By Karen Clark, Anatel Corp.

Introduction Methods Linearity Detection Limit; Limit of Quantitation Precision and Accuracy Swab Recovery Conclusions References

Introduction (Back to Top) In the pharmaceutical industry, Good Manufacturing Practices (GMP) require that the cleaning of drug manufacturing equipment be validated.1 Many different validation techniques can demonstrate that the manufacturing equipment is cleaned and essentially free from residual active drug substances and all cleaning agents.

Common analytical techniques in the validation process include HPLC, spectrophotometry (UV/Vis) and TOC. HPLC and UV/Vis are classified as specific methods that identify and measure appropriate active and substances. TOC is classified as a non-specific method and is ideal for detecting all carbon-containing compounds, including active species, excipients, and cleaning agent(s).

The disadvantage of specific methods, particularly HLPC, is that a new procedure must be developed for every active drug substance that is manufactured. This development process can be very time consuming and tedious, plus important sampling issues also must be considered. In addition, HPLC analyses must be performed in a relatively short time period after sampling to avoid any chemical deterioration of the active substance. Finally, the sensitivity of HPLC methods can be limited by the presence of degradation products.

TOC analysis can be adapted to any drug compound or cleaning agent that contains carbon. The method is sensitive to the ppb range and is less time consuming than HPLC or UV/Vis. USP TOC methods are standard for Water for Injection and Purified Water2, and simple modifications of these methods can be used for cleaning validation.

Methods (Back to Top) TOC analysis involves the oxidation of carbon and the detection of the resulting carbon dioxide. A number of different oxidation techniques exist, including photocatalytic oxidation, chemical oxidation, and high-temperature combustion. In this study, an Anatel A-2000 Wide-Range TOC Analyzer, equipped with an autosampler, was used. The Anatel A-2000 Wide-Range Analyzer measures TOC in accordance with ASTM methods D 4779-88 and D 4839-88. It measures TOC directly by adding phosphoric acid to the water sample to reduce the pH to approximately 2 to 3. At this low pH any inorganic carbon that is present is liberated as CO2 into a nitrogen carrier gas and is directly measured by a non-dispersive infrared (NDIR) detector. Any remaining carbon in the sample is assumed to be TOC. A sodium persulfate oxidant is then added to the sample, and in the presence of UV radiation, the remaining carbon is oxidized to CO2. The amount of CO2 generated is then measured by the NDIR to determine the amount of TOC originally present in the water.

For equipment cleaning validation there are two types of TOC sampling techniques. One is the direct surface sampling of the equipment using a swab. The second consists of a final rinse of the equipment with high-purity water (typically Linearity (Back to Top) TOC analysis should provide a linear relationship between the measured compound concentration and the TOC response of the analyzer. We evaluated four different types of cleaning agents for linearity: CIP-100 (alkaline), CIP-200 (acidic), Alconox (emulsifier), and Triton-X 100 (wetting agent). Results are shown in Figures 1–4. Correlation coefficients ranged from 0.9787 to 0.9998. Alconox and Triton-X 100 have a tendency to "foam" depending on the concentrations that are analyzed and this "foaming" phenomena can have a negative effect on the accuracy of the TOC result (reduced R2). Three representative examples of active substances were also tested for linearity: an excipient (sucrose), an antibiotic (vancomycin), and endotoxin. Results are shown in Figures 5–7. All three compounds demonstrated excellent linearity with correlation coefficients (R2) ranging from 0.9996 to 0.9998. Detection Limit; Limit of Quantitation (Back to Top) We determined the method detection limit (MDL) by measuring the TOC response of the method blank. A method blank consists of the sampling vial, swab, and recovery solution. In this study the recovery solution was low TOC (< 25 ppb) water. 10 pre-cleaned vials were filled with of the low TOC water. One swab was placed in each vial (Texwipe Alpha Swab TX761; tips cut off). Solutions were vortexed and allowed to stand for one hour prior to analysis. Four replicates from each vial were analyzed. The four replicates from each of the ten blank vials were averaged. These 10 values were averaged again and a standard deviation was calculated. The standard deviation was multiplied by the Student t number for n-1 degrees of freedom (3.25 for n=10), at 99% confidence levels to determine the method detection limit. The MDL was calculated to be 50 ppb. The LOQ was calculated by multiplying the MDL by 3. A value of 150 ppb was obtained (see Table 1). Precision and Accuracy (Back to Top) To demonstrate the precision and accuracy for this TOC method, a representative solution of CIP-100 as 1000 ppb, or 1 ppm as carbon, was analyzed sequentially ten times. This carbon concentration was chosen to evaluate these method parameters because, in general, TOC residual limits are typically around 1 ppm. Results are listed in Table 2. At this TOC level, the precision was ± 1% and the accuracy was ± 5%. Swab Recovery (Back to Top) Stainless steel plates were used in the swab recovery test to simulate manufacturing equipment. One side of each plate was spiked with a solution of active substance or cleaning agent. The plates were allowed to dry completely overnight at room temperature. A Texwipe alpha swab TX761 was moistened with low TOC (< 25 ppb) water and the spiked plate surface was swabbed both vertically and horizontally. The swab end was cut off, placed into a vial to which we added 40-mL of low TOC water. The vial was capped tight, vortexed, and allowed to stand for one hour prior to analysis. The same volume of each solution that was spiked onto the plates was separately spiked directly into 40-mL of low TOC water and analyzed. The percent recoveries of the different substances are listed in Table 3. Reported values are the average of three individual swab samples for each substance. The swab recoveries varied between 79.3% to 95.9% Conclusions (Back to Top) This study demonstrates that the Anatel A-2000 Wide-Range TOC Analyzer is suitable for measuring organic residues on stainless steel surfaces, and that it is a reliable tool for cleaning validation as demonstrated by surface residue recoveries of 79%–96%. This methodology shows that low limits of detection, excellent linearity, precision, and accuracy can be obtained. All of these TOC results, with the exception of Alconox and Triton-X 100, were generated using the same TOC method, making this TOC technology a low cost and less time consuming alternative for cleaning validation. References (Back to Top) Food and Drug Administration's Current Good Manufacturing Practice Regulations; 21 CFR 211.220 USP 23 Fifth Supplement; November 15, 1996 For more information: Karen Clark, Product Manager, Anatel Corp., 2200 Central Ave., Boulder, CO 80301. Tel: 303-442-5533. Fax: 303-447-8365.

CIP-100 (alkaline), CIP-200 (acidic), Alconox (emulsifier), and Triton-X 100 (wetting agent). Results are shown in Figures 1–4. Correlation coefficients ranged from 0.9787 to 0.9998. Alconox and Triton-X 100 have a tendency to "foam" depending on the concentrations that are analyzed and this "foaming" phenomena can have a negative effect on the accuracy of the TOC result (reduced R2). Three representative examples of active substances were also tested for linearity: an excipient (sucrose), an antibiotic (vancomycin), and endotoxin. Results are shown in Figures 5–7. All three compounds demonstrated excellent linearity with correlation coefficients (R2) ranging from 0.9996 to 0.9998.

Detection Limit; Limit of Quantitation (Back to Top) We determined the method detection limit (MDL) by measuring the TOC response of the method blank.

A method blank consists of the sampling vial, swab, and recovery solution. In this study the recovery solution was low TOC (< 25 ppb) water. 10 pre-cleaned vials were filled with of the low TOC water. One swab was placed in each vial (Texwipe Alpha Swab TX761; tips cut off). Solutions were vortexed and allowed to stand for one hour prior to analysis. Four replicates from each vial were analyzed. The four replicates from each of the ten blank vials were averaged. These 10 values were averaged again and a standard deviation was calculated. The standard deviation was multiplied by the Student t number for n-1 degrees of freedom (3.25 for n=10), at 99% confidence levels to determine the method detection limit. The MDL was calculated to be 50 ppb. The LOQ was calculated by multiplying the MDL by 3. A value of 150 ppb was obtained (see Table 1).

Precision and Accuracy (Back to Top) To demonstrate the precision and accuracy for this TOC method, a representative solution of CIP-100 as 1000 ppb, or 1 ppm as carbon, was analyzed sequentially ten times. This carbon concentration was chosen to evaluate these method parameters because, in general, TOC residual limits are typically around 1 ppm. Results are listed in Table 2. At this TOC level, the precision was ± 1% and the accuracy was ± 5%.

Swab Recovery (Back to Top) Stainless steel plates were used in the swab recovery test to simulate manufacturing equipment. One side of each plate was spiked with a solution of active substance or cleaning agent. The plates were allowed to dry completely overnight at room temperature. A Texwipe alpha swab TX761 was moistened with low TOC (< 25 ppb) water and the spiked plate surface was swabbed both vertically and horizontally. The swab end was cut off, placed into a vial to which we added 40-mL of low TOC water. The vial was capped tight, vortexed, and allowed to stand for one hour prior to analysis. The same volume of each solution that was spiked onto the plates was separately spiked directly into 40-mL of low TOC water and analyzed. The percent recoveries of the different substances are listed in Table 3. Reported values are the average of three individual swab samples for each substance. The swab recoveries varied between 79.3% to 95.9%

Conclusions (Back to Top) This study demonstrates that the Anatel A-2000 Wide-Range TOC Analyzer is suitable for measuring organic residues on stainless steel surfaces, and that it is a reliable tool for cleaning validation as demonstrated by surface residue recoveries of 79%–96%. This methodology shows that low limits of detection, excellent linearity, precision, and accuracy can be obtained. All of these TOC results, with the exception of Alconox and Triton-X 100, were generated using the same TOC method, making this TOC technology a low cost and less time consuming alternative for cleaning validation.

Food and Drug Administration's Current Good Manufacturing Practice Regulations; 21 CFR 211.220 USP 23 Fifth Supplement; November 15, 1996

For more information: Karen Clark, Product Manager, Anatel Corp., 2200 Central Ave., Boulder, CO 80301. Tel: 303-442-5533. Fax: 303-447-8365.

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