On-line sequential injection-capillary electrophoresis for near-real-time monitoring of extracellular lactate in cell culture flasks
Introduction
Cell culture of eukaryotic cells is widely applied throughout life sciences. At the small scale, cell based assays have replaced many in vivo assays because of ethical and regulatory restrictions on working with laboratory animals. Additionally, the production of biopharmaceuticals synthesized by living cells during fermentation or cell culture processes is a rapidly growing field. According to the FDA, approximately 30–40 percent of the authorized medical products in 2012 were biopharmaceuticals. Bioprocessing offers many advantages. For example, the production of vaccines by cell culture technology instead of conventional methods provides the capability for rapid manufacturing start-up in case of a pandemic because characterized cell lines can be stored and are thus readily available. Moreover, the risk of impurities can be reduced because vaccine production takes place in a highly controlled, closed and sterile environment [1]. Cell culture based technologies are intensively employed in drug discovery [2], and are significant tools for drug screening and new potential drug targets studies [3]. In 2012 alone, 35 novel biologics were developed by the biopharmaceutical industry and approved by the FDA [4]. In this context it is important to note that bioprocess monitoring in the production process of biopharmaceuticals is essential to ensure the safety of the product as well as to satisfy economic and regulatory demands.
At present, the majority of cell culture based monitoring is restricted to a few end point based assays that do not reflect the dynamic metabolic processes in cells that influence the final product. Therefore, a detailed and continuous monitoring of the bioprocesses in each production batch would significantly help manufacturers to control product quality, increase production yields and reduce production costs [5]. At the same time, online monitoring of bioprocesses will also significantly enhance our understanding of fundamental dynamic cellular metabolic reactions that cannot be easily ascertained by end point measurements.
Lactate is one of the major products of eukaryotic and prokaryotic cells and is one of the most important organic acids in extracellular media that can be used to monitor cellular metabolism and energy status. It is produced from glucose and glutamine in mammalian cells [6]. It is important to monitor because it affects the physicochemical stability of the bioprocess medium by reducing pH levels and is outright toxic to some cells. In addition, under certain conditions, cultured cells can use lactate as an alternative source for carbon, even in the presence of glucose [7]. At the same time, lactate can be used as an indicator of biological activity. For example, metabolic shifting from lactate production to lactate consumption was reported to result in improvements of process performance regarding productivity, scalability, process robustness and cell growth [8].
Due to the tight connection between extracellular and intracellular metabolic pathways, cellular bioprocesses are primarily controlled by manipulating the external environment in form of the composition of the cell culture medium [9]. Therefore, timely information about even small changes in the concentration of extracellular lactic acid during cell culture will directly help to control and improve the efficiency of the bioprocess. Realizing this depends on the availability of suitable and functional tools that can be used for monitoring [10]. Currently, a number of different methods are used for lactate detection, the most important being enzymatic assays [11], [12], [13], and analytical separations such as liquid chromatography (LC) [14], [15] and capillary electrophoresis (CE) [16], [17]. Enzymatic assays are highly specific, instrumentally simple, but time consuming – typically requiring hours per measurement [18], and are restricted to a single analyte. Chromatographic techniques are sensitive, versatile, and have excellent reproducibility. However, separations are slow and typically sample pre-treatment is required. Capillary Electrophoresis (CE) is a powerful alternative characterized by faster separation compared to LC and has been widely used to separate a diverse range of analytes, from small ions through to macromolecules in many fields [19], [20], in particular for monitoring bioprocesses. As discussed in a recent review, CE has been readily employed for the analysis of discrete samples, utilizing commercially available single or multiple capillary instruments [21]. It has also been employed for on line monitoring of a variety of analytes in different matrices and environments, but only a few reports present dedicated, online sampling interfaces for CE analysis [21]. One of those, a CE method with conductivity detection was applied for automated continuous on-line analysis of 23 ions in tap water over two days [22]. CE was also used for on-line determination of perchlorate in biological samples such as breast milk, human urine, serum, red wine and cow's milk using a supported liquid membrane [23]. A filter probe was integrated with a computerized pneumatic sampling system to monitor the bioaccumulation of Cu2+, Zn2+, Co2+ and Cd2+ in the bacteria species Rhodococcus sp. [24].
Another CE method using LIF detection was successfully employed for microbial analysis of water collected from two local streams through continuous electrokinetic injection under field-amplified conditions [25].
The low sample volume makes CE ideally suited for monitoring cell culture conditions and cell-based assays. In this study, an automated, robust and portable SI-CE setup was developed by modifying the experimental set up previously reported by Blanco et al. [26]. A flow-through interface was designed to sample cell-free media for monitoring lactate production by the human embryonic kidney cell line HEK293. The unique combination of the sampling interface and SI-CE system minimized sample consumption and analysis time. Using this system, 72 samples were analyzed per day using less than 700 μL of sample per day, enabling monitoring of lactate production by HEK293 cells in vitro over three days, using less than 10% of the total media volume.
Section snippets
Chemicals
All reagents were analytical grade reagent obtained from Sigma–Aldrich (Sydney, AUS) and were used as supplied unless otherwise stated. Solutions were prepared in Milli-Q water (Millipore, Bedford, MA, USA). Lactate standard solution (10 mM) was prepared weekly and stored at 8 °C by dissolution of its sodium salt. Chloride standard solution (2 M) was prepared monthly from sodium chloride and stored at room temperature.
The cationic polyelectrolyte poly(ethylenimine) (PEI) (ACROS organics, Geel,
SI-CE of lactate
A CE method for monitoring lactate in cell cultures must be a fast, efficient, selective, and automated. Most importantly, it should only use minute amounts of sample per analysis. As our previously described SI-CE system [22], [26] had the potential to meet the sampling requirements, this set-up was modified to develop a suitable separation method. The most significant change compared to our previous work is the use of peristaltic pumps instead of the MilliGAT piston pumps. Peristaltic pumps
Conclusion
An online, automated system for monitoring lactate in cell culture is presented, consuming less than 10% of media (1.99 mL), or only 8.73 μL of sample per run, over a three-day period (76 h). A flow-through sequential injection capillary electrophoresis system was connected to a sampling interface to inject cell-free media from an adherent cell culture into the analytical system and applied for near-time monitoring of the production of lactate in mammalian cell culture. The system is flexible and
Acknowledgments
AAA acknowledges Al-Zaytoonah University of Jordan for financial support. The authors would like to thank Yi Nai (ACROSS) for the photography and to John Davis (Central Science Laboratory, University of Tasmania) for technical support. MCB acknowledges the Australian Research Council for funding and provision of a QEII Fellowship (DP0984745).
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