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.

Membrane chromatography for AAV full capsid enrichment: Process development to scale up

The recent FDA approval of several adeno-associated virus (AAV)-based gene therapies is driving demand for AAV production. One of the biggest AAV manufacturing challenges is removing “empty” capsids, which do not contain the gene of interest. Anion exchange chromatography has emerged as the leading solution for scalable full capsid enrichment. Here we develop a process for the baseline separation of empty and full AAV capsids using anion exchange membrane chromatography. This process development approach utilized AAV serotypes 8 and 9 and traverses initial screening of separation conditions up to manufacturing-scale processes. Process development of a two-step elution was performed via response surface DoE, exploring conductivity and the length of the first elution step. The results from response surfaces were used to construct statistical models of the process operating space. These models provide optimal conditions for recovery and purity, both of which can exceed 70 %. Model predictions were then validated at small scale prior to scale-up. We present the results from our scale-up purification and show that purity and yield are consistent with the results obtained from the response surface model.

Purification of phage for therapeutic applications using high throughput anion exchange membrane chromatography

As cases of multidrug resistant bacterial infections increase, scientists and clinicians around the world are increasingly turning to bacteriophages as alternatives to antibiotics. Even though our understanding of phage has increased significantly since the early days of its discovery, over a century ago, the currently used tools and technologies for phage purification for therapeutic applications are severely limited. Bacteriophages are produced by bacterial cultures, and impurities such as endotoxins must therefore be removed before clinical use. We present an anion exchange bind-and-elute membrane chromatographic method for purifying T7 bacteriophage from Escherichia coli culture supernatant that removes undesirable impurities, while ensuring a high viable phage count in the purified product. Our method does not involve the use of chemicals such as organic solvents and caesium chloride that could typically leave residual toxicity in the final product. It also does not require expensive equipment, such as an ultracentrifuge. Using our method, that is based on an in-house designed membrane module, 65% of viable T7 phage was recovered, and up to 94% endotoxins could be removed. The method, which took approximately 15 min, is rapid and scalable, and produces quite pure bacteriophage samples in a single step. It therefore potentially represents a major improvement over the status quo, and shows the way ahead for streamlining phage manufacturing for therapeutic use.

Factors affecting robustness of anion exchange chromatography: Selective retention of minute virus of mice using membrane media

Mobile and stationary phase factors were investigated in order to identify conditions for effective capture of minute virus of mice (MVM), a potential adventitious contaminant in biomanufacturing, using anion exchange membrane chromatography (AEX). The initial study was conducted for Membrane A for a range of feed conditions using bovine serum albumin (BSA) as a model protein mimicking acidic host-cell proteins (HCPs) competitive for virus binding. The effects of pH (6–8), salt concentration (0–150 mM NaCl) and level of BSA (0–10 g/L) were systematically investigated. It was found that higher BSA concentration has the most negative impact on MVM binding followed by the increased conductivity of the feed solution. The effect of pH on MVM binding is also detected but has a less impact compared to other two factors in the range of feed conditions investigated. In addition to Membrane A, three other AEX membranes (Membrane B, C and D) were investigated for MVM binding at a selected feed condition. Based on properties of the membranes investigated, it was found that ligand charge density has the most significant impact on MVM binding performance of AEX membranes from stationary phase perspective.

Design and optimization of membrane chromatography for monoclonal antibody charge variant separation.

The manufacturing scale implementation of membrane chromatography to purify monoclonal antibodies has gradually increased with the shift in industry focus toward flexible manufacturing and disposable technologies. Membrane chromatography are used to remove process-related impurities such as host cell proteins (HCPs) and DNA, leachates, and endotoxins, with improved productivity and process flexibility. However, application of membrane chromatography to separate product-related variants such as charge variants has not gained major traction due to low-binding capacity. The work reported here demonstrates that a holistic process development strategy to optimize static binding (pH and salt concentration) and dynamic process (membrane loading, flowrate, and gradient length) parameters can alleviate the capacity limitations. The study employed high throughput screening tools and scale-down membranes for intermediate and polishing purification of the model monoclonal antibody. An optimized process consisting of anion exchange and cation exchange membrane chromatography reduced the acidic variants present in Protein A eluate from 89.5% to 19.2% with 71% recovery of the target protein. The membrane chromatography process also cleared HCP to below limit of detection with 6- to 30-fold higher membrane loading, compared to earlier reported values. The results confirm that membrane chromatography is effective in separating closely related product variants when supported by a well-defined process development strategy.

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.

Novel anion exchange membrane chromatography method for the separation of empty and full adeno-associated virus.

A challenge in the production of recombinant Adeno-Associated Virus (AAV) for gene therapies is the presence of capsids that lack the required gene of interest. The impact of these empty vectors in therapies is not fully understood, however the ability to control the ratio of empty to full particles, which contain the genetic payload, is a necessary step in the purification of these viruses. In this study, a novel anion exchange chromatography elution method for enrichment of full AAV particles is demonstrated. A step gradient with small conductivity increases of around 1 mS cm-1 provides more efficient separation of empty and full AAV serotype 5 across membrane media as compared to conventional linear gradient method. The use of this approach in optimizing a simpler method for manufacturing processes and scalability to a larger chromatographic volume is explored. With this approach, the authors achieved greater than 4-fold enrichment of full capsids, to give a total of ≈50%-60% full capsids, using a 25mM Bis-Tris Propane pH 9.0 buffer system with NaCl as the eluting salt. Results suggest that this elution method can be implemented into a scalable process and can provide insight into development of elution methods for other AAV serotypes.

Empty and full separation of adeno-associated virus vectors by anion exchange membrane chromatography

Empty and full separation of adeno-associated virus vectors by anion exchange membrane chromatography

Selection of chromatographic methods for the purification of cell culture-derived Orf virus for its application as a vaccine or viral vector

In recent years, the Orf virus has become a promising tool for protective recombinant vaccines and oncolytic therapy. However, suitable methods for an Orf virus production, including up- and downstream, are very limited. The presented study focuses on downstream processing, describing the evaluation of different chromatographic unit operations. In this context, ion exchange-, pseudo-affinity- and steric exclusion chromatography were employed for the purification of the cell culture-derived Orf virus, aiming at a maximum in virus recovery and contaminant depletion. The most promising chromatographic methods for capturing the virus particles were the steric exclusion- or salt-tolerant anion exchange membrane chromatography, recovering 84 % and 86 % of the infectious virus. Combining the steric exclusion chromatography with a subsequent Capto™ Core 700 resin or hydrophobic interaction membrane chromatography as a secondary chromatographic step, overall virus recoveries of up to 76 % were achieved. Furthermore, a complete cellular protein removal and a host cell DNA depletion of up to 82 % was possible for the steric exclusion membranes and the Capto™ Core 700 combination.The study reveals a range of possible unit operations suited for the chromatographic purification of the cell culture-derived Orf virus, depending on the intended application, i.e. a human or veterinary use, and the required purity.

Adsorption of beta-lactoglobulin in anion exchange membrane chromatography versus the contacting mode and temperature

Adsorption of beta-lactoglobulin to strong anion exchange membranes was investigated using three different contacting modes (batch adsorption, radial and tangential flow), and by equilibrium adsorption isotherm, as a function of temperature. The maximum binding capacity (qmax) was: (1) higher at lower temperature for the isotherm where qmax = 1.08 mg/cm2 at 10 °C versus qmax = 0.82 mg/cm2 at 50 °C, (2) independent of temperature for batch adsorption where the average qmax = 0.82 mg/cm2, (3) higher at lower temperature for radial flow where qmax = 0.87 mg/cm2 at 15 °C versus qmax = 0.38 mg/cm2 at 50 °C. For tangential flow at 10 °C, qmax = 0.65 mg/cm2. Differences in qmax between the three contacting modes were minor compared to the differences in the mass transfer coefficients that were tenfold higher for tangential flow than for batch adsorption. This was attributed to convective flow in the membrane pores closest to the flow channel, and diffusive mass transfer in the membrane pores farthest from the flow channel. The present work is the first report of mass transfer coefficients in tangential flow adsorbers, and the first report on the effect of temperature on the binding capacity of beta-lactoglobulin to adsorptive membranes.

Removal process of prion and parvovirus from human platelet lysates used as clinical-grade supplement for ex vivo cell expansion

Background aimsPooled human platelet lysate (HPL) is becoming the new gold standard as supplement for ex vivo cell culture for clinical protocols. However, the risk of pathogen contamination of HPL increases with the platelet pool size. We hypothesized that hollow fiber anion exchange membrane chromatography using QyuSpeed D (QSD) could remove resistant and untested bloodborne pathogens, such as parvoviruses and prions, from HPL-supplemented growth media without substantially affecting their capacity to support ex vivo cell expansion. MethodsFrozen or thawed platelet concentrates were serum-converted and centrifuged for obtaining HPL that was added to various growth media (ca. 100 mL), filtered through a 0.6-mL QSD membrane and characterized for proteins, growth factors and chemical composition. Capacity to expand Chinese hamster ovary, periodontal ligament, gingival fibroblast cells and Wharton's jelly mesenchymal stromal cells was studied. Removal of porcine parvovirus (PPV) and of the 263K prion strain of hamster-adapted scrapie was studied by spiking experiments following international guidelines. ResultsQSD had minimal impact on HPL-supplemented medium composition in proteins, growth factors and chemical content, nor capacity to expand and differentiate cells. In addition, QSD could remove ≥5.58 log10 [TCID50/mL] and ≥3.72 log10 of PPV and the 263K prion, respectively. ConclusionsQSD hollow fiber chromatography can be used to improve the virus and prion safety of HPL-supplemented media to safely expand cells for clinical protocols. These data bring new perspectives for increasingly safer use of pooled HPL in cell therapy and regenerative medicine applications.

Optimization of lactoferrin and bovine serum albumin separation using ion-exchange membrane chromatography

Lactoferrin (LF), which is a high value minor whey protein, has recently received extensive attention from research scientists and industry due to its multifunction and potential therapeutic applications. In this study, the separation of two similar-sized proteins: bovine serum albumin (BSA) and LF was investigated using strong cation and anion exchange membrane chromatography (MC). Single protein and BSA–LF mixture adsorption were performed on Sartobind Q75 and S75 at pH between the LF and BSA isoelectric points. Identical breakthrough curves were obtained for both single protein and binary protein mixture, which suggests that there is no protein adsorption competition at the binding sites. The process optimization was further studied to yield optimum buffer and operating conditions. The highest BSA flux per membrane area (728.00gm−2h−1) was obtained using 100mM phosphate buffer solution at pH 6.0 on the cation exchange membrane, whereas LF was bound to the membrane with the dynamic binding capacity at 10% breakthrough (DBC10%) of about 60mg. On the anion exchange membrane, LF was collected in the effluent at the flux of 287.46gm−2h−1 using 5mM phosphate buffer at pH 6.0, while BSA was retained on the membrane with DBC10% equal to 60.96mg. The combination effect between pH change and hydrophobic interaction improved the eluted protein mass for both anion and cation exchangers. Furthermore, the completed separation cycle was operated with the Sartobind S75 device with a short process time of 34.19min and optimal LF productivity over 2628.84mgmL−1h−1. This study confirms the advantage of MC for the separation of biomolecules with similar molecular weight and different isoelectric points, such as BSA–LF mixture separation. This fast and effective protein separation method could be applied at an industry scale.

Purification of biomolecules combining ATPS and membrane chromatography

Upstream processes for production of therapeutic proteins have been innovated and fermentation processes have been adopted for the use of recombinant microorganisms with high expression, but the downstream process is still the bottleneck in the biotechnological manufacturing process. A combined process consisting of aqueous two phase extraction (ATPE) and membrane chromatography is suggested to debottleneck downstream processing. ATPE has a large capacity, but the yield of the target product is from 74% to 97%. For this reason the product of ATPE waste stream is captured by membrane chromatography. In this work the binding capacity for the protein on protein A, ion exchange and hydrophobic exchange membrane chromatography was investigated experimentally with different concentration of polyethylene glycol (PEG), salt and protein. Protein A membrane was loaded with solutions resembling waste streams of ATPE for purifying IgG. For ion exchange and hydrophobic interaction membrane chromatography, the membrane was loaded with bovine serum albumin (BSA). PEG shows no significant effect on stability and capacity of membrane process. Even for small amount of BSA/IgG and high salt concentrations membrane adsorption is applicable. In this work it is demonstrated experimentally that a total product recovery of 99.9% for the purification of monoclonal antibody is possible.

Separation and purification of linear covalently closed deoxyribonucleic acid by Q-anion exchange membrane chromatography

We have constructed an in vivo system for rapid, scalable production of linear covalently closed (LCC) DNA from precursor circular covalently closed (CCC) plasmid DNA (pDNA) that offers a stronger safety profile compared to conventional CCC pDNA vectors. In the processing of LCC DNA products from the precursor CCC pDNA, LCC minivector DNA is produced in addition to other precursor DNA species and isoforms. DNA purification by anion exchange chromatography (AEC) attains high vector purity, making it an efficient and valuable approach to purification processes for the production of clinical grade DNA. Membrane chromatography offers significant advantages over traditional column chromatography including large convective pores, higher binding capacities, high throughput, scalable purification processes, and disposability. A hydrogel-based strong Q-anion exchange membrane for anion exchange chromatography can bind DNA with high capacity and recovery upon purification. We exploited these membrane properties in the separation of DNA sizes and isoforms to purify LCC DNA. We employed a NaCl concentration gradient at varying flow rates to successfully achieve effective separation of parental supercoiled CCC pDNA from processed isogenic LCC derivatives generated by the LCC DNA vector production system. We propose that anion exchange membrane chromatography is well positioned to play an integral role in large scale LCC DNA vector purification, successfully separating vectors by DNA isoforms.

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.

Cytokine production using membrane adsorbers: Human basic fibroblast growth factor produced by Escherichia coli

AbstractBasic fibroblast growth factor (FGF‐2) is a multifunctional cytokine that regulates various cellular processes both in vitro and in vivo. FGF‐2 is extensively used in embryonic stem cell cultures since it can maintain the cells in an undifferentiated state. However, the high price of FGF‐2 has limited its application in stem cell research. Here we present a fast and efficient process for the purification of FGF‐2 from recombinant Escherichia coli cultures using reusable membrane adsorbers. A high expression level of FGF‐2 (42 mg/g dry cell) was achieved by fed‐batch cultivation of E. coli BL21(DE3). A new combination of cation exchange membrane chromatography and heparin‐sepharose affinity chromatography was used for the purification of the protein. A novel anion exchange membrane chromatography was used in the polishing step to remove endotoxins and DNA. In this new process, about 200 mg soluble FGF‐2 was yielded from 1.9 L culture broth with a purity of 98%. The purified protein was identified to be endotoxin‐free and bioactive. It was successfully tested to keep primate embryonic stem cell and human‐induced pluripotent stem cell pluripotent. Our approach, in which a controlled cultivation process is combined with an optimized fast and versatile downstreaming process, is suitable for low‐cost preparation of bioactive FGF‐2 at bench‐scale and may be beneficial to the effective production of other cytokines.

Simplified production and concentration of HIV-1-based lentiviral vectors using HYPERFlask vessels and anion exchange membrane chromatography

BackgroundDuring the past twelve years, lentiviral (LV) vectors have emerged as valuable tools for transgene delivery because of their ability to transduce nondividing cells and their capacity to sustain long-term transgene expression in target cells in vitro and in vivo. However, despite significant progress, the production and concentration of high-titer, high-quality LV vector stocks is still cumbersome and costly.MethodsHere we present a simplified protocol for LV vector production on a laboratory scale using HYPERFlask vessels. HYPERFlask vessels are high-yield, high-performance flasks that utilize a multilayered gas permeable growth surface for efficient gas exchange, allowing convenient production of high-titer LV vectors. For subsequent concentration of LV vector stocks produced in this way, we describe a facile protocol involving Mustang Q anion exchange membrane chromatography.ResultsOur results show that unconcentrated LV vector stocks with titers in excess of 108 transduction units (TU) per ml were obtained using HYPERFlasks and that these titers were higher than those produced in parallel using regular 150-cm2 tissue culture dishes. We also show that up to 500 ml of an unconcentrated LV vector stock prepared using a HYPERFlask vessel could be concentrated using a single Mustang Q Acrodisc with a membrane volume of 0.18 ml. Up to 5.3 × 1010 TU were recovered from a single HYPERFlask vessel.ConclusionThe protocol described here is easy to implement and should facilitate high-titer LV vector production for preclinical studies in animal models without the need for multiple tissue culture dishes and ultracentrifugation-based concentration protocols.

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.

Development and validation of an enzyme-linked immunosorbent assay for vitellogenin in Chinese loach ( Misgurnus angaillicaudatus)

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for the quantification of vitellogenin (Vtg) in the plasma of Chinese loach ( Misgurnus angaillicaudatus). Vtg was isolated by anion exchange membrane chromatography from the plasma of 17β-estradiol (E 2)-treated loach. Polyclonal antibody against Vtg was raised in rabbits, and the specificity of the antibody was assessed by Western blotting analysis. No cross-reactivity was observed with plasma from male loach. Plasma samples from vitellogenic females and E 2-treated males were diluted parallel with the purified Vtg standard curve in the ELISA. The detection limit of the assay was 5.7 ng ml − 1 and a working range of the standard curve was between 15.6 and 1000 ng ml − 1. The intra- and inter-assay coefficients of variations were 6.9% and 10.4%, respectively. The recovery was 104.4%. The assay was applied for measuring vitellogenin concentration in the plasma from 24 wild Chinese loach from Lianyungang, Jiangsu Province, in October 2003. It was found that, in addition to females (plasma Vtg concentration ranging from 518.4 μg ml − 1 to 1922.3 μg ml − 1), male loach showed low plasma Vtg—from undetectable (two fish) to 2.9 ± 0.7 μg ml − 1 (mean ± S.D.). The results indicate that this method provides a valuable tool for the study of estrogenic effect in Chinese loach.