Failure of Cleaning Verification in Pharmaceutical Industry Due to Uncleanliness of Stainless Steel Surface

The overall goal of this work is to obtain high and reproducible recoveries of pharmaceutical residues from the surface of stainless steel coupons by applying the best practices for analytical cleaning method development for small molecules, therapeutic proteins and antibodies. This method can help answer key questions in the field of cleaning verification in the pharmaceutical industry, such as why cleaning verification of manufacturing equipment fails. The main advantage of this study is that it shows the procedure to properly clean the surface of the stainless steel coupons used for analytical method validation.

To start this experiment, prepare sample solutions of the drug dissolved in the diluent of choice after calculating the cleaning limit for a drug. Clean the stainless steel coupons by rinsing with water and wiping the surface twice for 10 to 15 seconds. Then, repeat this rinsing and wiping step with methanol to eliminate any residual deposit.

To perform the advanced cleaning approach using clean in place solutions, immerse the coupons in high performance liquid chromatography or HPLC grade water and sonicate for two minutes. Immerse the coupons in 0.1%alkaline detergent solution in HPLC grade water and sonicate for two minutes. Immerse the coupons in HPLC water again and repeat two minute sonication.

Then, immerse them in 0.1%acid detergent solution in HPLC water and sonicate for two minutes. Finish with two minute sonication in HPLC water. Next, using a double-sided tape, mount the stainless steel coupons on the bottom of a 250 milliliter plastic beaker.

Hold the beaker during the procedure to facilitate spiking and swabbing and to avoid accidents. Use a positive displacement pipette to infuse a defined drug volume, at a desired concentration and formulation, in a whirly pattern on the coupon surface. Once the coupon surface is dry, dip a dry swab in a vial with two milliliters of diluent.

Press the swab against the inside of the vial to remove excess solvent. Use even, overlapping side-to-side strokes to firmly wipe the coupon surface with one side of the wet swab, until the total 50 square centimeter test area is wiped. Use the same side of the swab to repeat the wiping process.

Swab each of the four edges of the coupon twice. Rotate the coupon 90 degrees and use the other side of the swab, now rotating the direction of wiping by 90 degrees. Repeat the cleaning using this side of the swab in the same fashion, making sure to wipe all the four edges of the coupon twice.

After the surface wiping, use scissors to cut the swab head into the solvent vial. After that, repeat the whole swabbing procedure using a second swab, while rotating the coupon 90 degrees toward the same direction chosen before. Next, sonicate the vial containing two swab heads for five minutes.

Then, vortex the vial for 10 seconds. Finally, transfer the solution into an HPLC vial and label it as the working solution. To recover the sample, prepare the control solutions by mixing 200 microliters of each coupon spiking or sample solution with 1, 800 microliters of diluent.

Use these solutions to run chromatography according to the text protocol. Calculate the recovery of the swab working solution based on the relative area under peaks of the working and control solutions. Repeat this process for three coupons to calculate the average recovery and the relative standard deviation.

Before the coupons have been cleaned, recovery results for different spike levels were inconsistent, regardless of active pharmaceutical ingredient or API excipient ratio, different analysts, or even different day for the same analyst. The majority of the recovery results had high relative standard deviation. The observed variability in the recovery was not eliminated when several different parameters were adjusted, including swabbing technique, the diluent, the spiking solvent, the pH of the spike and the diluent, the spiking technique and the extraction technique.

The difference between the individual recovery at each coupon surface and the average recovery shows that the average recovery is different from one coupon surface to another, therefore, it is very likely that the observed variability comes from the differences between the coupon surfaces. After following the coupon cleaning described in this protocol, the recovery results were reproducible from one trial to another, with minimal difference in recovery between the coupons. The coupon cleaning performed here resulted in the recoveries being higher than 90%Furthermore, relative standard deviation values for each spiking level were acceptable and smaller than the results obtained before the cleaning.

Once mastered, the cleaning of the coupons can be done in less than 30 minutes, if performed properly. While attempting this procedure, it's important to remember how to spike and recover properly without losing the analyze of interest. After watching this video, you should have a good understanding of how properly clean stainless steel surfaces to obtain high and reproducible recoveries of the residues.

After its development, this technique paved the way for researchers in the pharmaceutical industry to provide reliable cleaning data for manufacturing processes.