The confluence of FDA’s PAT initiative with the biotech boom has meant a flourishing of bioprocess monitoring sensors and products—and the use of NIR and other traditional analytical devices in more applications. Bioprocesses are difficult enough to understand, so why not leverage any and all technology at our disposal? Amgen’s scientific executive director Duncan Low, speaking at the ISPE annual meeting last fall, hinted at a coming explosion of online bioprocess data—a time when PAT might be used not just for dissolved oxygen, glucose, or cell culture mass, but also sub-visible aggregates, protein variants, process-derived impurities, and even immunogenicity.
A challenge that has confronted bioprocess monitoring is the increasing use of disposables and their seeming incompatibility with most sensor technology. Single-use and automation do not mix, it would seem. Fortunately, that view is shortsighted, as sensor developers look toward disposable technologies as well. Upstream, vendors are beginning to offer products that conform to disposable bioreactors—sensors enveloped in disposable sheaths that are compatible with biobag ports, and even those with completely disposable components.
A sign of things to come: GE was recently awarded a patent (#20090289792) for a precalibrated, disposable sensor incorporating RFID technology, to monitor various conditions upstream and down. The device “comprises a radio frequency based sensor; a support for positioning the sensor in operative proximity to the inside of the container, and a pick-up coil in operative association with the sensor,” the application states.
| SciLog's SciCon
As for what’s already on the market, SciLog, Inc. (Middleton, Wisc.) is offering fully disposable sensors for temperature, pressure, and conductivity measurements, primarily for downstream bioprocessing. The company produces automated, single-use platforms for filtration and liquid handling, and so it was a natural outgrowth to move into single-use sensors as well, says Karl G. Schick, Ph.D., co-founder and chairman of SciLog.
With the proliferation of PAT-ready sensors, a broad spectrum of quality has evolved. “Customers do not have the time, inclination, or background to fully analyze all the sensors being offered,” Schick says. “That’s why we’ve tried to be proactive in publishing information regarding performance, and to let customers know what the limitations are.” SciLog’s sensors are precalibrated, an increasing trend in the marketplace and a critical factor in assuring that manufacturers get the value they seek out of advanced sensors (Box below).
What does SciLog have in development? Schick says he is working on laser-based sensors to monitor the quality of filtrates, as a way of ensuring that filters conform to their porosity standards or have not leaked. Expect this product in a few months, he says.
Finesse Solutions (San Jose, Calif.) has benefited from a recent cooperative R&D agreement with Biogen Idec—Biogen tests and provides feedback on sensors and software before they hit the general market. Finesse now has sensors that monitor and measure dissolved oxygen, pH, temperature, and pressure, including those that support disposable processes.
| Finesse TruFluor pH
Sensor probes have not always been made with disposable bags in mind, notes co-founder and CTO Mark Selker, and thus Finesse has developed products to adapt specifically to the single-use products of Thermo Fisher Hyclone, or any single-use bioreactor that currently uses a port to insert traditional electrochemical probes. (Finesse’s software and control algorithms have been developed both on Emerson’s DeltaV platform as well as an Intel microprocessor-based platform; the software has been designed to allow sharing of vessel configurations and “recipes” between platforms.)
The photo (left) shows the TruFluor pH and temperature sensor—consisting of a disposable sheath, an optical reader, and a transmitter—combined with a Hyclone SUB (Single-Use Bioreactor). The reader (on the left) is inserted into the disposable sheath, which comes pre-installed in the SUB. The sheath is gamma radiated with the SUB, thus the sterile barrier is never broken, explains Selker. The reader can be re-used while the sheath is disposed of with each bag.
Also last year, Finesse released TruTorr, a pre-calibrated, gamma radiation resistant single-use sensor for measuring headspace pressure in single-use containers. “Our starting point for sensor design was that there had been no dramatic change in technology for bio-processing for 20 years,” says Selker. Technologies such as FRET (fluorescence resonance energy transfer) and surface-enhanced Raman scattering are very powerful but products made using these techniques can be improved upon, he believes.
Many of the company’s products for single use applications rely upon real-time phase fluorometric detection, but with specially designed equipment that minimized dye degradation, and thus stalls sensor drift. Fluorimetry is a “nice technology,” Selker says, but Finesse aims to push the envelope with new products—to bring a Silicon Valley approach to bioprocess monitoring and control, he says.
Polestar Technologies (Needham Heights, Mass.) has launched the iDOT (integrated Disposable Optical Transducer) single-use sensor, which it claims can monitor oxygen, pH and carbon dioxide. The pre-calibrated sensor is installed into biobag ports and sterilized with the whole system.
The Criticality of Calibration
While the whiz-bang aspects of today’s process sensors get most of the attention, it’s often their calibration that is critical to their success. Or failure—FDA continues to pay heed to calibration control. Last year, the Agency cited one manufacturer for: “failure to retain original calibration data”; “calibration data . . . do not correspond to the data observed by our investigators”; and “written records of calibration were not adequately verified.”
“You need to know what’s going on in your solution,” says Karl Schick of SciLog. “But that information has to be accurate. Otherwise, you’re flying blind.”
SciLog calibrates its sensors in an aseptic environment, packages them in a Tyvek bag along with a certificate of calibration, Schick explains. Most customers seek a standard setting, but customized calibrations are done as well. An example: Disposable bioprocess bags have been known to rupture, so some manufacturers are taking the precaution of installing a pressure sensor at fill bag inlets to ensure user-defined pressure levels are not exceeded, Schick says.
Precalibration becomes more critical in a single-use environment, Schick says. With bags, tubing, manifolds, and such all presterilized, performing field calibration is not an option. Manufacturers have been known to create a diverted loop from their process, using gamma-irradiated tubing and a glove box in which to calibrate a sensor on site, Schick says. But the risk of introducing a diversion from a process is obvious. Nor is calibration post-process typically viable, since it cannot guarantee the accuracy of the sensor as it being used.
Calibration requires traceability as well, and SciLog has introduced a proprietary (and gamma-stable) memory chip into its sensors to track calibration values and sensor response, as well as extractable reports, production lot numbers and various other data. (The data can later be uploaded to a PC post-use.)
In fermentation, particle tracking can be used to monitor increase in bio-mass, essentially the product of the reaction. Roche Innovatis’ (Bielefeld, Germany) Cedex HiRes is an automated cell culture analyzer, for advanced cell count including differentiation between live and dead cells. It’s the first fully automated Cedex, says Daniel Allen, account manager for Roche Applied Science.
HEL (Hertfordshire, U.K.) has released the LaserTRACK probe, based on laser reflection technology, providing on-line process measurement of particle size and distribution during fermentation.
Hamilton Company (Reno, Nev.), specialists in fluid measurement, recently introduced the ARC sensor family for process control that includes pH, dissolved oxygen and conductivity measurements. In its Visiferm DO optical sensor (photo), Hamilton claims to be the first company to offer a self-contained oxygen measurement in the 12-mm format common to process and pH electrodes and classical sterilizable oxygen sensors.
The DO uses luminescence technology where energy released from an excited luminiphore inside the sensor cap is quenched, indicating the presence of oxygen. The DO also relies on a 4-20 mAmp output, says Amber Ratcliff, product manager for sensors. “Data is much more robust than any data you would get from a traditional transmitter,” she says. And with no electrolytes or fragile membrane, there is no risk of leakage, she adds. The sensor is autoclavable, as well as steam-in-place and clean-in-place compatible.
Spectroscopy is being used more often for bioprocess analysis as well. There’s been plenty of work done with Near Infrared in bioprocessing, says Todd Strother, PhD, NIR application scientist for Thermo Fisher Scientific. “But most individuals or companies working with cell cultures haven’t really known about it,” he says.
Thermo has just begun to offer turnkey NIR systems for bioprocess monitoring—one lab-oriented and one process-scale—that include the instrument, probe, software, and training. (It’s also working with its Hyclone division to make them suitable for SUB’s.) Depending on the application, NIR is being used for real-time monitoring of glucose, lactate, ammonia, and even cell counts and pH, Strother says. The image (left) shows a transmission cell with fiber optic cables for data transmission; the holes in front serve as ports for cell media.
|Thermo's NIR Transmission Cell
Viscotek (a Malvern company) has just introduced OmniSEC software for gel permeation/size exclusion chromatography (GPC/SEC).OmniSEC has a Windows-based interface, supports most analysis methodologies, and works with varied detectors from multiple vendors. Analysis is streamlined by, for example, a unique proprietary algorithm that automatically determines an accurate and consistent baseline, and tools that make building a column calibration straightforward and intuitive.
Last November, Stratophase (Romsey, U.K.), in partnership with the Centre for Process Innovation (CPI), began marketing an insertion probe variant of its SpectroSens system, for tracking compositional changes and monitoring temperature. SpectroSens relies on an optical silicon chip which measures refractive index, analyzes changes in a liquid on the surface of the chip and directly measures binding of antigens to antibodies attached to the chip. The probe fits standard biomonitoring ports. The system is currently being trialed at the UK’s National Industrial Biotechnology Facility (NIBF).
Sensorin (Burlingame, Calif.) made a splash last year when it introduced a self-calibrating pH sensor and platform for bioprocesses. The company still does not have a product on the market, but recently added several high-profile staff, including CTO Dr. James Wilkins, formerly of Genentech. Founder and president Carolyn Kahn says that she will announce more about the product by this spring’s Interphex show in New York.
The sensor is based upon amperometric technology and has advantages over glass electrodes, optical, and other sensors, Sensorin claims. It is “self-calibrating” in that the internal reference is pH insensitive, Kahn explained at last year’s Interphex show in New York. Both the proprietary pH sensitive and insensitive molecules are chemically stable (ie, they won’t leach) and thermally stable (withstanding temps up to 250 °C) she says.
Cole-Parmer is now offering in-line, single-use conductivity sensors and pressure sensors in lab- and process-scale sizes. Each PendoTECH system consists of a monitor and disposable in-line sensors, and can be used during tangential flow filtration (TFF), cross flow filtration, depth filtration, bioreactor monitoring, or chromatography. Though the sensors are disposable, they can withstand some sterilization and reuse.
Finally, AdvantaPure has been using RFID with disposable tubing, but to monitor the equipment rather than the process. The company’s P•E•T (Process Equipment Tracking) uses to track usage and cleaning cycles for all process equipment (pumps, hoses, bags, valves, etc.) involved with a particular batch, providing user details, product wear data, and validation support.
Stepping Up Automation
Bayer Technology Service's
BaychroMAT from Bayer Technology Services is a platform that aims to automate all bioprocess sampling into one system, for improved time monitoring, modeling and titer prediction. Via a single valve (photo), BaychroMAT distributes specified sample volumes to whatever analyzers the manufacturer uses, then retrieves data from each, collects information in a central database, and makes adjustments to the fermentation process as needed.
The system “does its thing all by itself,” says Allen of Roche Innovatis (which as partnered with Bayer), and can take up to 200-300 samples without operator involvement.
“Companies are warming to it” as new PAT projects come online, he adds, and as Bayer has worked with HPLC vendors like Shimadzu, Agilent Technologies, and Dionex to ensure integration with their systems for titer and amino acid data. It has also partnered with YSI for glucose and lactate determination, and Innovatis AG for online cell counting with a Cedex analyzer
Groton Biosystems' Automated Reactor Sampling (ARS) system can sample from up to eight reactors and deliver to up to four analytical instruments. Groton also has taken to partnering with other vendors to improve its offerings—with DASGIP for advanced sampling interfaces to that company’s parallel reactor system, with Agilent for bioprocess HPLC, and with YSI for interfacing with a nutrient monitor.
The partnership with Agilent—the ARS and the Agilent 1200 Series LC together form a closed-loop system—is another indication that the PAT initiative is encouraging creativeness in bringing HPLC closer to the production process. Biogen IDEC has used the system to monitor 21 amino acids in a CHO cell culture during a a 14-day bioreactor run.
YSI has its own automated sampler in the 7100 MBS (Multiparameter Bioanalytical System), with the capability of measuring six parameters for nutrient and byproduct monitoring, from glucose to lactate to ethanol and methanol.
In October, Mettler Toledo began offering a solution combining its InPro6800 dissolved/gaseous oxygen sensors with its M300 two-channel multi-parameter transmitter. The InPro6800 sensors are often used in inertization applications or gas-phase nitrogen blanketing. The M300’s two-channel transmitter makes it possible to track either gas and liquid phase oxygen, or oxygen and conductivity.