Elsevier

Journal of Chromatography A

Volume 1249, 3 August 2012, Pages 1-7
Journal of Chromatography A

Design of a microfluidic platform for monoclonal antibody extraction using an aqueous two-phase system

https://doi.org/10.1016/j.chroma.2012.05.089Get rights and content

Abstract

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 μL/min) and PEG-rich phases (0.2–0.5 μL/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.

Highlights

► We developed an ATPS microfluidic device for mAbs extraction. ► PDMS microfluidic device fabricated using soft lithography techniques with three inlets/three outlets design was used. ► A stable interphase between the phases was obtained. ► The diffusion of IgG into PEG-rich phase was complete for a length of 16.8 cm. ► The partition behavior of IgG at the macroscale can be translated into the microscale.

Introduction

The need for therapeutic proteins, such as monoclonal antibodies, for the treatment of age and society-related diseases, such as Alzheimer's, Parkinson's, cancer, or diabetes, continues to increase. Presently more than 250 of these therapeutic products are in clinical trials and hundreds more are at the preclinical stage of development [1]. Downstream processing (dsp) of therapeutic proteins has however failed to keep up with the throughput of the upstream stage, resulting in a production bottleneck and in escalating costs [2]. Improved dsp processes are therefore actively sought, among them aqueous two phase systems (ATPS), which rely on the selective partitioning of a molecule in a biocompatible polymer (salt)/polymer environment. The widespread use of ATPS has been hampered by poorly understood partition mechanisms, empirical and time-consuming method development, and the need to evaluate a large number of variables for process optimization [2], [3], [4], [5].

Microscale process techniques have appeared as effective tools for accelerating bioprocess design in a cost-effective manner since the 1990s [6] nevertheless the trend to a widespread use has only emerged more recently [7]. This approach allows for a large number of variables to be evaluated in a parallel process, reducing the time and sample/reagent volumes required. Microliter scale systems can be designed in microwell or microfluidic format. Microfluidic systems process microliter quantities of fluid within channels with dimensions of tens to hundreds of micrometers, and are compatible with continuous flow processing [8]. Continuous flow is often the mode of operation in large-scale processing, so the use of microfluidic devices brings high-throughput, lab-scale process optimization closer to the final large-scale production processes [9]. Given the small dimensions of the microchannels, the flow regime in a microfluidic environment is laminar, with mixing relying on diffusion, and interfacial forces dominating over gravitational forces [10]. Microfluidic devices furthermore take advantage of high surface-to-volume ratios, which minimize heat and mass transfer distances, and can allow a high level of parallelization, thus providing useful tools for gaining early information on the performance of production systems. Most of the work with microfluidic devices has centered on the reaction/metabolite production stage and there has been relatively little work in the dsp. Titchener-Hooker et al. published pioneering work on microscale dsp [11]. Current microfluidic approaches to protein purification tend to focus on chromatography, affinity capture, and electrophoresis, which are predominantly batch systems, requiring the injection of small and precise amounts of sample and whose efficiency is established only at the end of the separation. ATPS is particularly suitable for continuous operation in microfluidic devices [12], where the laminar regime and the low interfacial tension favor interface stabilization, a key requirement in ATPS [3], [13].

Although the use of ATPS in a microscale is still relatively limited, examples in the recovery of whole cells [14], [15], [16], [17], [18], tagged proteins [19] with recovery of 75% of activity and crude membrane proteins with recovery yields of 91% [20] have been reported. An ATPS-microfluidic setup was also used for the purification of bacteriorhodopsin, a light-sensitive membrane protein, from crude cell extract [21]. For cell separation, a PEG/dextran system was used to extract live and dead CHO K-1 cells from a culture medium [15] and to extract leukocytes from whole blood samples [16], [18]. Microfluidic extraction of a genetically tagged recombinant protein has also been reported using a ATPS and about 85% of the contaminants were successfully removed [19]. An overview of the use of aqueous two-phase systems in microfluidics was recently published highlighting the opportunities for future developments of using of ATPS in microfluidic devices as an automated high-throughput platform for separation, purification and analysis of biomolecules [12].

In this paper, a microfluidic device was designed and tested for the partition of immunoglobulin G (IgG) tagged with fluorescein isothiocyanate (FITC) in polyethylene-glycol (PEG)/phosphate buffer with NaCl in ATPS. The PDMS microfluidic devices were fabricated using soft lithography techniques. The optimized chip design was a three inlets/three outlets serpentine structure. This design allowed the formation of a stable interphase and the complete extraction of IgG from the salt-rich phase to the PEG-rich phases. Fluorescence microscopy image allowed the monitoring of the IgG concentration both across and along the microchannel. The extraction process was simulated to allow the modeling of the IgG diffusion and partitioning behavior. The application of microfluidics to ATPS 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.

Section snippets

Chemicals and biologicals

Polyethylene glycol (PEG, MW 3350 Da) and sodium chloride (NaCl) were purchased from Sigma (St. Louis), while potassium phosphate dibasic anhydrous (K2HPO4) and sodium phosphate monobasic anhydrous (NaH2PO4) were purchased from Panreac. A rabbit anti-goat IgG conjugated with FITC was purchased from Sigma as an aqueous solution with a concentration of 3.6 mg/mL. Polydimethylsiloxane (PDMS) was obtained from Dow Corning, under the trade name of Sylgard® 184.

Aqueous two-phase systems preparation

Stock solutions of 50% (w/w) PEG and 40%

Preliminary microfluidic structure design studies

The first structure fabricated was a two inlets and two outlets straight microchannel with 200 μm of width, 20 μm of height and 3.14 cm of length. The devices were made with PDMS, an elastomeric polymer commonly used in microfluidics with biological samples in aqueous solutions. Micron-sized features can be reproduced by PDMS using the replica molding technique. Furthermore (i) it is optically transparent above 280 nm; (ii) it can be deformed reversibly; (iii) it is cured at low temperatures; (iv)

Conclusions

A microfluidic device with a three inlets/three outlets design was fabricated using soft-lithography techniques based on PDMS and successfully demonstrated for the extraction of IgG with ATPS. It was possible to establish a stable, laminar flow of the two aqueous phases in the microfluidic network. Partition studies with FITC-labelled IgG performed in PEG/phosphate ATPS showed a decrease of the fluorescence of the salt-rich phase, indicating partioning of the IgG–PEG-rich phase, as observed in

Acknowledgements

The authors acknowledge funding from FCT through the Associated Laboratories IN – Institute of Nanoscience and Nanotechnology and IBB – Institute of Biotechnology and Bioengineering. A. Azevedo and P. Fernandes acknowledge FCT for funding under program Ciência 2007.

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