Riding a Workhorse Method
Process monitoring using high-performance liquid chromatography, arguably the workhorse analytical method in biology, is an intriguing idea that is just now catching on. Traditional HPLC has for years been deemed too slow for in-line or online measurements, but wider adoption of fast LC techniques like uHPLC (ultra-HPLC) and monolithic LC columns is changing the equation.
The utility of fast LC monitoring depends on the application, says Helmut Schulenberg-Schell, Ph.D., worldwide marketing manager for LC at Agilent Technologies. For amino acids and small peptides, uHPLC’s one-minute cycle time can be quite efficient for monitoring runs in near-real-time. “But for proteins or larger molecules, runs are significantly longer and fast feedback more difficult to achieve.”
Nevertheless quite a few large pharmaceutical companies, according to Dr. Schulenberg-Schell, are working toward the goal of real-time uHPLC-based monitoring. Moving uHPLC equipment into process suites is easy, and the equipment is robust.
The challenge, he says, is to combine knowledge from several disciplines, for example the process engineer running a bioreactor, sampling, chromatography, and data analysis. Automation can be a tremendous help but analyzing chromatograms, for purposes of real-time control, will probably have to wait until the first shift crew arrives.
HPLC is more likely found in a PAT-like role during process development. Shelly Pizarro at Genentech has published a technique for confirming the validity of dissolved oxygen on a protein refolding reaction using reverse-phase HPLC. The overriding quality issue was to achieve sufficient levels of active protein while avoiding oxidative degradation.
Working with a 3 L bioreactor, Pizarro correlated folding and degradation levels to DO concentrations as measured directly by in-line sensing. She found that DO profiles and refolding correlated and were comparable across scales, thus validating her PAT approach.
Traditional process monitoring is based on life-size sensors, transmitters, and probes familiar to most bioprocessors. Most of these simply don’t fit the form factors of process-development equipment, which creates a disconnect between PAT-worthy large-scale processes and the smaller vessels in which process ideas are evaluated.
In fact Govind Rao, Ph.D., of the Center for Advanced Sensor Technology at the University of Maryland Baltimore County, argues that lack of adequate sensors has hamstrung process development.
Early process development is characterized by extensive experimentation on process parameters that is generally carried out in many small, un-instrumented “scouting devices” such as flasks, small bioreactors, and microtiter plates. “Ideally, one would want to employ sensing and monitoring of quality-related parameters at the very earliest stages,” Dr. Rao explains.
The cost of “instrumenting” small-scale devices has been prohibitive, however, and the underlying sensing technology inadequate. “Current technology does not permit sufficient control of process scouting devices to adequately replicate large-scale processes.” Part of the problem is integration, and the need for conventional sensors to come into contact with process fluids.
The introduction of disposable sensor devices is beginning to change this picture, Dr. Rao says. In particular, he mentions low-cost, disposable, semi-invasive optical sensor patches that are easily calibrated and provide through-space readout.
These sensors reside within the process but transmit signals through the vessel wall. Several such devices, for example for measuring pH, dissolved oxygen, pCO2 have been developed in Dr. Rao’s lab. These sensors have been commercialized by Fluorometrix and sub-licensed to Sartorius Stedim Biotech. Presens, Finesse Solutions, and other manufacturers offer similar sensors. In addition to being used with process scouting devices, these sensors are finding use in conventional and disposable bioreactors ranging from a few milliliters to several hundred liters.
Issues still remain with microsensors. Foremost is their proprietary status. End-users are uncomfortable with being locked in to a single supplier, while the lack of complete information on sensor composition causes problems with toxicology departments and potentially with regulatory agencies.
To circumvent these issues, Dr. Rao’s lab has developed next-generation technology where the sensors are placed outside the bioprocess vessel with a suitable barrier. Sartorius Stedim Biotech has acquired the exclusive license to this technology, which can allow for entire bioprocess trains to be integrated and monitored using noninvasive sensors.
Ultimately, Dr. Rao feels that a broad technology sharing consortium approach is essential for widespread adoption of the sensor technology and for its incorporation into disposables. He likens the development of the USB standard for computers as an example that the bioprocess instrumentation community could follow.
Such an approach could revolutionize the field with plug-and-play sensors that would be available from several vendors and yet offer standard outputs for connectivity to control systems. This evolution will greatly facilitate PAT approaches that will drive QbD.
Cleaning and cleaning validation, while not technically manufacturing steps, nevertheless factor into processing time. Hyde Engineering has introduced a technique, based on total organic carbon (TOC) analysis, for verifying the effectiveness of clean-in-place (CIP) operations. TOC should drop as cleaning proceeds.
The technique collects rinse water from the CIP return lines and delivers it to a high-throughput GE 900 Turbo TOC analyzer. A pump pulls rinse samples for systems under vacuum, while the return pump suffices for CIP systems under positive pressure.
The GE instrument takes a data point every 4.5 seconds, but the technique is not quite that fast. Due to residence times within the instrument and sample lines, and instrument equilibration, there is a two- to five-minute delay between sampling and readout.
“This is not quite an in-line sensor, it’s more online,” comments Keith Bader, director of technical and quality services at Hyde.
Timeliness is approximately the same as that of an at-line analyzer where samples are pulled manually. The advantage lies in the automation, which greatly reduces the likelihood of sample contamination.
Bader believes the the TOC technique, which has been tested at a large biopharmaceutical manufacturer in a microbial fermentation, will eventually be used as a release test, or verification of CIP effectiveness. “Users can determine within five minutes of completing a cleaning cycle if the equipment is clean,” he says, based on a method that is orthogonal to traditional criteria such as conductivity.
Another essential but nonproduction step, buffer preparation, has come under increasing scrutiny due to the capital expense for holding/mixing tanks, related cleaning and cleaning validation, and the serious demands these activities place on floor space. Disposable buffer handling bags can eliminate the first two concerns, at least up to the bag’s working volume, but concerns over where to put these tanks—whether full or empty—remain.
In-line buffer dilution can reduce the need for storage tanks but precision metering of buffer concentrates has traditionally been a problem. Michael Li, Ph.D., process sciences manager at Asahi Kasei Bioprocess, has devised a solution based on real-time conductivity measurements that is fully PAT worthy.
Because it is real-time and controls the flow of both concentrate and water, the technique does not require precise composition of the buffer concentrate to achieve exact final buffer concentrations. “Once you know the proper conductivity of the end solution, the feedback control assures consistency every time,” Dr. Li says.