Ruggedness of Visible Residue Limits for Cleaning Validation

Visual inspection is always used in cleaning validation programs and for routine inspections of cleaning effectiveness, but its use as a sole criterion for equipment cleanliness has not been implemented successfully as a valid cleaning validation approach. Visible residue limits (VRLs), however, can be established for processed active pharmaceutical ingredients (APIs), excipients, and detergents. VRLs for APIs are generally of greatest concern because the API is the most potent component of a drug's formulation. If the VRL is lower than the health-based and adulteration-based acceptable residue limit (ARL) as determined by the company, then the VRL is a viable approach to assessing equipment cleaning within a facility (1).

Sample preparation and viewing parameters for VRL use have been established for both pilot-and commercial-manufacturing facilities (1, 2). A solution or suspension of API applied at different concentrations to stainless-steel coupons results in residues of uniform size. Examination of these viewing parameters consisted of viewing distance, viewing angle, light intensity, residue composition, and observer subjectivity. These paremeters provided optimal viewing conditions to detect visible residues. Viewing conditions in the pilot plant were set at 18 in., 30°, and >200 lux. For commercial facilities with larger, fixed equipment, the viewing inspection parameters were more restricted. Optimal viewing conditions were set at <10 ft, >30°, and >200 lux. A discussion of VRL applications and associated risks concluded that the potential for cleaning failure was small under a well-controlled VRL program (3–5).

AUTHOR

The subjectivity of observers and the appearance of dried residues were potential limitations to a VRL program. The original VRL work used a small group of four to six observers (1, 2). For subsequent work, a larger pool of observers determined VRLs of APIs in development at one site. In addition, a study of VRL determinations for five APIs at multiple sites helped address the issue of observer subjectivity and further defined the ruggedness of residue sample preparation.

The complete set of VRL data was examined for distribution and trends. Original VRL data were compared with the subsequent data set to assess improvements in process and technique. VRLs of the APIs, excipients, and formulations were also compared to determine whether a correlation exists between the VRL of a formulation and its components. Theoretically, the VRL of the formulation should be the same as the lowest component VRL. Analysis of VRL data showed differences between the early data and later VRL determinations. VRL data of the APIs, excipients, and formulations also proved to be worthwhile for future considerations and VRL policy definition.

VRL residues appear as a ring or as uniform residue. The appearance depends on the amount of API residue being spotted and the volume of spotting solvent, which translates to the concentration of the resulting residue. Other physical parameters include drying the solvent, and any physical or chemical interaction between the solvent and the residue. Methanol is consistently used as a spotting solvent because its low surface tension allows it to spread to a uniform spot size and its high volatility allows it to dry quickly.

The API, however, is consistently different, making control difficult. The amount of residue around the VRL is extremely low with an API, though, so it is more likely to fall out of the solution last. As methanol and other solvents dry, the perimeter of the wetted area is the last place to dry, which favors the ring-type residue appearance.

Experience gained through ongoing equipment inspections and direct soiling of manufacturing equipment with test soils confirmed that potential residue on cleaned equipment would be similar in appearance to the experimental VRL residues. Varying the application volumes and concentrations of the residue spots addressed the issue of residue appearance.

Methods and materials

As the VRL program expanded at the author's single site, a larger pool of observers came in. The expanded pool of observers included 20–30 scientists. In addition, equipment washers and supervisory personnel were trained in VRLs and visual inspection of clean equipment. Data from the additional VRLs were compared with the original work. The data set was also analyzed for API, excipient, and formulation correlations.

The multisite study consisted of the company's three international sites: West Point in the United States, Montréal in Canada, and Hoddesdon in England. Each site determined VRL for the same five APIs. Visual determinations were based on two grades of stainless-steel coupons from a common source: milled or 316-finish stainless steel (the grade of material on most manufacturing equipment) and mirror-finish stainless steel. The experimental conditions, as defined previously, were the same as those used for the pilot plant: a distance of 18 in., a viewing angle of 30°, and a light intensity level of >200 lux.

Personnel at each site weighed approximately 20 mg of the respective test material and placed it into a 25-mL volumetric flask. They dispersed the material using a newly opened bottle of high-performance liquid chromatography (HPLC) grade methanol, added the methanol to completely dissolve or disperse the material, and brought the flask to volume. The resulting sample concentration was approximately 800 μg/mL.

After verifying that the coupons were visually clean before use, personnel cleaned the coupons with methylene chloride, chloroform, or methanol to remove any residue from the coupon that might interfere with the residue-spotting process or visual observations. Spot residues were prepared according to conditions listed in Table I. The range of spots was targeted to span between ARL at 4 μg/cm² to below VRL and intended to cover approximately 25 cm². The target spot size was a 5-cm diameter circle, or 20 cm². The consistency of spotting areas was important to the eventual spot concentration and, therefore, personell made every attempt to keep residue spots consistently close to the intended size. Residues were arranged in order of descending concentration for consistency among sites and spotted by pipetting 100 μL of the appropriate sample onto the stainless-steel coupon. The residue was then dried and the area of each residue spot was determined.

Table I: Residue target concentrations.

If prepared correctly, each of the residue spots generally approximated a circle. The area was calculated by measuring the circle's diameter. If the spot resembled more of an oval, two diameter measurements provided the area. The amount of residue was divided by the spot area to find the concentration of residue.

The coupons were placed on a flat background and the ambient light level was measured to verify accuracy. The identity and concentration of each spot was unknown to the observers. The coupons were oriented so that each spot could be observed from the same distance and angle, and the ability of each observer to see each of the residue spots was recorded.

The highest standard spot for APIs was 100 μL of an 800-μg/mL solution or suspension in methanol. This resulted in a circular residue with an approximate diameter of 5 cm, which was approximately the size of the swab area (25 cm²). Various decreasing concentrations with the same volume resulted in VRL when the residue was no longer visible to all observers. If the lowest spotted residue was visible to the observers, then the VRL was reported as "less than" the lowest spotted residue concentration. For VRLs with the "less than" designation, the lowest tested residue was sufficiently lower than the ARL as to not pose a significant risk of cleaning failure.

To address the phenomenon of residue appearance, various volumes of the same concentration solution near to VRL were spotted to complement existing data and to determine if there was an effect on the appearance of residue and subsequent VRL determination.

Results and discussion

Initial versus later VRL determination. Of the original 59 VRLs established (1), the average VRL in this experiment was 1.6 μg/cm². Three of the four observers agreed on most residue levels, but one observer differed, which resulted in slightly higher VRLs. Of the original set of VRLs, 78% were <2 μg/cm², but 14% were >4 μg/cm² (see Table II and Figure 1).

Table II: Comparison of visible-residue limit (VRL) data.

Additional VRL determinations increased the experience level, widened the observer pool, and refined the experimental technique. Instead of a small, dedicated group of observers, 20–30 scientists worked on the development compound to establish VRLs. The most significant difference in subsequent VRL determinations, however, was the standardization of the residue-spot preparations using lower spotted residue levels, which resulted in lower VRLs.

Figure 1: Visible-residue limit (VRL) distribution. (ALL FIGURES ARE COURTESY OF THE AUTHOR.)

Interestingly, variability among the observers decreased even as the observer pool increased. Three major factors led to greater consistency: the initiation of a VRL training program for observers, equipment cleaners, and inspectors; overall increased experience level of the observers; and a more consistent residue-spot preparation technique. Of the additional VRLs established, the average VRL dropped to 0.9 μg/cm² and 89% of determinations were <2 μg/cm². Ninety-six percent of the total determinations were <3 μg/cm², and only 2% were >4 μg/cm² (see Table II). A t-test comparison of the original and additional VRL data in Figure 1 showed that the data distributions were not equivalent. The additional VRL data with a lower average were statistically different when compared with the original VRL data, which confirmed the effects of experience and technique refinement. The overall average VRL was ultimately <1.1 μg/cm² for the 253 VRLs (see Table III) because 63% of the VRLs determined were less than the lowest level tested.

Table III: Current visible-residue limit (VRL) data.

API versus excipient versus formulation determinations. VRL data were broken into API, excipient, and formulation categories and analyzed to determine VRL correlation between a formulation and its components. Earlier work (1) compared VRL of 12 formulations with VRLs of the formulation components. Logically, the VRL of the formulation would be the same as the lowest component VRL—this was the case in 7 of the 12 comparisons. In three of the cases, however, the formulation VRL was higher, and in the other two cases, it was lower than the component VRLs.

The more important comparison was between VRL of the formulation and its API. In a pilot plant, it is not practical to perform VRL on every development formulation because the formulation compositions continually evolve up to the final market formulation selection. In the VRL comparison of formulations against components, 9 of the 12 formulation VRLs were lower than the VRL of the respective API. In one case, they were equal, and in the remaining two the formulations, the VRL was higher. The data concluded that VRL of API is not a good indicator for VRL of a formulation.

The data, however, were generated during the original VRL work where the residue concentrations and observer variability were higher. The subsequent VRL work generated additional data with increased experience and refined technique. The data gap narrowed between the formulations and APIs. The final average VRL of the 64 formulations was 0.7 μg/cm². Of the 113 API determinations, it was 1.0 μg/cm². Average VRL of the 64 excipients tested to date was 1.6 μg/cm², and data showed significant overlap among formulations, APIs, and excipients.

A t-test comparison of API and formulation VRL data in Figure 2 shows that data distributions were equivalent. The formulation VRL data, despite its lower average value, was not statistically different when compared with API VRL data. The expanded data set analysis demonstrates that VRL of API is a good indicator for VRL of a formulation. VRL of a development API can be determined and safely used as the VRL for the development formulations.

Figure 2: Visible-residue limit (VRL) distribution.

Multisite study

VRL data from the three sites are shown in Table IV. Data from the Hoddesdon facility was generally lower than data from the other sites. Data from the West Point facility was slightly higher, which correlated to smaller spot sizes and resulted in higher spot concentrations (see Table V). Observers in Hoddesdon and West Point typically detected the lowest or next-to-lowest residue level. Data from the Montréal site resulted in three VRLs that were higher than VRLs from other sites; observer variability at Montréal was also greater. Of the three higher levels, one was comparable with the established VRL, and the other two were higher. All three of the higher levels were still well below the adulteration limit of 4 μg/cm². A review of observer data showed that, in all cases, the higher levels were based on one observer not detecting the residue. Otherwise, the data more closely agreed with that of the other sites.

Table IV: Multisite visible-residue limit (VRL) data.

Several factors led to variability in the multisite data. The sample solution concentrations, spot sizes, and the resulting residue concentrations influenced VRL determination. The Hoddesdon site's lowest residue level was lower than the other sites, explaining their overall lower VRL levels. Observer variability at the Montréal site was similar to the early West Point data; a single observer skewed the results compared with the other observers and sites.

Table V: Multisite residue concentration comparison.

Overall, VRL determination was comparable at all three sites, and the experimental variability from sample preparation and observer subjectivity posed no risk for a potential cleaning failure because all VRL values were well below the ARL. The study also highlighted the value of the VRL training program and the experience gained through ongoing visual equipment inspections.

Residue appearance

Standard preparation for residue spots involved pipetting 100 μL of sample solution or suspension onto a material coupon. This volume of methanol consistently supplied a circular residue spot of approximately 5 cm. in diameter, which was nearly equal to the swabbed 25-cm² area. As sample concentrations decreased, residue appearance changed from being uniform to that of a ring (see Figures 3 and 4).

Figure 3: Effect of concentration on residue appearance.

To determine the effect of spotting volume, 60 μL of the lowest spotted solution was pipetted along with 0, 20, 40, 60, 80, and 100 μL of methanol. The lowest concentration was used because the appearance of the residue near VRL was the primary area of interest. The appearance of the different volumes had little effect on the appearance of the residue around VRL. All of the residues were similar, but, as expected, the rings became larger with the increased volume (see Figure 5). Eventually, larger volumes of spotting solvent would make the ring too dilute to detect, but the area of the ring at that point would be significantly larger than the swab area of 25 cm².

Figure 4: Effect of concentration on residue appearance.

The appearance of residues near VRL concentration, it was concluded, are expected to take on the appearance of a ring. If the residue has a uniform appearance, it is most likely well above the VRL limit.

Figure 5: Effect of volume on residue appearance (6-μg sample).

Conclusion

The ruggedness of VRL viewing conditions has been tested, and optimal viewing conditions have been defined. Studies established the ruggedness of VRL determination among multiple observers at different sites, showed the relationship between VRLs of formulations and individual components, and assessed the effects of residue appearance on VRL preparation parameters. The research also highlighted the value of a VRL training program for all involved personnel.

Acknowledgment

The author gratefully acknowledges colleagues at the respective sites who generated VRL data and participated in the multisite study.

Richard Forsyth is a consultant for Cleaning Validation and GMP Topics, tel. 610.948.2970, mr.rich.forsyth@gmail.com

Submitted: June 25 2008. Accepted: June 30, 2008.

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References

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