification of enterotoxigenic Escherichia coli fimbriae

Enterotoxigenic Escherichia coli (ETEC) are endemic pathogens in the developing world. They frequently cause illness in travelers, and are among the most prevalent causes of diarrheal disease in children. Pathogenic ETEC strains employ fimbriae as adhesion factors to bind the luminal surface of the intestinal epithelium and establish infection. Accordingly, there is marked interest in immunoprophylactic strategies targeting fimbriae to protect against ETEC infections. Multiple strategies have been reported for purification of ETEC fimbriae, however none is ideal. Purification has typically involved the use of highly virulent wild-type strains. We report here a simple and improved method to purify ETEC fimbriae, which was applied to obtain two different Class 5 fimbriae types of clinical relevance (CFA/I and CS4) expressed recombinantly in E. coli production strains. Following removal from cells by shearing, fimbriae proteins were purified by orthogonal purification steps employing ultracentrifugation, precipitation, and ion-exchange membrane chromatography. Purified fimbriae demonstrated the anticipated size and morphology by electron microscopy analysis, contained negligible levels of residual host cell proteins, nucleic acid, and endotoxin, and were recognized by convalescent human anti-sera.

Enablers for QbD implementation: Mechanistic modeling for ion-exchange membrane chromatography

Monoclonal antibody (mAb) based therapeutic products presently dominate the pipelines of most biopharmaceutical companies. Due to the large manufacturing requirements for these products, higher productivity is desirable and membrane chromatography has emerged a possible solution. In this study, a mechanistic model has been proposed for membrane chromatography for predicting product breakthrough at different mAb concentrations. First, Linear, Freundlich, Langmuir, and Temkin isotherms have been evaluated for their suitability with respect to modeling of adsorption of a monoclonal antibody therapeutic on an ion- exchange membrane adsorber. Next, pseudo-first-order, pseudo-second-order, modified Langmuir and steric mass action (SMA) adsorption kinetic models have been evaluated and compared. The results indicate that a combination of Langmuir isotherm and modified Langmuir or the pseudo-first order kinetic model provides the best prediction of mAb adsorption on ion-exchange membrane adsorbers. The final model could be successfully used to understand the adsorption kinetics between complex protein and ligand within the membrane device. The proposed approach would be of significant value to the biotech industry in ensuring the better development of future membrane adsorber devices with optimal sizing.

Synthesis and functionality of proteinacious nutraceuticals from casein whey—A clean and safe route of valorization of dairy waste

Due to stricter environmental legislation, coagulation and precipitation technologies have been practiced for whey protein sedimentation from a long prior. Nowadays, in pharmaceutical and dairy industries, individual whey proteins have been isolated by size-exclusion membrane separation technology, ion-exchange membrane chromatography, gel-permeation chromatography, affinity-peptide ligand chromatography and electro-chromatography. Furthermore, whey protein-derived peptides have been synthesized by conventional batch or catalyst-immobilized packed-bed bioreactors or membrane-assigned bioreactor with high throughput and purity, considering raw whey or isolated individual protein as feedstock. Different types of enzymes, such as trypsin, chymotrypsin, alcalase, pepsin and chymosin, have been popularly used for enzymatic hydrolysis of whey proteins. Whey proteins, such as β-lactoglobulin, α-lactalbumin, bovine serum albumin, lactoperoxidase, lactoferrin and immunoglobulins, and enzymatic hydrolysis of whey proteins have their unique physiological, medicinal, functional and nutritional values. It could be expected that without going for mere treatment of whey (coagulation or sedimentation of whole protein), synthesis of proteinacious bioactives (separation of individual whey proteins and synthesis of their derivatives) will lead to zero effluent discharge within a domain of whey upgradation. The present review pretends to highlight the different process-related technological aspects for the synthesis of different proteinacious bioactives from whey, as well as their functionality.

Simultaneous clarification of Escherichia coli culture and purification of extracellularly produced penicillin G acylase using tangential flow filtration and anion-exchange membrane chromatography (TFF-AEMC)

Downstream purification often represents the most cost-intensive step in the manufacturing of recombinant proteins since conventional purification processes are lengthy, technically complicated, and time-consuming. To address this issue, herein we demonstrated the simultaneous clarification and purification of the extracellularly produced recombinant protein by Escherichia coli using an integrated system of tangential flow filtration and anion exchange membrane chromatography (TFF-AEMC). After cultivation in a bench-top bioreactor with 1L working volume using the developed host/vector system for high-level expression and effective secretion of recombinant penicillin G acylase (PAC), the whole culture broth was applied directly to the established system. One-step purification of recombinant PAC was achieved based on the dual nature of membrane chromatography (i.e. microfiltration-sized pores and anion-exchange chemistry) and cross-flow operations. Most contaminant proteins in the extracellular medium were captured by the anion-exchange membrane and cells remained in the retentate, whereas extracellular PAC was purified and collected in the filtrate. The batch time for both cultivation and purification was less than 24h and recombinant PAC with high purity (19U/mg), yield (72% recovery), and productivity (41mg of purified PAC per liter of culture) was obtained. Due to the nature of the non-selective protein secretion system and the versatility of ion-exchange membrane chromatography, the developed system can be widely applied for effective production and purification of recombinant proteins.

Purification of a recombinant baculovirus of Autographa californica M nucleopolyhedrovirus by ion exchange membrane chromatography

Significant progress in the application of viral vectors for gene delivery into mammalian cells and the use of viruses as biopesticides requires downstream processing that can satisfy application-specific demands on performance. In the present work the stability and ion exchange membrane chromatography of a recombinant of Autographa californica M nucleopolyhedrovirus is studied. To adjust the degree of purification the effect of ionic conductivity or pH on the viral infectivity was assessed (0.77-78.00mS/cm, pH 3-8). Infectivity decreased rapidly by several orders of magnitude at below 5mS/cm (i.e., 0.49MPa osmotic pressure change) or at below pH 5.5 (rationalized with particle aggregation). The virus was concentrated and purified via adsorption (0.2-1.1×10(16)pfu/m(3) chromatographic bed volume, 0.6-1.1×10(12)pfu/m(2) membrane area facing the incident fluid flow) and elution at pH 6.1 and 6.35mS/cm from three strong anion exchange membranes. Virus recovery and concentration in accord with the volume reduction were obtained using a polyether sulfone-based membrane with quaternary ammonium ligands. The level of host cell protein (down to below the detection limit) and suspended DNA (below 93pg DNA per 10(6)pfu) are reported for each membrane employed, for the purpose of comparability, under equal adsorption or elution conditions respectively.

Zonal rate model for stacked membrane chromatography part II: Characterizing ion‐exchange membrane chromatography under protein retention conditions

The Zonal Rate Model (ZRM) has previously been shown to accurately account for contributions to elution band broadening, including external flow nonidealities and radial concentration gradients, in ion-exchange membrane (IEXM) chromatography systems operated under nonbinding conditions. Here, we extend the ZRM to analyze and model the behavior of retained proteins by introducing terms for intra-column mass transfer resistances and intrinsic binding kinetics. Breakthrough curve (BTC) data from a scaled-down anion-exchange membrane chromatography module using ovalbumin as a model protein were collected at flow rates ranging from 1.5 to 20 mL min(-1). Through its careful accounting of transport nonidealities within and external to the membrane stack, the ZRM is shown to provide a useful framework for characterizing putative protein binding mechanisms and models, for predicting BTCs and complex elution behavior, including the common observation that the dynamic binding capacity can increase with linear velocity in IEXM systems, and for simulating and scaling separations using IEXM chromatography. Global fitting of model parameters is used to evaluate the performance of the Langmuir, bi-Langmuir, steric mass action (SMA), and spreading-type protein binding models in either correlating or fundamentally describing BTC data. When combined with the ZRM, the bi-Langmuir, and SMA models match the chromatography data, but require physically unrealistic regressed model parameters to do so. In contrast, for this system a spreading-type model is shown to accurately predict column performance while also providing a realistic fundamental explanation for observed trends, including an observed increase in dynamic binding capacity with flow rate.

Impact of ligand density on the optimization of ion‐exchange membrane chromatography for viral vector purification

The effect of ligand density on anion-exchange membrane chromatography (AEXmc) for the purification of recombinant baculoviruses (rBVs), potential viral vectors in clinical applications, is studied by surface plasmon resonance on customized AEX surfaces and gradient elution experiments on Sartobind D membrane prototypes with different diethylamine ligand densities, complemented by dynamic light scattering analysis for estimation of the hydrodynamic particle size of the various biologics. A chromatographic-column model based on the steric mass action model of ion exchange is employed to analyze the gradient-elution AEXmc experiments, extrapolate the results to other operating conditions, and provide directions for process improvement. Although counterintuitively, the experimental evidence provided in this study shows that the lowering of ligand density is beneficial for rBV purification by AEXmc in bind-and-elute-mode, because it decreases the residual concentrations of host cell protein, dsDNA, and non-infective rBVs in the eluted product cut, and increases the overall yield by roughly 20% over current standard values. Overall, we present a case study on how rational design can streamline downstream process development.

Purification of monoclonal antibody from tobacco extract using membrane-based bioseparation techniques

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 antibody purification schemes rely on the use of Protein A as the affinity ligand for antibody capture. The main objective of our work was to develop non-Protein A-based purification methods to avoid some of the problems and limitations associated with this ligand, e.g. cost, immunotoxicity, and antibody aggregation during elution. Ion exchange membrane chromatography (IEMC) was used for primary capture and preliminary purification of the mAb from tobacco juice. Hydrophobic interaction membrane chromatography (HIMC) was then used for high-resolution purification, followed by ultrafiltration for polishing, desalting and buffer exchange. Using this scheme, both high mAb purity (single peak in size exclusion chromatogram, i.e., ca. 100% purity) and high recovery (77% of mAb spiked into the tobacco extract) were achieved. Membrane chromatography is generally considered unsuitable for resolving bound proteins by gradient elution and is therefore commonly used in the bind and elute mode with a single-step change of mobile phase. We show that the gradient elution process in the HIMC step can be optimized to increase the resolution and thereby obtain product of high purity.

Ion-Exchange Membrane Chromatography Method for Rapid and Efficient Purification of Recombinant Baculovirus and Baculovirus gp64 Protein

Significant progress in the application of baculoviral vectors for gene delivery into mammalian cells calls for the development of powerful methods for virus purification and concentration. We report here a novel and efficient method based on membrane chromatography to prepare baculoviral stocks. On a strong cation-exchange membrane unit, baculovirus in insect cell culture supernatants was captured at a flow rate of 3 ml/min and efficiently eluted at the same flow rate with a physiological buffer containing 150 mM NaCl as a desorption reagent. The procedure allowed for a final recovery of 78% of infective viral particles from the original supernatant and 30-fold enrichment. The high purity of the viral preparation was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Baculovirus gp64 proteins could be purified by the same method, indicating the importance of the protein in mediating the binding of baculovirus to the cation-exchange membrane. The method developed should be suitable for preparing baculoviral stocks, and probably other gp64-pseudotyped viral vectors, for gene therapy applications.

Studies on the fractionation of β-lactoglobulin from casein whey using ultrafiltration and ion-exchange membrane chromatography

The objective of this work is to obtain a relative separation of β-lactoglobulin (β-LG) from whey protein concentrate by fractionation of protein using two-stage ultrafiltration (UF) with 30 and 10 kg mol −1 molecular weight cut-off (MWCO) flat-disk membranes in stirred rotating disk module followed by ion-exchange membrane chromatography (IEMC) using Vivapure™ Q Mini-H column. Prior to UF, centrifugation, microfiltration and a four-stage discontinuous diafiltration (DD) were carried out to obtain whey protein concentrate from raw casein whey. Centrifugation and microfiltration were carried out to remove the major foulants like colloidal matters, suspended casein particles and lipid. DD using 5 kg mol −1 polyethersulphone (PES) membrane with volume concentration ratio (VCR) 2 in each stage was employed with an objective to enrich the whey proteins by removing most of lactose, minerals and salts. The effects of various parameters, like solution pH, trans-membrane pressure (TMP), stirrer speed and membrane rotation speed on UF flux and rejection were studied thoroughly. A 36% higher flux was obtained with a feed pH of 2.8 compared to that at pH of 5.6 at a fixed TMP of 5.0 × 10 5 Pa which has been explained with the help of the effect of pH on monomer–dimer equilibrium of β-LG and prevailing protein charge. A 33% enhancement of flux was observed at 1 min with membrane disc rotating at 300 rpm compared to the corresponding value of stationary membrane due to reduction in concentration polarization, the main limiting phenomenon for flux decline. A suitable loading buffer pH was investigated during IEMC runs so as to get a facilitated transport of β-LG over α-LA through the strong anion exchanger membrane. An 87.6% purity of β-LG (on total protein basis) was obtained in the filtrate of ion-exchange membrane chromatography.

793. Adenopure LS™ Large Scale Kit for Purification of Recombinant Adenovirus

Recombinant adenovirus vectors are still the preferred choice for many types of gene therapy. However, the availability of a process to purify larger quantities of these vectors at high purity and on a consistent basis for screening and small animal experiments remains a challenge. Classic 2X CsCl gradients are labor-intensive and time-consuming. Alternatives to CsCl-gradient purification based on ion-exchange membrane chromatography have been developed, but results are not consistent and the procedures cumbersome. We report the development of the Adenopure LS™ Large Scale Adenovirus Purification Kit. This kit is membrane adsorber-based and produces high-quality adenovirus in as little as two hours.

Overexpression of Functional Hydrolase Domain of Rat Liver 10-Formyltetrahydrofolate Dehydrogenase inEscherichia coli

Rat liver 10-formyltetrahydrofolate dehydrogenase (FDH) is a tetrameric enzyme composed of four identical 902-amino-acid-residue (99 kDa) monomers. We expressed the enzyme and its 310-amino-acid-residue amino-terminal domain, which is 10-formyltetrahydrofolate hydrolase, inEscherichia coliBL21 (DE3) cells using the pRSET expression vector. We removed the entire translated region of the vector including the polyhistidyl tag and the recombinant proteins were expressed, not as a fusion constructs, but as unmodified sequences. The expressed full-length enzyme was found to be an insoluble protein and was not purified and characterized, while the amino-terminal domain was expressed as a soluble protein possessing hydrolase activity. The recombinant amino-terminal domain was purified in one step on a DEAE MemSep 1000 HP Ion-Exchange Membrane Chromatography Cartridge (Millipore) using a ConSep LC100 chromatographic system (Millipore). The chromatography gave a homogenous and active preparation of the recombinant protein with a yield of about 2 mg per 100 ml of bacterial culture. Kinetic parameters of the hydrolase reaction displayed by the amino-terminal domain expressed inE. coliwere similar to those of the recombinant full-length enzyme and its amino-terminal domain previously expressed in insect cells. The purified recombinant enzyme remained active for at least 4 weeks at 4°C. These results show that the hydrolase amino-terminal domain of FDH can be overexpressed as a functional enzyme inE. colicells and purified in one step by a simple chromatographic procedure.