Many biologic drugs, such as antibodies and proteins, and vaccines are produced through the large-scale culturing of mammalian cells. From R&D through screening and production, bioprocess cell culturing must be carefully monitored and regulated to optimize growth conditions, control feeding and prevent contamination. To do so, genetic, physical and environmental parameters are continuously tested. Environmental parameters measured include temperature, pH, Dissolved Oxygen (DO), Dissolved Carbon Dioxide (DCO2), nutrients (such as glucose and glutamine), and waste (such as ammonia and lactate). Many of these parameters have traditionally been monitored using in-situ optochemical and amperometric probes that measure a single parameter and lab-based testing of off-line samples. The latest generation of analytical systems for bioprocess monitoring of environmental parameters (in situ and on line) employ a wider variety of analytical techniques, perform multiparameter testing and provide time and cost savings. These systems are experiencing increased utilization due to rising sales of biologic drugs and growing interest in approaches supported by the FDA’s PAT initiative.

Among the latest techniques to be applied to bioprocess monitoring is Fluorescence Lifetime-Based Sensing (FLBS). FLBS is an optical method based on reading dye-excitation wavelengths to measure DO, pH and DCO2. One supplier of such instruments is Fluorometrix. Founded in 1999, Fluorometrix provides the disposable, patch-based Cellphase, capable of detecting DO, pH or DCO2, as well as the automated Cellstation HTBR-1 bioreactor with integrated sensors for the measurement of all three parameters. Fluorometrix’s technology is also offered by Sartorius with its bioreactors under a 2006 exclusive licensing agreement. The Cellphase consists of a parameter-based patch stuck to the inside of a vessel, which is monitored externally by a phase-based fluorometer. The Cellstation HTBR-1 employs multiple vessels and patches in an automated sensing system, allowing for multiparameter measurement. According to the company’s promotional material, Cellphase systems cost about $2,000.

Joe Qualitz, president and CEO of Fluorometrix, described to IBO how, up until recently, it has been tough for the company to make headway in the bioprocess monitoring market. “At the start, people were looking at this thing as the sort of thing they might buy out of curiosity. They had no faith in it, so it took us about two years to sell our first 25 units. Even now, adoption out of the country is going better than adoption in the country, although it is picking up in both places.”

As for FLBS’s current competition, Mr. Qualitz explained that many new bioprocess monitoring techniques are up against the same adversary: “We do have a few competitors in the optical market, but basically we’re competing with the conventional instrumentation.” He continued, “once [drug manufacturers] get to the production stage, they’re using conventional reactors and conventional probes, so they have a certain confidence level [with the equipment]. That may change in the future, because now there’s a push towards disposable systems, even in the large scale.” Fluorometrix’s sensors require minimal calibration and their disposability reduces sterilization and validation time.

The company’s products are most heavily used during culture-media screening for bioprocess development, where cell media is formulated to optimize cell viability and product output. During development, media is tested in milliliter-volume vessels. Patch technology allows real-time continuous measurement of DO, pH and DCO2 under such conditions, compared to having to periodically sample the media off line. “It turns out that there’s a wealth of information to be had if you can tell what’s going on during the culture. That’s something that hasn’t been practical because it’s very difficult to stick conventional probes into a shaker flask,” explained Mr. Qualitz.

Fluorometrix soon plans to release a number of new products. This includes a dual DO and pH sensor patch, which is currently being beta tested and will be released by the end of summer. In an effort to digitize its sensor technology, the company will introduce a new line of self-adjusting sensors with built-in microprocessors at the beginning of fall, as well as wireless sensors, with the capacity to network up to 256 sensors. Fluorometrix is also creating patches for glucose and ammonia measurement.

Like FLBS, NIR spectroscopy is used in bioprocess monitoring to measure DO, pH and DCO2. In addition, commercialized systems can also measure glucose, lactate, glutamine and ammonia. An in-situ technique, NIR takes spectral readings using transmission and reflectance fiber optics placed into a bioreactor or bioreactor probe slot. Transmission spectroscopy enables measurements when the cell culture broth is clear, and reflectance spectroscopy enables measurements when it is opaque, upon the formation of biomass.

Asked about competing techniques, Robert Mattes, an applications scientist at FOSS NIRSystems, explained: “While there may be some competition from other NIR providers, biopharmaceutical customers are already using other monitoring technologies off line, at line and in situ, so the status quo is a large competitor.” He added, “however, these other technologies either do not give the advantage of real-time analysis, or do not have the ability to monitor multiple parameters and constituents that NIR is capable of.” NIR does come with some drawbacks. It is more expensive than other optical methods and requires chemometric analysis. According to Mr. Mattes, FOSS NIRSystems’ products are mostly used at the R&D and cell-line development stages of bioprocess monitoring. “So far, most of biopharmaceutical applications for NIR have been for bench-top bioreactors and fermentors, from 1 L to 20 L, or pilot scale up to 100 L for R&D and cell line development.”

FOSS NIRSystems offers multiple NIR systems for bioprocess monitoring, including the smaller, laboratory-oriented XDS Rapid Liquid Analyzer, and the XDS Process Analytics Microbundle Analyzer, designed for multiple point analysis. The Microbundle Analyzer comes in three models: for single point, four channel and nine channel multiplexer. The company has been involved in the microbial cell culture monitoring market for over a decade, but it was only until 2002 that the company began to notice a substantial amount of interest in mammalian cell culture bioprocess monitoring. However, Mr. Mattes expects future growth. “It is expected that multiple PAT-type tools will be utilized in large-scale production, as well as NIR to best control and optimize production,” said Mr. Mattes.

Although single-parameter probes, such as optochemical probes and amperometric sensors, are commonly used for bioprocess cell culture monitoring, some instrument manufacturers now offer systems that present these probes and sensors in novel ways. These automated near on-line monitoring systems withdraw samples from a bioreactor with the use of an autosampler, and take environmental measurements with multiple probes and return them to the reactor, forming a closed-loop. “The biosensor technology has a proven track record, and we have added more analyte capabilities to our instruments over the years,” explained Sara Veth, biotechnology consultant at YSI Life Sciences. YSI Technologies provides multiple systems for near on-line measurement using amperometric-based sensors. “YSI uses immobilized enzyme sensors for amperometric measurements to measure many nutrients and byproducts of fermentation and cell culture,” explained Ms. Veth. The company’s systems can measure are glucose, lactate, ammonium, glutamine, potassium, ammonium and xylose. YSI’s biosensor technology is nearly 25 years old, and is based on electrical measurements received when hydrogen peroxide is produced upon oxidation.

YSI offers the 2700 M, a coupled system composed of the 2700 Select environmental analysis system and the YSI 2730, the company’s aseptic monitoring and control accessory. The 2730 pulls a sample from a bioreactor, delivers it to the Select for analysis, and returns the sample to the bioreactor, to form an analysis loop. Other automated options include the 2700 ARS and YSI Flownamics, which are based on respective collaborations with Groton and Flownamics for closed-loop near on line monitoring. Some automated systems can both perform environmental measurements and feeding processes based on the measurement readings. The 2700 and 7100 ARS systems combine the 2700 and 7100 Select (YSI’s more comprehensive system for standard environmental analysis) and Groton Biosystems’ ARS autosampling system. The combined systems can monitor up to four bioreactors, and YSI soon plans on adding a feed control pump option for the system. The YSI Flownamics combines YSI’s Select systems with the Flownamics Seg-Flow 804M autosampling system to monitor up to eight bioreactors. The Flownamics Seg-Flow software can control reactor feeding if a feeding system is added to the setup. All systems come with a choice of sensor bundles, and the most popular for bioprocess monitoring are the glucose and lactate bundle and the six-parameter glucose, lactate, ammonium, glutamine, glutamine and potassium bundle. “We recently added the xylose measurement capability to the YSI 2700 and the YSI 7100 in 2008. We also added ion selective electrodes to the YSI 7100 to measure ammonium and potassium in 2003,” said Ms. Veth, describing the company’s latest technical innovations. According to Ms. Veth, the company’s systems are most commonly used in R&D and process development.