Continuous extraction strategies for monoclonal antibodies: From macro- to micro- scale

Aqueous two-phase extraction (ATPE) have shown to be a valuable option for the downstream processing of biopharmaceuticals, combining a high biocompatibility and selectivity with an easy and reliable scale up. Moreover, ATPE can overcome some of the technical drawbacks currently encountered using established purification platforms, such as batch operation, diffusional limitations and scale-related problems. We have developed a continuous ATPE process incorporating three different steps (extraction, back-extraction and washing) for the capture of monoclonal antibodies (mAbs). The ATPE process was set up and validated in a pump mixer-settler battery and successfully applied to a Chinese hamster ovary and a PER.C6® cell supernatant. The limited predictive design of ATPE, however, has restrict its applicability to current downstream processing. Microscale process techniques have recently emerged as effective tools for expediting bioprocess design in a cost-effective manner. ATPE in a microfluidic platform was therefore designed and tested for mAbs extraction, as an effective tool to accelerate bioprocess design and optimization. Furthermore, this platform has the potential of combining the process efficiency of ATPS with the reduced times and volumes associated with microfluidics, as well as the possibility to multiplex and parallel process in real downstream processes. In this way, we have develop a microfluidic channel-based toolbox for the rapid screening of antibody extraction conditions. Several microfluidic structures have been designed including a multiplexed structure that allows a simple negative-pressure driven rapid screening of up to 8 continuous extraction conditions simultaneously, using less than 20 μL of each phase forming solution per experiment. Results obtained from this device can be further applied in a second microfluidic structure (Figure 1) that integrates multiple-step continuous extraction protocols

Effect of ionic strength and presence of serum on lipoplexes structure monitorized by FRET

<p>Abstract</p> <p>Background</p> <p>Serum and high ionic strength solutions constitute important barriers to cationic lipid-mediated intravenous gene transfer. Preparation or incubation of lipoplexes in these media results in alteration of their biophysical properties, generally leading to a decrease in transfection efficiency. Accurate quantification of these changes is of paramount importance for the success of lipoplex-mediated gene transfer <it>in vivo</it>.</p> <p>Results</p> <p>In this work, a novel time-resolved fluorescence resonance energy transfer (FRET) methodology was used to monitor lipoplex structural changes in the presence of phosphate-buffered saline solution (PBS) and fetal bovine serum. 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/pDNA lipoplexes, prepared in high and low ionic strength solutions, are compared in terms of complexation efficiency. Lipoplexes prepared in PBS show lower complexation efficiencies when compared to lipoplexes prepared in low ionic strength buffer followed by addition of PBS. Moreover, when serum is added to the referred formulation no significant effect on the complexation efficiency was observed. In physiological saline solutions and serum, a multilamellar arrangement of the lipoplexes is maintained, with reduced spacing distances between the FRET probes, relative to those in low ionic strength medium.</p> <p>Conclusion</p> <p>The time-resolved FRET methodology described in this work allowed us to monitor stability and characterize quantitatively the structural changes (variations in interchromophore spacing distances and complexation efficiencies) undergone by DOTAP/DNA complexes in high ionic strength solutions and in presence of serum, as well as to determine the minimum amount of potentially cytotoxic cationic lipid necessary for complete coverage of DNA. This constitutes essential information regarding thoughtful design of future <it>in vivo </it>applications.</p

Oscillatory flow reactor: A solution for continuous bioprocessing

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Cutinase structure, function and biocatalytic applications

This review analyses the role of cutinases in nature and their potential biotechnological applications. The cloning and expression of a fungal cutinase from Fusarium solani f. pisi, in Escherichia coli and Saccharomyces cerevisiae hosts are described. The three dimensional structure of this cutinase is also analysed and its function as a lipase discussed and compared with other lipases. The biocatalytic applications of cutinase are described taking into account the preparation of different cutinase forms and the media where the different types of enzymatic reactions have been performed, namely hydrolysis, esterification, transesterification and resolution of racemic mixtures. The stability of cutinase preparations is discussed, particularly in anionic reversed micelles considering the role of hexanol as substrate, co-surfactant and stabilizer. Process development based on the operation of cutinase reactors is also reviewed

Rapid optimization of chromatography operating conditions using a nano- liter scale column on a microfluidic chip with integrated pneumatic valves and optical sensors

Purification of monoclonal antibodies (mAbs) is traditionally achieved by chromatographic separations, which are very robust but require time-consuming optimization on a case-by-case, particularly if a non-affinity step is used. In this context, multimodal chromatography has been explored as a versatile and cost-effective alternative to the established affinity step employed for capturing mAbs. However, selective capture/polishing of a target mAb using such multimodal ligands comes with the need for extensive and time-consuming optimization, due to the multitude of interactions that can be simultaneously promoted in the ligand. In this work, we developed a novel microfluidic platform comprising multimodal chromatography beads inside micro-columns for rapid screening of operating conditions. Sequential liquid insertion in the device was achieved by using integrated pneumatic valves and the chromatographic assays were combined with a signal acquisition module for on-chip fluorescence measurements. Please click Additional Files below to see the full abstract

Miniaturization of aqueous two-phase extraction for biological applications

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. Studies of ATPSs at nanoliter-scale are further extending the range of applications of these systems by taking advantage of rapid diffusion times, increased degree of control of individual liquid streams and droplets, continuous flow and the integration of multi-dimensional separation modes. Several examples of microfluidic ATPS platforms are described. The partition of molecules between two co-flowing liquid streams confined within a microchannel was successfully demonstrated by the on-line extraction of a fluorescein isothiocyanate (FITC) labeled immunoglobulin G (IgG) from a salt rich flow to a PEG rich flow. IgG diffusion to the PEG-rich phase was complete after 16 cm of channel using flow rates of 1 and 0.2 μL/min for the salt and PEGrich phases respectively. Besides proteins, ATPS have also been used to separate other more complex biomolecules in microfluidics such as virus-like particles. The potential of miniaturization as a high-throughput screening tool has also been explored. The developed setup allowed the screening of a wide range of concentrations inside the microchannel by varying the flow rates of the solutions while using sub-mL volumes for each ATPS-forming system. As a novel demonstration of the integrative potential of ATPE as a microfluidic sample preparation module, a microfluidic device comprising two modules was developed and used to perform a complex matrix clean-up in-line with an immunoassay. References: Silva, D. F., Azevedo, A. M., Fernandes, P., Chu, V. et al., Design of a microfluidic platform for monoclonal antibody extraction using an aqueous two-phase system. J. Chromatogr. A 2012, 1249, 1–7. Jacinto, M. J., Soares, R. R. G., Azevedo, A. M., Chu, V. et al., Optimization and miniaturization of aqueous two phase systems for the purification of recombinant human immunodeficiency virus-like particles from a CHO cell supernatant. Sep. Purif. Technol. 2015, 154, 27–35. Silva, D. F. C., Azevedo, A. M., Fernandes, P., Chu, V. et al., Determination of aqueous two phase system binodal curves using a microfluidic device. J. Chromatogr. A 2014, 1370, 115–120. Soares, R. R., Novo, P., Azevedo, A. M., Fernandes, P. et al., On-chip sample preparation and analyte quantification using a microfluidic aqueous two-phase extraction coupled with an immunoassay. Lab Chip 2014, 14, 4284–429

LYTAG-driven purification strategies as a key to integrate and intensify the downstream processing of monoclonal antibodies

Monoclonal antibodies (mAbs) are currently the most important class of recombinant protein therapeutics in the biotechnological and biopharmaceutical industry with more than 250 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 (LYTAG) fused with the synthetic antibody Z domain (LYTAG-Z) as a tool to integrate and optimized the downstream processing of mAbs. Upon addition of this ligand to an animal cell culture broth, antibody-LYTAG-Z complexes are formed which can be easily captured and separated from host cell impurities by affinity partitioning in aqueous two-phase systems (ATPS) composed of polyethylene glycol –PEG, as PEG molecules have the ability to binding to the choline binding sites of LYTAG. Integration of clarification and primary mAbs recovery was successfully accomplished using a system composed of 6% PEG 3350 Da and 7% dextran 500,000 Da in which an extraction yield of 89% and a clarification higher than 95% were achieved. IgG-rich phases were further processed by chromatography, using three different strong anion exchange matrices charged with quaternary methyl amines (a choline analogue) – CIMmultus QA, HiTrap Q FF and gPore NW Q. A two-elution method was developed for the separation of the antibody-LYTAG-Z complexe, allowing simultaneous purification of the antibody and recovery of the ligand. The process was successfully scale-up 10000 times allowing a global antibody recovery of 70% with a purity of 89% and enabling 100% cell removal

Analytical affinity chromatography-on-a-chip for selective capture and sensitive detection of protein and polynucleotide biomarkers

Affinity Chromatography is a powerful technique which has been applied to the highly selective purification of several biomolecules from complex mixtures. This technique is currently a core technology in the industrial purification of disruptive biopharmaceuticals such as monoclonal antibodies. The use of high affinity ligands, together with densely functionalized three-dimensional solid-phase supports, confers a remarkable analytical potential, making it a current standard for the quantification of several compounds in certified laboratories, ranging from health biomarkers to environmental contaminants. Aiming at extending the application of affinity chromatography to a portable setup, we report the miniaturization of this system down to nL-scale, by trapping Q-sepharose or protein-A agarose beads in microfluidic channels with total volumes ranging from 60 to 210 nL. This versatile and simple platform combined the high surface area and robust surface chemistry provided by the chromatographic media with the high degree of fluidic control, portability, improved reaction kinetics and low reagent expenditure inherent to microfluidics. Furthermore, the microfluidic structures are simple in terms of microfabrication and can be sequentially operated using standard pipette tips and a negative pressure source at the outlet (Figure A). This system was tested within the scope of prostate cancer diagnostics for the capture of protein and polynucleotide biomarkers. Along these lines, prostate specific antigen (PSA) was selectively captured from unprocessed human serum and a 23 bp polynucleotide (ssDNA analogous to micro RNA MIR145) in fetal bovine serum as model matrix, by coupling a monoclonal anti-PSA IgG2a with protein-A beads or a complementary ssDNA strand with Q-sepharose beads, respectively. The assay schematics are described in Figure A. Clinically relevant sensitivities below 10 ng/mL PSA (Figure B) and 10 pM polynucleotide were achieved using a horseradish peroxidase-labelled reporter and measuring chemiluminescence directly on the bead surface. The results demonstrate a high potential for the miniaturization of analytical affinity chromatography, providing good sensitivities in a portable setup, particularly considering the amenability of integrating miniaturized thin-film sensors for optical transduction, as previously demonstrated by our group. Please click Additional Files below to see the full abstract