Available online at www.sciencedirect.com ScienceDirect Procedia Engineering 168 (2016) 1426 – 1429 30th Eurosensors Conference, EUROSENSORS 2016 Integration of photosensors in a nano-liter scale chromatography column for the online monitoring of adsorption/desorption kinetics of a fluorophore-labeled monoclonal antibody Inês F Pintoa,b*, D.R Santosa,b, R.R.G Soaresa,b, M.R Aires-Barrosb,c, V Chua, A.M Azevedob,c, J.P Condea,c a Instituto de Engenharia de Sistemas e Computadores – Microsistemas e Nanotecnologias (INESC-MN) and IN – Institute of Nanoscience and Nanotechnology, Rua Alves Redol, 9, 1000-029 Lisbon, Portugal b IBB – Institute for Bioengineering and Biosciences, Instituto Superior Técnico, Universidade de Lisboa, Avenida Rovisco Pais, 1, 1049-001 Lisbon, Portugal c Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Avenida Rovisco Pais, 1, 1049-001 Lisbon, Portugal Abstract Chromatography is a robust purification technique, but requires time-consuming optimization on a case-by-case basis, particularly for monoclonal antibodies (mAbs) This work presents a novel microfluidic platform that significantly speeds up the optimization process and reduces the amount of reagent needed Binding kinetics for a fluorescent conjugate mAb-Alexa 430 were measured in real time at resin level by integration of the microfluidic chip with 200u200 Pm a-Si:H photodiodes on glass substrates aligned with micro-columns Screening studies were performed using reduced amounts of reagents (a50 PL), mAb molecules (a2.5 PL) and resin (a70 nL) in a rapid (a2 min/condition) and simple manner © Published by Elsevier Ltd This © 2016 2016The TheAuthors Authors Published by Elsevier Ltd.is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Peer-review under responsibility of the organizing committee of the 30th Eurosensors Conference Peer-review under responsibility of the organizing committee of the 30th Eurosensors Conference Keywords: On-chip chromatography; Microfluidics; a-Si:H photodiodes; Fluorescence; Monoclonal Antibodies; Binding Kinetics * Corresponding author Tel.: +351-21-3100237; fax: +351-21-3145843 E-mail address: ines.f.pinto@tecnico.ulisboa.pt 1877-7058 © 2016 The Authors Published by Elsevier Ltd This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) Peer-review under responsibility of the organizing committee of the 30th Eurosensors Conference doi:10.1016/j.proeng.2016.11.405 Inês F Pinto et al / Procedia Engineering 168 (2016) 1426 – 1429 1427 Introduction Monoclonal antibodies (mAbs) are a highly important class of biopharmaceuticals, providing valuable treatment options for diseases such as cancer and auto-immune disorders For typical chronic administration, mAbs are required in high doses for long periods of time, resulting in an increasing demand for large quantities of purified mAbs at affordable prices The production and economical constrains in meeting this demand are mostly located at the downstream processing, meaning the purification of mAbs from clarified fermentation broths Current techniques such as protein A affinity chromatography, while being robust and efficient in terms of purity, represent up to 25% of total production costs [1] This motivates the research of alternative low-cost synthetic multimodal ligands, which comprise multiple types of chemical interactions with the target, thus having a high potential for selectivity and tenability [2] However, in order to effectively screen and optimize a particular ligand in terms of binding kinetics, the development of high-throughput screening techniques becomes essential This work presents a novel integrated microfluidic platform that significantly speeds up the optimization process and reduces the amount of reagents needed In this platform, the chromatography multimodal ligand Capto™ MMC, which is negatively charged and capable of hydrophobic, tiophilic and hydrogen bond interactions, was studied for the capture of a fluorophore conjugate anti-IL8 mAb-Alexa 430 under different pH conditions Binding kinetics were measured in real time at resin level by integration of 200u200 Pm a-Si:H photodiodes fabricated on glass substrates, aligned with micro-columns fabricated in PDMS Experimental Section 2.1 Fabrication of the micro-columns and optical photosensors The microfluidic devices (Figure 1-A) were fabricated using standard polydimethylsiloxane (PDMS) soft lithography The fabrication process involved three main steps: (1) fabrication of an aluminum hard mask through direct write optical lithography (DWL) of a CAD design of the micro-columns and wet chemical etching of the Al; (2) fabrication of an SU-8 mold; and (3) fabrication of PDMS structures and sealing The mold fabrication required a two-level process, in which a layer of 20 µm was first defined by spin coating SU-8 2015 on top of a clean Si substrate, and then a second layer of 100 µm was defined by spin coating SU-8 50 on top of the previously fabricated layer [3] The 200x200 µm a-Si:H p-i-n photodiodes were fabricated using standard cleanroom techniques [4] For fluorescence measurements, the excitation light (blue-violet laser, O = 405 nm, Figure 1-B) was filtered by an integrated amorphous silicon carbon alloy (a-SiC:H) layer with a thickness of 1.6 µm, whereas the emission light passed through the filter and impinged the photodiode through an indium tin oxide (ITO) transparent top contact (Figure 1-C) 2.2 Device operation and fluorescence monitoring The micro-columns were designed to allow the packing of chromatography beads (average size of 70 µm) in a region confined by the height difference of two microchannels, preventing the downstream flow of the beads The liquid flow was driven at 15 µL/min by applying a negative pressure at the outlet using a syringe pump, which provided a gradual increase in liquid velocity with minor deformation of the beads against the 20 µm gap The binding kinetics of a target monoclonal antibody (anti-IL8 mAb) conjugated to an Alexa Fluor® 430 dye were monitored by continuously measuring the fluorescence emission of the packed beads under different pH values using (1) a fluorescence microscope or (2) integrated photosensors In the first case, the measurements were performed using an Olympus CKX41 inverted microscope equipped with a 50W mercury short-arc lamp and a filter cube for excitation in a band pass between 460 and 490 nm and an emission long pass above 520 nm In the second case, the PDMS microfluidic devices were first aligned with the 200 µm square a-Si:H photodiodes and the excitation laser beam was focused on the end-part of a single micro-column The current measurements were performed at V bias voltage using a picoammeter (Figure 1-B) 1428 Inês F Pinto et al / Procedia Engineering 168 (2016) 1426 – 1429 Fig 1: (A) Schematic of the single micro-column and a detail of the interface region between the two channels with different heights Agarose beads functionalized with a chromatography ligand (Capto™ MMC) were evaluated in their ability to bind a fluorescently labeled target mAb; (B) Alignment of the microfluidic structure sealed with a 200 Pm PDMS membrane on top of the photodiode array The laser excitation light is reflected from the surface of a mirror and focused on the micro-column (C) Cross-sectional view of the a-Si:H p-i-n photodiode used in fluorescence detection The excitation light is filtered by an integrated a-SiC:H layer (1.6 Pm) and the emission light enters the photodiode through an indium tin oxide transparent (ITO) contact Results and Discussion 3.1 Evaluation and validation of photodiodes for online fluorescence measurements The current generated by the p-i-n photodiode at V bias at different stages is represented in Figure 2-A The signal acquisition was initiated by measuring the current in the absence of excitation light (dark current) The excitation light was then turned on, resulting in a minimal increase of the measured current, highlighting the low transmission of the excitation light through the a-SiC:H filter Subsequently, the mAb-Alexa 430 solution, at a concentration of 50 µg/mL prepared in buffers at pH 5.5, 6.5 and 7.5, was flowed through the micro-columns During this stage it is possible to observe that the current increased steadily above 20-fold the initial current of about pA after about 100 s of adsorption Between stopping the pump and the flow of a concentrated NaOH solution to regenerate the column by removing all bound antibodies, it is important to highlight that photobleaching effects are negligible at this time-scale as the fluorescence signal remains stable for at least 50 s Following the regeneration procedure, the signal readily returns to values comparable to those measured before mAb adsorption This sequence of steps thus validates the fabricated photodiodes as an effective fluorescence monitoring tool for further experiments 3.2 Comparison of integrated filtered photodiodes with a standard fluorescence microscope In order to further confirm the robustness of the photodiodes to monitor the fluorescence increase on the packed beads, these were compared with a conventional inverted fluorescence microscope The results are shown in Figure 2-B As expected, based on previous findings [3], there is an overall trend for the binding kinetics of the mAb to decrease as the pH of the adsorption buffer increases, justified by a decrease in the total charge of the mAb as the pH approaches its isoelectric point of It is also clear that the curves obtained using both methods not only show the same trend, but are also comparable in terms of slope Interestingly, the data also suggests a slightly higher sensitivity for the photodiodes compared to the fluorescence microscope, as observed by the lack of signal obtained with the latter when measuring the adsorption of the mAb at a sub-optimal pH of 7.5 Inês F Pinto et al / Procedia Engineering 168 (2016) 1426 – 1429 1429 Fig 2: (A) Curve obtained by measuring the current generated by the p-i-n photodiodes at V bias as a function of the assay time The signal was continuously acquired in a 2-stage assay: (i) monitoring of the adsorption of the target mAb labeled with Alexa Fluor® 430 (mAb-A430) and (ii) regeneration of the micro-column by flowing a NaOH solution Dark current acquisition was performed in the absence of excitation light and the fluorescence signal was measured by illuminating the beads using a 405 nm blue-violet laser (B) Photoresponse of the a-Si:H p-i-n photodiode as a function of the assay time for the binding of mAb-A430 to the chromatography beads at different pH values (left axis) The same conditions were also tested using a fluorescence microscope, by continuously monitoring the signal of the beads with an exposure time of 500 ms, 5u gain and 10u magnification (right axis) Conclusion The integrated microfluidic-based approach reported in this work presents a novel analytical method to effectively screen operating conditions for early stage optimization of bioseparation processes This general technique can be used with any target molecule or chromatography beads, assuming a previous labeling procedure with an appropriate fluorophore, in order to obtain a stable signal upon continuous excitation Fluorescence measurements at bead-level in real-time allowed to successfully evaluate the optimal conditions to promote the capture of a target mAb by a novel chromatography multimodal ligand (Capto™ MMC), with minimal consumption of antibody molecules and within a few minutes Furthermore, by coupling thin-film photosensors to the microfluidic device, it was possible to have an integrated and simple operation, without resorting to complex and expensive instrumentation Acknowledgements I.F Pinto acknowledges Fundaỗóo para a Ciờncia e Tecnologia (FCT) for the PhD fellowship SFRH/BD/96442/2013 INESC-MN and IBB acknowledge FCT for support through research units UID/NAN/50024/2013 and UID/BIO/04565/2013, respectively; and the projects OptLoc (PTDC/BBBNAN/5927/2014) and PureMab (PTDC/QEQ-PRS/0286/2014) References [1] J.G Elvin, R.G Couston, C.F van der Walle, Therapeutic antibodies: Market considerations, disease targets and bioprocessing, Int J Pharm 440 (2013) 83-98 [2] I.F Pinto, M.R Aires-Barros, A.M Azevedo, Multimodal chromatography: debottlenecking the downstream processing of monoclonal antibodies, Pharm Bioprocess (2015) 263-279 [3] I.F Pinto, R.R.G Soares, S.A.S.L Rosa, M.R Aires-Barros, V Chu, J.P Conde, A.M Azevedo, High-throughput nanoliter-scale analysis and optimization of multimodal chromatography for the capture of monoclonal antibodies, Anal Chem in press (2016), DOI: 10.1021/acs.analchem.6b00781 [4] A.C Pimentel, D.M.F Prazeres, V Chu, J.P Conde, Fluorescence detection of DNA using an amorphous silicon p-i-n photodiode, J Appl Phys, 104 (2008) 054913 ... provided a gradual increase in liquid velocity with minor deformation of the beads against the 20 µm gap The binding kinetics of a target monoclonal antibody (anti-IL8 mAb) conjugated to an Alexa Fluor®... obtained by measuring the current generated by the p-i-n photodiodes at V bias as a function of the assay time The signal was continuously acquired in a 2-stage assay: (i) monitoring of the adsorption. .. The results are shown in Figure 2-B As expected, based on previous findings [3], there is an overall trend for the binding kinetics of the mAb to decrease as the pH of the adsorption buffer increases,