In 1998, the US Pharmacopeial

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1 B IOP ROCESSTECHNICAL On-Line TOC Analyzers Are Underused PAT Tools José E. Martínez In 1998, the US Pharmacopeial Convention (USP) implemented changes to its water monographs used in the pharmaceutical industry. One change was the inclusion of total organic carbon (TOC) analysis and deletion of the oxidizable substance test. USP test monograph <643> calls for TOC tests to be performed using a calibrated instrument, with periodic demonstration of its suitability (1). It also requires analytically dependable low limits of quantitation for TOC analysis and a true TOC measurement for standardization and validation. USP test monograph <645> calls for measuring conductivity accurately using calibrated instrumentation (2). It also states that the frequency of recalibration is a function of instrument design, degree of use, and so on. Implemented 1 July 1999, European Pharmacopoeia (EP) method is the equivalent of USP <643> and has the same analytical requirements (3). PRODUCT FOCUS: ALL PRODUCTS PROCESS FOCUS: MANUFACUTURING WHO SHOULD READ: ANALYTICAL LABORATORIES, MANUFACTURING, AND FACILITY PLANNING/DESIGN KEYWORDS: PAT, UTILITIES,TOC ANALYSIS, IN-PROCESS WATER LEVEL: INTERMEDIATE 30 BioProcess International DECEMBER 2004 TOC and WC analytical tests can be performed off-line and/or online. Of course, both analytical and economical advantages are realized in performing both tests on-line. By design, on-line TOC analyzers perform both tests at the same time with the same sample. Such analyzers are easier to use and less trouble-prone than desktop (offline) units, but they are not used as much for release testing of pharmaceutical waters as they should be. This is probably due to qualification uncertainties as well as the fact that online TOC is relatively unfamiliar. As a result, companies miss out on the huge savings in time and money that on-line TOC analyzers can provide not to mention their higher accuracy and precision in readings. Results of a recent survey by one TOC measurement equipment manufacturer suggests that fewer than 10% of companies use on-line TOC analyzers for release testing (4). By quantifying carbon, TOC provides an indirect measure of organic molecules present in pharmaceutical waters. Those organics come from source water, from purification and distribution system materials, and from biofilm growing in the water utility system. TOC can also be used as a tool for monitoring the performance of different steps in water purification for production of purified water (PW) and water for injection (WFI). Such processes may include sand An example TOC analysis system from Ionics Instrument Business Group ( filtration, water softening, membrane filtration, reverse osmosis, distillation, storage and distribution systems, and periodic sanitation. Several methods are acceptable for analyzing TOC. In the basic standard solution method, a theoretically easy-to-oxidize solution gives an instrument response at the attribute limit. Analytical technologies are qualified by challenging their capability using a theoretically difficult-to-oxidize solution to determine system suitability. All analytical technologies used to measure TOC share the objective of completely oxidizing the organic molecules in an aliquot of sample water to yield carbon dioxide (CO 2 ). A detector then measures the amount of CO 2 present and applies the result to a calibration curve that gives a corresponding TOC value. These technologies must discriminate between inorganic carbon (IC), which may come from sources such

2 as dissolved CO 2 and bicarbonate, and the CO 2 generated by oxidation of organic molecules in the sample. Regarding pharmaceutical waters, USP <643> presents two welldescribed analytical approaches. The first one determines TOC by subtracting measured IC from the measured total carbon (TC), which represents the sum of both organic and inorganic carbon: TOC = TC IC. The second approach first purges IC from a sample before any carbon measurement is performed. However, that purging step also purges some organic molecules, which can be retrapped, oxidized to CO 2, and then quantitated as purgeable organic carbon (POC). Remaining organic matter in the sample is also oxidized to CO 2 and quantitated as nonpurgeable organic carbon (NPOC). In the latter approach, TOC = POC + NPOC. For pharmaceutical waters, the amount of POC is negligible and can be discounted, so NPOC = TOC. PROCESS ANALYTICAL TECHNOLOGY The goal of the FDA s recent Process Analytical Technology (PAT) initiative is to understand and control manufacturing processes (5). This goal is consistent with the current agency philosophy that quality cannot be tested into products; it should be designed or built in. The FDA Process Analytical Technology Team defines PAT as a system for designing, analyzing, and controlling manufacturing through timely measurements (during processing) of critical quality and performance attributes of raw and in-process materials and processes with the goal of ensuring final product quality. (5) Improvements in product quality and safety, as well as manufacturing efficiency, can be expected after successful implementation of a PAT process control system. Some typical improvements include reducing production cycle times by using on-, in-, and/or at-line measurements and controls; increasing automation to reduce the incidence of human error; facilitating continuous processing to manage variability and improve efficiency; and improving energy and material use. Because they already fulfill its requirements, on-line TOC analyzers are ideally suited to be under the PAT umbrella. Such analyzers therefore should be frontrunners in the PAT initiative. OXIDATION TECHNOLOGIES Over the years, many TOC analyzers have been introduced by different manufacturers using different oxidation technologies. All instruments currently on the market use either the combustion method or low-temperature oxidation. The latter involves chemical oxidation aided by 100 C heat or ultraviolet radiation and a persulfate (ammonium persulfate, for example) or by UV irradiation alone. These major oxidation technologies dominate the market: combustion, ultraviolet, and UV persulfate.

3 Table 1: Official TOC testing methods Oxidation Detection Analytical Range Official Method Technology mg/l Methods UV/persulfate NDIR ,000 EPA 415.1, 9060A and heated Standard Methods 5310C persulfate ASTM D2579 ISO 8245, USP 643 AOAC Combustion NDIR ,000 EPA 415.1, 9060A Standard Methods 5310B ASTM D2579 ISO 8245, USP 643 AOAC UV persulfate Membrane/ Standard Methods 5310C, USP SP 643 conductivity ASTM D5997, ASTM D5904 UV Conductivity or USP 643 NDIR and membrane + conductivity Table 2: Savings per year by implementing on-line testing of TOC and conductivity Cost/hour Yearly Item Quantity ($) Savings ($) Sampling time of WFI 2,100 hours 21 44,100 and/or PW per year Conductivity analysis time/year 920 hours 21 19,320 TOC analysis time/year 2,940 hours 21 61,740 Materials/reagents used $31,198 N/A N/A in off-line tests per year Estimated materials to be used $18,560 N/A N/A in on-line tests per year Estimated savings in N/A N/A 12,638 materials/reagents per year Total 137,798 Combustion Oxidation: The combustion technique uses temperatures of at least 680 C in a stream of air, oxygen, or nitrogen (N 2 ) usually in the presence of a catalyst: cupric oxide (CuO), cobalt oxide (CoO), or platinum (Pt). Both particles with organics and dissolved organics oxidize fully to CO 2 under these conditions. Persulfate and Other Wet-Chemical Oxidization: Particles in a sample present a problem to all three lowtemperature technologies. With PW and WFI samples, however, intrinsically low particle levels make it less of an issue. Typically, organics in particles are more difficult to oxidize because some molecules escape exposure to reagents by 32 BioProcess International DECEMBER 2004 being inside them. High molecularweight compounds such as proteins may be slow to oxidize with lowtemperature techniques. But those technologies analyze large sample volumes, thereby improving the low limit of detection. Also, blank values will be very low if pure reagents are used, therefore improving accuracy and precision. A blank is the contribution to TOC by the dilution medium (water), carrier media (e.g., nitrogen) and reagents if used. The blank contribution is subtracted from a final TOC reading. With ultraviolet irradiation alone, a sample is exposed to UV light from a mercury vapor lamp source. The mechanism of UV-promoted oxidation relies on negatively charged hydroxyl (HO ) radicals generated from the irradiation of water with UV energy. With sufficient exposure, all dissolved organics can be oxidized to yield CO 2, detection of which is usually by a change in the sample s conductivity. A direct measurement of conductivity is made, or the CO 2 is allowed to permeate through a membrane into low-conductivity water (thereby changing its conductivity). TOC analyzers using direct measurement include the Anatel A1000 and A643 ( and the Thornton 502P and 550 ( Sievers TOC analyzers (400 DI, 400 ES, 800, and 900) from Ionics Instrument Business Group ( use the membrane technique. Alternatively, CO 2 can be purged into a nondispersive infrared (NDIR) detector. In practice, the maximum amounts of TOC measured range from 1 to 2 ppm. The direct conductometric method can involve significant positive interference from chlorinated organics produced during periodic increases in chlorination due to special conditions (storms, spring run-off, municipal water distribution sanitation, and so on) (6). The semipermeable membranes used in the membrane-based methods exclude from measurement all foreign ionic species present in the sample or produced during oxidation. For the heated persulfate method, a sample is mixed with a quantity of persulfate solution and heated to about 100 C for digestion. After a set period, the resulting CO 2 is purged out by a carrier gas (typically nitrogen, N 2 ) and detected either by NDIR or by allowing the gas to permeate into a conductivity detector. Heated persulfate oxidation is better than UV alone, but users may need to optimize their digestion times for complete oxidation. With persulfate + UV irradiation, a sample is simultaneously exposed to

4 both before the resulting CO 2 is purged out by a carrier gas and detected by NDIR. This oxidation method offers significantly better results than either the UV- or persulfate-only method because it simultaneously ionizes dissolved organics and produces highly reactive sulfate and hydroxyl free radicals. With this methodology, all available organic carbon is oxidized, allowing for readings that are accurate in the parts-per-billion range. ANALYTICAL CONSIDERATIONS Requirements for official TOC analytical methods are well defined, as Table 1 describes. All TOC analyzers today convert sample organic carbon to CO 2. A variety of detection technologies are used to measure the results. But only two detection technologies are used to measure TOC in PW and WFI: conductivity and NDIR. The range of TOC measurements varies by oxidation method and detection technique. A combustion NDIR method can quantify ,000 ppm of carbon in a sample, whereas quantification by UV and UV persulfate methods with NDIR or conductivity detectors ranges from ppm to 50 ppm. In general, NDIR detectors are sensitive to low amounts of TOC, and conductivity detectors can measure very low levels. But they are sensitive to ionic interference, a BENEFITS OF ON-LINE TESTING Minimization of Sample Contamination: Elevated TOC levels can be observed in grab samples due to contamination by organic compounds from the sample bottles or during collection. The same applies to conductivity measurements. This is demonstrated by Figures 1 and 2, in which on-line TOC and WC tests give values about one-third of those from the off-line tests. The difference is believed to result from contamination of the off-line samples. Continuous on-line determinations of TOC and conductivity enable users to profile TOC and conductivity changes in their water systems and minimize the risk of missing deficiency that can be minimized by using semipermeable membranes that are selectively permeable to CO 2. NDIR technologies for gas measurement rely on the energy absorption characteristics of each gas in the infrared region of the spectrum. A simple NDIR instrument passes infrared energy through two identical tubes to a detector. The first tube serves as a reference cell and is filled with a nonabsorbing gas such as N 2 ; the second is a measurement cell containing the gas sample to be analyzed. IR energy in the region of excursions and out-of-specification results. Laboratory labor costs associated with collecting and analyzing samples for both TOC and conductivity are reduced. No Glassware Required: In off-line TOC testing, vials that have not been precleaned can contribute significant background TOC. If no alternative is available, the vials should be rinsed thoroughly with low-toc water before use to minimize contamination. Less Variability: Figures 1 and 2 clearly show less variability in online tests than in off-line tests of both TOC and WC. interest is absorbed by the sample, attenuating the energy passing through the cell and falling on the detector. That attenuated energy is compared to the unattenuated signal from the reference cell, with a difference proportional to the amount of absorbing gas in the measurement cell. NDIR analyzers can operate at the low TOC levels required for meeting USP and EP requirements, but they require much more effort from user technicians to keep blanks under control. Unfortunately, it is difficult to produce CO 2 -free blanks. Just the act of processing a Figure 1: Water-for-injection (WFI) total organic carbon (TOC) Figure 2: WFI conductivity data 34 BioProcess International DECEMBER 2004

5 sample can contaminate it with environmental CO 2. These limitations are exacerbated in NDIR technology because it measures CO 2 directly rather than bicarbonate. Field experience has demonstrated that both direct and membranebased conductometric methods produce better TOC results with less calibration, maintenance, and support than the NDIR analyzers at the TOC levels found in pharmaceutical water systems. ANALYTICAL PARAMETERS Four analytical parameters are extremely important to consider when choosing a TOC analyzer: recovery, blank contribution, detection limits, and particles with organics. For PW and WFI samples, the particles with organics are not an issue. Recovery: The technical literature is equally divided on whether it is better to oxidize dissolved organic compounds by persulfate digestion or combustion. Only when testing difficult particles such as crystallized cellulose does the combustion method show a significant advantage. With high levels of organic particles or TOC, it is clearly the best approach. The latest UV and UV persulfate TOC analyzers achieve much higher recoveries than older-generation models because of higher persulfate concentrations and/or advances in UV lamp design. So both methods are perfectly suited for routine tests of PW and WFI samples. Blank Contribution: In most cases, this analytical parameter is important only for low-level TOC measurement such as in potable water, high-purity water, PW, and WFI. Blank contribution is carbon associated with the TOC analyzer itself. Some sources include persulfate, dilution water, and the catalysts used in combustion-based TOC analyzers. The amount and stability of a blank has a major effect on an analyzer s detection limits and reproducibility. Blank contribution to sample analysis by UV, heatedpersulfate, and UV persulfate 36 BioProcess International DECEMBER 2004 With >90% of pharmaceutical water systems running at <70 ppb TOC, it is scientifically and economically SOUND to use on-line TOC analyzers for release testing of PW and WFI. methods is well known to be smaller than that of the combustion method. Detection Limits: TOC testing of PW and WFI samples requires a very low instrument background. Analyzers that use UV, UV persulfate, or heated-persulfate oxidations typically show low background interference. High blanks are the primary disadvantage of combustion methods. The UV, heated-persulfate, and UV persulfate methodologies can easily quantify TOC below USP <643> requirements (<50 ppb). COMPARING ON-LINE AND OFF-LINE TESTING The Benefits box lists several benefits associated with on-line sampling and testing of PW and WFI. Figures 1 and 2 compare two on-line units (Sievers 400-ES) with desktop units (Sievers 800-AS) for TOC and conductivity test results from a WFI system. Small differences in conductivity values between supply and return units (seen in Figure 2) were expected. The reason is simple: WFI acquires a very small amount of ions that normally leach from stainless steel pipes. So an on-line TOC unit installed at the return of the distribution system should give higher WC values than one installed on the supply side. Such results are not seen in on-line TOC values. THE ECONOMIC PICTURE There are economic reasons to adopt on-line methods in addition to the quality aspects (lowered variability and better accuracy) of removing the potential for off-line contamination of samples. A typical off-line TOC test costs $20 50 per sample, depending whether the tests are performed in-house or forwarded to an outside laboratory. The numbers of daily and weekly tests determine the overall cost per year for a given pharmaceutical or biotechnology company. Table 2 summarizes a typical example of TOC WC testing expenses for one of two PW WFI generation trains at a medium-sized biotech company. The yearly savings can be substantial. Total cost of implementation for on-line Sievers 400-ES TOC analyzers was about $120,000 including installation, calibration, and qualification. Note that in this case a total of four on-line TOC analyzers were installed, two in the PW system and two more in the WFI system. In each water system, one unit was installed on the supply side and the second at the return. If only two units are installed, then the implementation cost should be about $60,000. The payback time for the four units is about a year. QUALIFICATION OF ON-LINE TOC ANALYZERS In general, on-line TOC analyzers should be qualified as laboratory instruments (like desktop TOC analyzers and conductivity meters) regardless of where they are installed. Performance qualification (PQ) should be performed in parallel with desktop analyzers. Equivalency between off-line and on-line units should not be pursued; on-line measurements of TOC and conductivity are usually lower than those of desktop units. Although the behavior is the same in on-line and off-line test results (Figures 1 and 2), the on-line test results are about onethird the value of the off-line results. A lot of planning and care should be taken when installing and qualifying on-line TOC analyzers in

6 FOR FURTHER READING Wallace B. Common Total Organic Carbon Terms (Application Note). Tekmar-Dohrmann: Cincinnati, OH, Clesceri LS, Eaton AD, Greenberg AE. Standard Methods for the Examination of Water and Wastewater (19th Edition Supplement). American Public Health Association American Water Works Association, Water Environment Federation, Washington, DC Furlong J, Wallace B. Total Organic Carbon Analysis for Difficult Matrices: An Ultraviolet Persulfate Approach (Application Note). Tekmar- Dohrmann: Cincinnati, OH, Srikanth B. Recent Advancements in UV Technology Yield Enhanced TOC Reduction Performance. Ultrapure Water 15(6) July/August 1998: Furlong J, Booth B, Wallace B. Selection of a TOC Analyzer: Analytical Considerations (Application Note) Tekmar-Dohrmann: Cincinnati, OH, Cooper K. A New Approach to Total Organic Carbon Analysis Using Closed-Loop Photocatalytic Oxidation. Presented at ISA Analytical Division Symposium, April 2002 (Denver, CO) and ISA National October 2002 (Chicago, IL); Anatel Corporation, Loveland, CO; cold-water systems. Ideally, these instruments work better and more reliably in hot-water systems because hot water sanitizes the tubing connections between them. That inhibits the growth of microorganisms and biofilm development inside the tubing that carries water to the TOC analyzer. In the world of TOC analysis, microbial contamination means a lot of trouble. However, on-line analyzers can be implemented and qualified successfully in cold-water systems if special precautions are taken during installation and the right preventive maintenance is in place. WORTH THE INVESTMENT For monitoring purposes, the US and European pharmaceutical industries have implemented on-line analyzers almost exclusively with UV and direct-conductometric or UV and membrane-conductometric technology. In pharmaceutical laboratories, however, the UV persulfate and membraneconductometric are the methods of choice. For USP and EP compliance (release testing), the choice seems to be either UV persulfate and NDIR or combustion NDIR TOC analyzers. The strength of both conductometric methods (UV and UV persulfate), either direct or membrane based, is that they CIM recover functional protein with AthenaES protein refolding kit TM Accelerate Your»Viruses are purified extremely fast and maintain Infectivity and Immunogenicity.«Professor Alois Jungbauer, University of Natural Resources, Austria Plasmid DNA purification is one order of magnitude more efficient than using traditional resins. Dr. Roman Necina, Head of Biopharmaceutical Production, Boehringer Ingelheim*»The specific activity of Factor IX was increased by one order of magnitude.«professor Djuro Josi}, Brown University, RI, USA *Genet Eng News, 2003, 19 (23), with CIM P r o d u c t i v i t y Monoliths BIA Separations d. o. o. Teslova 30 SI 1000 Ljubljana Phone: Fax: AthenaES Protein Refolding Kit lets researchers determine the optimal refolding buffer for their proteins. Unlike similar products, our refolding kit allows you to examine a wider range of conditions in one experiment. The AthenaES Protein Refolding Kit is the choice of leading researchers around the world for its: Robust Design: uses a fractional factorial matrix Rapid Response: refolding in one hour Simplicity: single-step screening technique Convenience: pre-made buffer solutions using high quality reagents Disclosed Formulations: no hidden or secret ingredients Visit or call our scientific staff at for more information or to order a kit. Restore protein function with the AthenaES TM Protein Refolding Kit. TM AthenaES

7 produce better TOC results with much less calibration, maintenance, and support than are needed by NDIR TOC analyzers. Nevertheless, the direct conductivity methods are prone to false positives by chlorinated compounds (6). Membrane-based methods are superior in that regard. Based on a gross estimate that >90% of the pharmaceutical water systems worldwide are running with less than 70 ppb of TOC, it is scientifically and economically sound to use on-line TOC analyzers for release testing of PW and WFI. It is logical to assume that compliance with USP and EP requirements should be straightforward wherever those analyzers are properly qualified and maintained. In fact, compliance should be improved by the better accuracy and precision of online units. Field experience with four on-line TOC analyzers supports these statements. Operational expenses with on-line TOC analyzers are much lower than with their desktop counterparts. Another source of savings would be a reduction in the number of investigations due to false positives and outliers. So resources formerly used in off-line sampling and testing for TOC analysis can be reallocated to more productive activities. ACKNOWLEDGMENTS My special gratitude to Richard Godec and Francisco Baco for reviewing this manuscript. In addition, my appreciation to Lymarie Caballero for her contributions. REFERENCES 1 <643> Total Organic Carbon. United States Pharmacopeia 27th Edition and National Formulary 22nd Edition (USP 27 NF 22). US Pharmacopeial Convention, Inc.: Rockville, MD 2003; 2 <645> Water Conductivity. United States Pharmacopeia 27th Edition and National Formulary 22nd Edition (USP 27 NF 22). US Pharmacopeial Convention, Inc.: Rockville, MD 2003; 3 European Pharmacopoeia Method : Total Organic Carbon in Water for Pharmaceutical Use. European Pharmacopoeia 4. European Pharmacopoeial Commission, 1 July 1999; 4 Godec R. Pharmaceutical Industry Strategies for Releasing Water to Production Using Automated On-Line TOC Instrumentation. Presented at Ultrapure Water Europe 2004, September 2004 (Amsterdam, The Netherlands). Tall Oaks Publishing, Littleton, CO; 5 FDA Process Analytical Technology Team. Process Analytical Technology (PAT) Initiative. Office of Pharmaceutical Science, HFD- 003, Center for Drug Evaluation and Research, 14 May 2004: 6 McCurdy L. Implementing TOC Testing for USP 23: A Case Study. Pharm. Engin. November December 1997: 2 7. José E. Martínez is a biopharmaceutical consultant at JEM Consulting Services, PO Box 7526, Caguas, Puerto Rico 00726, , mobile , jemartin@coqui.net.