Separation & Purification Reviews, 2016
Over the last years, aqueous two-phase systems regained an increasing interest due to their poten... more Over the last years, aqueous two-phase systems regained an increasing interest due to their potential in the downstream processing of biomolecules. After many years with only a few articles published, a lot of effort and work has been put into studying these systems for partitioning of a range of molecules including proteins, organic low-molecular weight compounds or metal ions. Although many research articles and very interesting reviews have been published in this area, there seems to be a lack of a background review on ATPS partitioning fundamentals. In this article, partitioning theories and main effects of several important factors in partitioning are extensively reviewed. These are complemented with and motivated by 5-years trends established based on research articles published over the same period.
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Biotechnology Journal, 2015
For half a century aqueous two-phase systems (ATPSs) have been applied for the extraction and pur... more For half a century aqueous two-phase systems (ATPSs) have been applied for the extraction and purification of biomolecules. In spite of their simplicity, selectivity, and relatively low cost they have not been significantly employed for industrial scale bioprocessing. Recently their ability to be readily scaled and interface easily in single-use, flexible biomanufacturing has led to industrial re-evaluation of ATPSs. The purpose of this review is to perform a SWOT analysis that includes a discussion of: (i) strengths of ATPS partitioning as an effective and simple platform for biomolecule purification; (ii) weaknesses of ATPS partitioning in regard to intrinsic problems and possible solutions; (iii) opportunities related to biotechnological challenges that ATPS partitioning may solve; and (iv) threats related to alternative techniques that may compete with ATPS in performance, economic benefits, scale up and reliability. This approach provides insight into the current status of ATPS as a bioprocessing technique and it can be concluded that most of the perceived weakness towards industrial implementation have now been largely overcome, thus paving the way for opportunities in fermentation feed clarification, integration in multi-stage operations and in single-step purification processes.
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Biotechnology Journal, 2016
Aqueous two-phase extraction (ATPE) is a biocompatible liquid-liquid (L-L) separation technique t... more Aqueous two-phase extraction (ATPE) is a biocompatible liquid-liquid (L-L) separation technique that has been under research for several decades towards the purification of biomolecules, ranging from small metabolites to large animal cells. More recently, with the emergence of rapid-prototyping techniques for fabrication of microfluidic structures with intricate designs, ATPE gained an expanded range of applications utilizing physical phenomena occurring exclusively at the microscale. Today, research is being carried simultaneously in two different volume ranges, mL-scale (microtubes) and nL-scale (microchannels). The objective of this review is to give insight into the state of the art at both microtube and microchannel-scale and to analyze whether miniaturization is currently a competing or divergent technology in a field of applications including bioseparation, bioanalytics, enhanced fermentation processes, catalysis, high-throughput screening and physical/chemical compartmentalization. From our perspective, both approaches are worthy of investigation and, depending on the application, it is likely that either (i) one of the approaches will eventually become obsolete in particular research areas such as purification at the preparative scale or high-throughput screening applications; or (ii) both approaches will function as complementing techniques within the bioanalytics field.
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Pharmaceutical Bioprocessing, 2013
Downstream processing is currently the major bottleneck for bioproduct generation. In contrast to... more Downstream processing is currently the major bottleneck for bioproduct generation. In contrast to the advances in fermentation processes, the tools used for downstream processes have struggled to keep pace in the last 20 years. Purification bottlenecks are quite serious, as these processes can account for up to 80% of the total production cost. Coupled with the emergence of new classes of bioproducts, for example, virus-like particles or plasmidic DNA, this has created a great need for superior alternatives. In this review, improved downstream technologies, including aqueous two-phase systems, expanded bed adsorption chromatography, convective flow systems, and fibre-based adsorbent systems, have been discussed. These adaptive methods are more suited to the burgeoning downstream processing needs of the future, enabling the cost-efficient production of new classes biomaterials with a high degree of purity, and thereby hold the promise to become indispensable tools in the pharmaceutical and food industries.
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Biotechnology Journal, 2013
An aqueous two-phase extraction (ATPE) process based on a PEG/phosphate system was developed for ... more An aqueous two-phase extraction (ATPE) process based on a PEG/phosphate system was developed for the capture of human immunoglobulin G and successfully applied to a Chinese hamster ovary and a PER.C6® cell supernatant. A continuous ATPE process incorporating three different steps (extraction, back-extraction, and washing) was set up and validated in a pump mixer-settler battery. Most of the higher molecular weight cell supernatant impurities were removed during the extraction step, while most of the lower molecular weight impurities were removed during the subsequent steps. A global recovery yield of 80% and a final protein purity of more than 99% were obtained for the IgG purification from a CHO cell supernatant, representing a 155-fold reduction in the protein/IgG ratio. For the purification of IgG from a PER.C6® cell supernatant, a global recovery yield of 100%, and a host cell protein purity were attained, representing a 22-fold reduction in the host cell protein/IgG ratio. These results, thus, open promising perspectives for the application of the developed ATPE process as a platform for the capture of antibodies. In fact, this new process has shown the ability to successfully recover and purify different antibodies from distinct cell culture supernatants. This technology can also overcome some of the limitations encountered using the typical chromatographic processes, besides inherent advantages of scalability, process integration, capability of continuous operation, and economic feasibility.
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Separation & Purification Technology, 2017
Monoclonal antibodies (mAbs) are currently the most important class of recombinant protein therap... more Monoclonal antibodies (mAbs) are currently the most important class of recombinant protein therapeu-tics in the biotechnological and biopharmaceutical industry with more than 250 therapeutic mAbs currently undergoing clinical trials. High titer producing cultures and complex mixtures containing high cell densities, together with an increasing growing demand for highly pure mAbs is making recovery and purification processes hot targets for improvement and opens important technological challenges in mAbs manufacturing platforms. This work explores the use of an affinity dual ligand based on a choline binding polypeptide tag (C-LytA) fused to the synthetic antibody binding Z domain (LYTAG-Z) in aqueous two-phase systems (ATPS) composed of phase forming polymers able to bind to the choline binding site of C-LytA (polyethy-lene glycol-PEG-and thermosensitive polymers-EOPO) for mAbs selective extraction. Integration of harvesting and ATPS affinity extraction steps were evaluated with ATPS proving to be an alternative strategy for integrating the clarification and the primary recovery of mAbs. An extraction yield of 89% and a clarification higher than 95% were achieved using a system composed of 7% PEG 3350 and 6% dextran 500,000.
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The purification of monoclonal antibodies anti-CD34 produced in hybridoma cells was accomplished ... more The purification of monoclonal antibodies anti-CD34 produced in hybridoma cells was accomplished by aqueous two phase extraction, using an integrated process that allowed to clarify and partially purify the produced mAb in just one step. The feasibility of using polyethylene-glycol (PEG)/dextran systems was studied at different ionic strengths (0-300 mM NaCl) and at different pH values (pH 3,4 and 7). The effect of molecular weight (MW) of PEG (3350 and 6000 Da) was also evaluated. For all the conditions studied, it was observed that antibodies partition preferentially to the PEG-rich phase, cells to the interface and soluble proteins to the bottom dextran-rich phase. The best recovery yield was obtain with an ATPS composed by 7% PEG 6000 Da, 5% dextran 500,000 Da, 150 mM NaCl at pH 3. In this system, it was possible to recover 84 +/- 6.5% IgG with 0.1 +/- 0.2 % of cells in the top phase. (C) 2014 Elsevier B.V. All rights reserved.
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Revista Mexicana de Ingeniería Química, 2014
Antibodies play an important role in both medicine and analytical biotechnology. Recently, the de... more Antibodies play an important role in both medicine and analytical biotechnology. Recently, the demand for monoclonal antibodies (mAbs) has been increasing exponentially, since their potential for therapeutic use was disclosed and improved techniques to produce them at higher titers have become available. Although upstream processing of mAbs has suffered a tremendous improvement, in the last decades, the downstream processing has been considered the bottleneck in providing antibodies at reliable costs, being, in fact, the major cost factor with 50 to 80% of total production costs. Therefore, alternative methods for mAbs's purification may be required. Aqueous two-phase systems (ATPS) have been described, for the last decade, as a viable alternative to the current establish platform, since clarification, concentration and purification can be achieved in just one step using a biocompatible environment. Therefore, the use of ATPS for integrative purification of mAbs it will be reviewed.
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Computer Aided Chemical Engineering, 2007
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Multimodal Chromatography by Ana Azevedo
The commercial potential of monoclonal antibodies (mAbs) has been continuously increasing during ... more The commercial potential of monoclonal antibodies (mAbs) has been continuously increasing during the last years alongside with the number of approved mAb-based drugs and clinical trials. Despite their effectiveness and safety, the general access to this class of biopharmaceuticals is barred by high selling prices. Downstream processing is now considered the bottleneck in the manufacturing of mAbs. Therefore, the design of novel and economic operations and their implementation in the current technology platforms constitutes a pressing need. This review provides an insight into the current state-of-the-art in mAbs purification, focusing on multimodal chromatography as one of the viable options to upgrade the established purification train.
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Separation and Purification Technology, 2016
This work addresses the feasibility of scaling-up phenylboronic acid (PBA) chromatography for the... more This work addresses the feasibility of scaling-up phenylboronic acid (PBA) chromatography for the purification of human mAbs directly from Chinese Hamster Ovary cell culture supernatants. Column performance optimization regarding superficial velocity and feed volume was performed with the best results being achieved with superficial velocities of 5.1, 15.3 and 25 cm/min in the adsorption, wash and elution steps, respectively. The column volume was scale-up 100 times from 0.4 cm 3 (laboratory scale) to 4, 16 and 40 cm 3 (preparative scale). This 100-fold scale-up was successfully achieved with a recovery yield of 97.7% and a protein purity of 82.6%. Following purification, purified IgG fractions were characterized in terms of isoelectric point, size, CHO proteins and genomic DNA content. Overall results suggest that using ProSep Ò-PB for mAbs purification is simple, reproducible, robust and scalable without compromising the target molecule integrity and purity.
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In this work, phenylboronic acid (PBA) was thoroughly investigated as a synthetic ligand for the ... more In this work, phenylboronic acid (PBA) was thoroughly investigated as a synthetic ligand for the purification of an immunoglobulin G (IgG) from a clarified cell supernatant from Chinese Hamster Ovary (CHO) cell cultures. In particular, the study was focused on the development of a washing step and in the optimization of the elution step using a serum containing supernatant. From the different conditions tested, best recoveries - 99% - and purifications - protein purity of 81% and a purification factor of 16 out of a maximum of 20 - were achieved using 100 mM D-sorbitol in 10 mM Tris-HCl as washing buffer and 0.5 M D-sorbitol with 150 mM NaCl in 10 mM Tris-HCl as elution buffer. The purification outcome was also compared with protein A chromatography that revealed a recovery of 99%, 87% protein purity and 29 out of a maximum of 33 purification factor. Following the main purification, purified IgG was characterized in terms of isoelectric point, size and activity. In the end, a proof of concept was performed using two different mAbs from serum-free CHO cell cultures.
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Journal of Molecular Recognition, 2010
In this work, we investigated the feasibility of using phenyl boronate (PB) chromatography for th... more In this work, we investigated the feasibility of using phenyl boronate (PB) chromatography for the direct capture of monoclonal antibodies from a CHO cell supernatant. Preliminary results, using pure protein solutions have shown that PB media can bind to human antibodies, not only at strong alkaline conditions but also at acidic pH values. In fact, antibodies have been found to bind in the pH range 5.5–8.5. On the other hand, insulin and human serum albumin did not bind at alkaline pH but at lower pH, which reflects the importance of non-specific interactions with the matrix. Different binding and eluting buffers were evaluated for the capture of immunoglobulin G (IgG) from a CHO cell supernatant and the most promising results were obtained using 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid at pH 8.5 as binding buffer and 1.5 M Tris–HCl as eluting buffer. Using a step elution, all IgG was recovered in the elution pool with a maximum purification factor of 56. A gradient elution allowed a further increase of the final purity, yet achieving a slightly lower yield. IgG recovery was around 85% and the purification factor was 76. The highest purity was obtained when the pH of the cell supernatant feed was previously adjusted to 8.5. Starting from an initial protein purity of 1.1% and high-performance liquid chromatography (HPLC) purity of 2.2%, after PB adsorption, a final protein purity of 85% and a HPLC purity of 88% was achieved.
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Biotechnology Journal, 2015
Phenylboronate chromatography (PBC) has been applied for several years, however details regarding... more Phenylboronate chromatography (PBC) has been applied for several years, however details regarding the mechanisms of interactions between the ligand and biomolecules are still scarce. The goal of this work is to investigate the various chemical interactions between proteins and their ligands, using a protein library containing both glycosylated and nonglycosylated proteins. Differences in the adsorption of these proteins over a pH range from 4 to 9 were related to two main properties: charge and presence of glycans. Acidic or neutral proteins were strongly adsorbed below pH 8 although the uncharged trigonal form of phenylboronate (PB) is less susceptible to forming electrostatic and cis-diol interactions with proteins. The glycosylated proteins were only adsorbed above pH 8 when the electrostatic repulsion between the boronate anion and the protein surface was mitigated (at 200 mM NaCl). All basic proteins were highly adsorbed above pH 8 with PB also acting as a cation-exchanger with binding occurring through electrostatic interactions. Batch adsorption performed at acidic conditions in the presence of Lewis base showed that charge-transfer interactions are critical for protein retention. This study demonstrates the multimodal interaction of PBC, which can be a selective tool for separation of different classes of proteins.
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Non-viral gene therapy and DNA vaccination currently hold great potential for treatment and preve... more Non-viral gene therapy and DNA vaccination currently hold great potential for treatment and prevention of genetic and acquired diseases. The large-scale manufacturing of plasmid DNA (pDNA) vectors, and the downstream processing in particular, is one of the key aspects of process development required to move non-viral gene therapy to the clinic. To address the problematic isolation and purification of pDNA molecules , an alternative and cost effective process based on multimodal chromatography was developed. In particular, the possibility of using a cationic multimodal ligand (Capto TM adhere) to remove RNA impurities from E. coli pre-purified lysates and to isolate supercoiled (sc) pDNA isoforms from open circular (oc) pDNA was explored. The process involves E. coli culture, cell harvesting and alkaline lysis followed by iso-propanol, ammonium acetate and PEG-8000 precipitation. Finally, sc pDNA is isolated by multimodal chromatography using a stepwise NaCl elution method that includes washing of unbound oc pDNA at 830 mM NaCl, sc pDNA elution at 920 mM and removal of RNA at 2 M. The method provided baseline separation of isoforms and yielded sc-rich fractions (>90%) that are virtually free from RNA and have levels of gDNA (1.3 ± 0.3% mg gDNA/mg sc pDNA) and protein (4.97 ± 1.34 mg/mL) impurities within specifications. Approximately 376 lg of sc pDNA were obtained from 200 mL of bacterial cell culture (OD 600nm % 19). The process was reproducible and performed similarly with differently sized plasmids (2686 bp, 3696 bp and 10,410 bp).
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The ability to analyze the distribution of topoisomers in a plasmid DNA sample is important when ... more The ability to analyze the distribution of topoisomers in a plasmid DNA sample is important when evaluating the quality of preparations intended for gene therapy and DNA vaccination or when performing biochemical studies on the action of topoisomerases and gyrases. Here, we describe the separation of supercoiled (sc) and open circular (oc) topoisomers by multimodal chromatography. A medium modified with the ligand N-benzyl-N-methyl ethanolamine and an elution scheme with increasing NaCl concentration are used to accomplish the baseline separation of sc and oc plasmid. The utility of the method is demonstrated by quantitating topoisomers in a purified plasmid sample.
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Journal of Separation Science, 2012
This study addresses the feasibility of scaling-up the removal of host cell impurities from plasm... more This study addresses the feasibility of scaling-up the removal of host cell impurities from plasmid DNA (pDNA)-containing Escherichia coli lysates by phenyl-boronic (PB) acid chromatography using columns packed with 7.6 and 15.2 cm 3 of controlled porous glass beads (CPG) derivatized with PB ligands. Equilibration was performed with water at 10 cm 3 /min and no conditioning of the lysate feed was required. At a ratio of lysate feed to adsorbent volume of 1.3, 93–96% of pDNA was recovered in the flow through while 66–71% of impurities remained bound (∼2.5-fold purification). The entire sequence of loading, washing, elu-tion, and re-equilibration was completed in 20 min. Run-to-run consistency was observed in terms of chromatogram features and performance (yield, purification factor, agarose electrophoresis) across the different amounts of adsorbent (0.75–15.2 cm 3) by performing successive injections of lysates prepared independently and containing 3.7 or 6.1 kbp plas-mids. The column productivity at large scale was 4 dm 3 of alkaline lysate per hour per dm 3 of PB-CPG resin. The method is rapid, reproducible, simple, and straightforward to scale-up. Furthermore, it is capable of handling heavily contaminated samples, constituting a good alternative to purification techniques such as isopropanol precipitation, aqueous two-phase systems, and tangential flow filtration.
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High-Throughput Process Development by Ana Azevedo
This paper describes microbead-based microfluidic systems. Several aspects of bead assays in micr... more This paper describes microbead-based microfluidic systems. Several aspects of bead assays in microflu-idics make them advantageous for bioassays in simple microchannels, including enhanced surface-to-volume ratio, improved molecular recognition reaction efficiency, and the wide range of surface function-alization available with commercial microbeads. Two-level SU-8 molds are used to fabricate PDMS microchannels that can hydrodynamically trap different types of microbeads, with characteristic dimensions of tens of microns. The use of these microbead-based microfluidic systems in the biosensing of anti-bodies, toxins and nucleic acids, as well as in antibody purification will be presented and discussed in this paper.
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Multimodal ligands are synthetic molecules comprising multiple types of interactions that have be... more Multimodal ligands are synthetic molecules comprising multiple types of interactions that have been increasingly used for the capture of different biopharmaceutical compounds within complex biological mixtures. For monoclonal antibodies (mAbs) in particular, these ligands have shown the possibility of direct capture from cell culture supernatants in native conditions, as well as enhanced selectivity and affinity compared to traditional single-mode ligands. However, performing the capture of a target mAb using multimodal chromatography comes with the need for extensive optimization of the operating conditions, due to the multitude of interactions that can be promoted in parallel. In this work, a high-throughput microfluidic platform was developed for the optimization of chromatographic conditions regarding the capture of an anti-interleukin 8 mAb, using a multimodal ligand (2-benzamido-4-mercaptobutanoic acid), under a wide range of buffer pH and conductivities. The interaction of the ligand with the fluorescently labeled target mAb was also analyzed with respect to the individual contribution of the hydrophobic (phenyl) and electrostatic (carboxyl) moieties using fluorescence microscopy. The results were further validated at the macroscale using prepacked columns in standard chromatography assays, and recovery yield values of 94.6% ± 5.2% and 97.7% ± 1.5% were obtained under optimal conditions for the miniaturized and conventional approaches, respectively. In summary, this study highlights that a microfluidic-based approach is a powerful analytical tool to expedite the optimization process while using reduced reagent volumes (<50 μL), less resin (∼70 nL), and delivering results in less than 1 min per assay condition.
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The use of monoclonal antibodies (mAbs) in medical treatments and in laboratory techniques has a ... more The use of monoclonal antibodies (mAbs) in medical treatments and in laboratory techniques has a very important impact in the battle against many diseases, namely in the treatment of cancer, autoimmune diseases and neural disorders. Thus these biopharmaceuticals have become increasingly important, reinforcing the demand for efficient, scalable and cost-effective techniques for providing pure antibodies. Aqueous two-phase systems (ATPS) have shown potential for downstream processing of mAbs. In this work, an ATPS in a microfluidic platform was designed and tested for mAbs extraction. The system demonstrated the potential to be an effective tool to accelerate bioprocess design and optimization. The partition of immunoglobulin G (IgG) tagged with fluorescein isothiocyanate (FITC) in an ATPS of polyethylene-glycol (PEG)/phosphate buffer with NaCl was investigated using a PDMS microfluidic device fabricated using soft lithography techniques. Different structures were tested with different values of microchannel length (3.14–16.8 cm) and flow rates of the salt (1–2 L/min) and PEG-rich phases (0.2–0.5 L/min). A stable interphase between the phases was obtained and the phenomena of diffusion and of partition of the IgG from the salt-rich phase to the PEG-rich phase were measured by fluorescence microscopy. Process simulation allowed the modeling of the IgG diffusion and partitioning behavior observed in the microstructure. The reduction to the microscale does not greatly affect the antibody extraction yield when compared with macroscale results, but it does reduce the operation time, demonstrating the potentiality of this approach to process optimization.
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Aqueous Two-Phase Extraction by Ana Azevedo
Multimodal Chromatography by Ana Azevedo
High-Throughput Process Development by Ana Azevedo
RESULTS: MPs were synthesized via co-precipitation and exhibited a spherical-like physical aspect, with an average hydrodynamic diameter of 473 nm and a zeta potential of –26 mV. The adsorption and elution of IgG on these adsorbents was thoroughly studied. Adsorption of human IgG was enhanced at pH 6, for which a qmax of 244 mg IgG g−1 MPs and Kd of 25 mg L−1 were obtained. Increasing salt concentrations at a basic pH (1 mol L−1 NaCl at pH 11) were found to improve the elution of bound IgG. The MPs were challenged with an artificial protein mixture containing human IgG, albumin, insulin and apo-transferrin. An overall yield of 84% was achieved, retrieving 92% of bound IgG.
CONCLUSIONS :MPs were successfully used for the capture of monoclonal antibodies from two distinct mammalian cell cultures, a Chinese hamster ovary (CHO) and a hybridoma cell culture supernatants. The elution yields were high, ranging between 84% and 94%, with overall yields ranging from 72% to 88%. Final purities of 85% were reached for hybridoma cell supernatants. © 2014 Society of Chemical Industry