On the Process Analytical Technology group on LinkedIn recently, a member suggested that a turbidity sensor can serve as a good PAT tool for bioprocess fermentation. One of those who questioned this assumption was Mike Ponstingl, owner of Custom Sensors & Technology (CST), which promotes an alternative solution. He wrote: “Turbidity may be a good sensor as a PAT tool but not necessarily in fermentation, especially if you are going to use its output to control feed rate of nutrient solutions. The problem with turbidity is that it measures both live and dead cells including cell debris.”
So what would Ponstingl recommend, then? We spoke with him recently about monitoring fermentation and other bioprocesses:
PhM: What’s the state of real-time monitoring of biopharma processes, and how does turbidity sensing fit into that?
M.P.: Biopharma is starting to embrace the idea of online, at-line or in-situ measurement as a means of real-time control. Most of the applications can be solved by either a forward or backscatter method depending on the concentration of the suspended solids and the reflectivity of the solids. Additionally, a high percentages of the application are done in the NIR region.
Most of the probes on the market are transmission-type probes which measure the optical density of the fluid between two windows. The issue with this design is that, since it is based on pathlength, if that pathlength is too large to accommodate measuring low numbers of cells, then as the concentration increases and the optical density becomes high, the signal saturates and it is difficult to determine the upper level. In addition, with transmission type measurements, you cannot determine the death phase of the fermentation process. This method is not able to see any difference between a living or dead cell.
We are developing a fluorescence approach that utilizes front-surface technology and sees viable vs. cell density. This is a new direction that the technology is moving toward.
PhM: How extensively are turbidity probes used as online, PAT tools in biopharma, and for what applications?
M.P.: The value of turbidity as an online PAT tool is balanced between successful vs. unsuccessful outcomes. Proper application engineering work must precede any installation to insure success. Determination of a successful outcome must be tied to return on investment (ROV). This requires that customers must have a complete knowledge of the costs they incur before the installation in order to make the comparison after installation.
PhM: You suggest that turbidity measurements are not necessarily appropriate in fermentation, especially in adjusting nutrient feed rates. Can you explain a bit more?
M.P.: Turbidity only describes that a particle is scattering or reflecting light. It says nothing about the viability of the cells and viability is everything in a fermentation process. Why would one feed expensive nutrients if the cells were not viable? Viability is an indication of the health of the cells. To a biochemist knowing that the cells can metabolize nutrient feed stock is paramount to having a successful outcome. Employing a fluorescence method will insure throughout the entire fermentation process that the microenvironment surrounding each cell is ideal for its growth.
PhM: As such, what other recommendations do you have for fermentation control? (You mentioned fluorescence biomarkers like Tryptophan and NADH?)
M.P.: Depending on the type of fermentation (aerobic, anaerobic, etc.), one would be inclined to not only monitor grown biomarkers like tryptophan and NADH but may also want to measure the dissolved oxygen, temperature and pH. Each organism will have an optimal growth rate for a given growth media and micro-environmental conditions. Having more information from the above respective PAT tools will allow the end user a more comprehensive knowledge of the fermentation process to which an optimal control strategy can be developed.
PhM: Can the biomarkers provide real-time data? Also, how do they complement other key parameters (DO, pH, temp, nutrient availability) critical to monitoring cell growth?
M.P.: Yes, utilizing a front-surface fluorescence probe will allow the end user to monitor biomarkers in real time. In any of the biosynthetic pathways it is the interaction of all of the above variables (pH, DO, Temp, Tryptophan and NADH) that are indications that the goals of fermentation are being met.
Biopharma manufacturers are just now realizing the value of employing other methodologies like this. Simple adaptation of the method (fluorescence), a reasonable price, and a small footprint are the three goals we have for our customers.
PhM: Would you say that the biomarkers are relatively underutilized by industry for bioprocess monitoring? How are you making inroads?
M.P.: Yes, at this time I believe CST is the only company that has solved the sample interface issues that allow in-situ measurements and can monitor biomarkers in real time. We have successfully worked with other technology providers like Pendotech to develop total solutions for their customers. We supply a very simple, low-cost unit that will fit in the palm of your hand and one that can be integrated with their other products (temp, pressure, flow, etc.).
PhM: Finally, you’ve worked with GSK and other manufacturers on other fluorescence applications. What can you tell us about new developments and what you’re working on?
M.P.: Expansion of the smart sensor platform to include multi-wavelength fluorescence and UV-VIS absorbance are our new projects.