Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
In the world of biotechnology and biopharmaceuticals, cells produce everything from ethanol to high-value drugs. They are, quite literally, cellular factories. As in brick-and-mortar industrial plants, the factory generally cannot run unattended. Someone needs to ensure the machines are well maintained and functioning properly, that there is adequate supervision and that lines of communication remain open.
The extent of that supervision depends on scale. A research technician can visually inspect the media in a T-175 triple flask daily to determine density and pH. Yet, especially in larger and more industrialized operations, more needs to be monitored than a simple indicator dye can relate. The data also must be logged, catalogued and analyzed. Changes must sometimes be made.
There is a range of tools—from tried-and-true indicator dyes to cutting-edge spectroscopy—to monitor what’s going on inside your bioreactor. Many of these can be linked together with analysis and control systems, enabling the cell factories within to operate at their full potential. Here we review some of your options.
What to measure?
Temperature, pH and dissolved oxygen (DO, important for fast-growing bacterial and yeast cultures) are relatively easy to monitor with probes directly inserted into the bioreactor. Nutrients and by-products, like glucose, lactate, glutamine and ammonium, typically are monitored by sampling and assaying the culture medium. More cell-based measures, like density, viability and morphology, which are most often assessed optically, may also be of interest.
Many factors influence the extent to which instrumentation and automation play roles in monitoring bioprocess reactors, not the least of which are scale and resources. Biochemical assays exist, or can be cobbled together from lab reagents, to test for a variety of common nutrients and by-products.
Many of these same assays also can be performed by dedicated instruments, says Alex Lecorps, product manager at YSI Life Sciences. The benchtop YSI 2900 Biochemistry Analyzer, for example, uses enzyme electrode biosensor technology to automatically query up to six analytes (from a menu of 15 choices) from samples in tubes or microplates.
Bioanalyzers don’t look at the cells themselves, just the media around them, “the juice they’re floating in,” says Lecorps. “We test the by-products and other analytes and metabolites to make sure … the cultures are healthy.”
If a reading occurs that indicates a problem with the culture, the customer can then take a look at the cells themselves, she adds.
This can be performed manually in several ways, ranging from a simple trypan-blue exclusion assay for cell viability to a host of more nuanced assays. Enzo Life Sciences markets live-cell analysis kits suitable for flow cytometry and microscopy applications to detect autophagy, apoptosis and necrosis, hypoxia and oxidative stress. Other assays probe cytotoxic mitochondrial and lysosomal perturbations or monitor changes in gene expression related to copy-number variation or DNA methylation. “We can certainly sell those in bulk for larger-volume needs,” says technical marketing manager Randy Strube. “But the dye itself wouldn’t be engineered any differently for a basic cell-viability assay in the lab vs. for large-scale use.”
Automated instrumentation such as the Cedex HiRes Analyzer from Roche Applied Science use image-based analyses to query cell viability, concentration and aggregates as well as such parameters as cell diameter and compactness. Nova Biosciences also offers several instruments, including the BioProfile Flex, which, according to its website, can measure up to 15 key cell-culture attributes, including ion concentrations and osmolality.
Data management
In addition to performing measurements on media or cells, many analyzers can process and archive the data, chart and compare growth and compile regulatory documentation.
There is a trend toward integration, and customers are demanding that equipment be compatible with their process-management systems, notes Lecorps. “Some labs will have a DeltaV [from Emerson Process Management], where you can hook up several different instruments to it, and it manages all the different inputs coming in. You can hook up your YSI analyzer, your blood analyzer, your pH meter, everything.” That information is used to automatically provide feedback to bioreactor-control systems telling them, for example, to add more glucose.
Falk Schneider, a managing director of DASGIP Information and Process Technology GmbH, an Eppendorf Company, points to the benefits of using online measurement. Most labs, though, don’t have all the probes they need to accomplish this, so they likely still use at-line sampling and analytics (in which the sampling is distinct from analysis), either manually collecting samples or using robotics. In either case, systems like DASGIP’s “can provide a data interface between the analyzer and the bioprocess system, when after doing the sample you can send the data automatically back into your bioprocess-control software.” This eliminates many time-consuming and error-prone steps.
Promising probes
In-line measurement using Ramen technology or other spectral methods has been gaining attention lately, Schneider notes.
Probes for variables like DO, temperature and pH are established ways to measure culture parameters in-line. “But you’re getting one measurement at a time for whatever probe you’re putting in there. The benefit of NIR [near-infrared] spectroscopy is that we can continuously measure a whole suite of different parameters simultaneously, just within the fermentation media, without disturbing the process,” notes David Drapcho, Thermo Fisher Scientific’s marketing manager for NIR imaging products.
The NIR light travels through a fiber-optic cable to a stainless-steel probe with sapphire window. After interacting with the media, it returns to the detector and spectrophotometer where it can be used to determine concentrations of, for example, glucose, lactate, ammonia and lactate dehydrogenase, as well as cell count (by extrapolating from optical scattering). Similar measurements can be accomplished using a recirculating loop to direct the media through a flow cell in the spectrophotometer before returning to the reactor.
Drapcho points out that bioreactor monitoring using NIR could be turnkey, in terms of hardware. “But a big part of the analysis is the chemometrics. You have to do a calibration for that process for the conditions you’re using. So that’s where you still need to do the up-front work, basically on-site.”
Mini measurements
When setting up your own bioprocesses, it’s sometimes better to run a host of mini reactors in parallel rather than one or more larger ones. Several miniature bioreactor systems are available that use small volumes to quickly optimize processes at lower operating costs and higher information content.
Among these is m2p-labs’ BioLector®, a benchtop system based on 48-well microtiter-plate formats that offers online monitoring of common fermentation parameters, including pH, DO and biomass concentration, using noninvasive optode optical sensors. Because it features a fluorescence reader, fluorescent molecules such as NAD(P)H, riboflavin and “fluorescent proteins engineered to detect product expression” also can be queried, says managing director and cofounder Frank Kensy.
However you set up your cellular manufacturing plants, you need to know what’s happening on the factory floor. These days, that may mean anything from eyeballing a sample, to looking at an LED or touch-screen readout, to having the process-management system send a text to your mobile. Your bioreactor has never been so transparent.