Revolutionary Technology in pH Measurement Elicia Wong, PhD Mar 17-19, 2009

pH Control Wide Applications: Why is it Important in bioprocessing? Pharmaceutical/biotechnology (medicine) Cosmetic (perfume) Food and beverage (wine, sugar) Water purification & wastewater treatment Petroleum/deep sea/outer space exploration Why is it Important in bioprocessing? Critical quality parameter for cell and bacterial growth and metabolic activity, productivity and consistency. 2 L 80 L 400 L 2,000 L 10,000 L Upstream Process Downstream Process Why is it a problem in bioprocessing? Drifting Fouling Calibration

Outline Potentiometric pH measurement Glass electrode Existing pH measurement technologies Potentiometric pH measurement Glass electrode Ion-selective field effect transistor (ISFET) Optical pH measurement New technology Amperometric pH measurement

Potentiometric Approach – Glass electrode External reference electrode Measuring electrode High impedance pH meter Glass frit Glass membrane Inner solution Metal wire Glass frit Inner solution External reference electrode High impedance pH meter Glass membrane Glass electrode Voltage difference builds up on the sides of the glass membrane due to difference between H+ activities on both sides This voltage difference is measured with the help of an external reference electrode Often reference and measuring electrodes are combined in one single structure Fragile, wet storage, long term drift, requires frequent calibration

Ion-Selective Field Effect Transistor Vgs External reference electrode Analyte solution Solid state device Insulator Ion-selective interface Insulator Source Drain Electron path Vds Current flows Four major components: Source (s), Drain (d), Ion-selective interface and Gate (g). Hydrogen ions at or near the ion-selective interface will cause a variable voltage potential (Vgs – Vds) Cathode (-) No current flows Anode (+) H+ - - - - - +

Optical pH Measurement System Photo-detector Photodetector, optical fiber, sensor Sensor = fluorophore on solid support An incident beam of light is passed to the sensor, where it interacts with the fluorophore. The emitted light intensity is pH dependent Keep in dark (photo-bleaching) Sensor Optical light guide Incident beam Emitted light

Comparison Feature Glass Electrode ISFET Optical Robustness Glass (fragile) Solid state Calibration Required (frequently) Required (longer calibration interval) Required (factory+on site) Stability Drift Maintenance Intensive (wet storage) Easy (dry storage) Intensive (kept in dark (photo-bleaching) Temperature Range Wide Narrow pH range 0-14 5-9 (in ranges) Biological Fouling Occur Sensitivity  Low  Medium Good 

Amperometric pH Sensor Power supply Reference electrode Counter electrode V i Counter electrode Analyte solution pH-sensitive interface Internal reference pH-sensitive interface + Internal reference External reference electrode V V V V i i i i H+ H+ H+ H+ pH-sensitive interface semiconductor H+ H+ H+ H+ pH-sensitive interface semiconductor

Self-Calibrating Internal reference = pH insensitive molecule pH 10 pH sensitive molecules Internal reference

Stability Both the proprietary pH sensitive and insensitive molecules are chemically stable (won’t leach) and thermally stable (able to withstand high temperature up to 250 C). No degradation in performance with continuous operation and after heat sterilization. No degradation in signal with 2-day continuous operation after autoclaving Responses were observed after multiple autoclaving cycles (measurement performed after each autoclaving cycle) Continuous operation Multiple Autoclaving

Free from Biological Fouling pH detection relies purely on chemical interaction between hydrogen ion and sensing interface rather than membrane diffusion effect (glass electrode) - H+ Clotting of glass membrane H+ Continuous measurement in medium containing living cells pH sensitive molecules Detect change in pH from pH 7.82* to 7.01* Without pH control pH insensitive molecules *pH verified by off-line pH measurements

Ideal for bioprocess pH measurement Summary Chart Ideal for bioprocess pH measurement Feature Glass Electrode ISFET Optical Amperometric Sensor Robustness Glass (fragile) Solid state Calibration Required (frequently) Required (longer calibration interval) Required (factory) Self calibrated Stability Drift No drift Maintenance Intensive (wet storage) Easy (dry storage) Intensive (Kept in dark (photo-bleaching) Temperature Range Wide Narrow pH range 0-14 5-9 (in ranges) Biological Fouling Occur NO Sensitivity  Low Medium Good  Good