No funding agencies supported this work.

1. Sample Solution
<ol>
	<li>Calculate the cleaning limit (CL) for a drug based on the maximum allowable carry-over (MACO) according to the therapeutic dose criteria. 
	NOTE: Here, the cleaning limit (CL) for drug A has been calculated to be 2.4 &#181;g per 50 cm2 based on the maximum allowable carry-over (MACO) according to the therapeutic dose criteria. The cleaning verification is executed at the 50%, 100%, and 150% cleaning limit. The cleaning verification has been assessed for two formulations (2.5% and 60% w/w drug load).</li>
</ol>
2. Cleaning Procedure for Coupons
<ol>
	<li>Initial approach
	<ol>
		<li>Clean the coupons by rinsing and wiping the surface for 10 - 15 s twice with water and twice with methanol to eliminate any residual deposit. Perform this process in the hood as methanol is toxic and highly volatile.</li>
	</ol>
	</li>
	<li>Advanced approach
	<ol>
		<li>Using clean-in-place solutions
		<ol>
			<li>Set the sonicator at room temperature. The sonicator has no power setting adjustment, therefore the time is set to give adequate cleaning results.</li>
			<li>Immerse the coupons in high performance liquid chromatography (HPLC) grade water and sonicate for 2 min.</li>
			<li>Immerse the coupons in 0.1% alkaline detergent solution in HPLC grade water and sonicate for 2 min.</li>
			<li>Immerse the coupons in HPLC water and sonicate for 2 min.</li>
			<li>Immerse the coupons in 0.1% acid detergent solution in HPLC water and sonicate for 2 min.</li>
			<li>Immerse the coupons in HPLC water and sonicate for 2 min.</li>
		</ol>
		</li>
		<li>Using acid-base-peroxide 
		&#8203;NOTE: This is an alternative method included using acid, base, and peroxide rather than alkaline and acid detergents.
		<ol>
			<li>Set the sonicator at room temperature. The sonicator has no power setting adjustment, therefore the time is set to give adequate cleaning results.</li>
			<li>Immerse the coupons in water and sonicate for 2 min.</li>
			<li>Immerse the coupons in 0.1 M sodium hydroxide and sonicate for 2 min.</li>
			<li>Immerse the coupons in water and sonicate for 2 min.</li>
			<li>Immerse the coupons in 0.1 M hydrochloric acid and sonicate for 2 min.</li>
			<li>Immerse the coupons in water and sonicate for 2 min.</li>
			<li>Immerse the coupons in 0.1 mg/mL NaNo2 and sonicate for 2 min.</li>
			<li>Immerse the coupons in water and sonicate for 2 min.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Swabbing Procedure
<ol>
	<li>Mount the stainless steel coupons on the bottom of a 250 mL (or 500 mL) plastic beaker using a double sided tape.</li>
	<li>Hold onto the beaker to make spiking and swabbing processes easy. This also minimizes some undesirable accidents such as overshooting the corner when swabbing.</li>
	<li>Infuse a defined volume at a specific concentration and formulation (e.g., 200 &#181;L of 12 &#181;g/mL at 2.5 % w/w drug A load) in a whirly pattern on the surface of the coupon using a positive displacement pipette.</li>
	<li>Wait until the surface of the coupon is dry (~ 3 - 10 min depending on the volatility of the sample).</li>
	<li>Dip a dry swab in a vial with 2 mL of diluent (methanol:formic acid 100:0.2 volume/volume).</li>
	<li>Remove excess solvent by pressing the swab against the inside of the vial.</li>
	<li>Firmly wipe the surface of the coupon with even, overlapping side-to-side strokes until the total 50 cm2 test area is wiped with one side of a wet swab.</li>
	<li>Repeat the wiping process (step 3.7) using the same side of the swab.</li>
	<li>Swab the four edges of coupon twice.</li>
	<li>Flip the swab to the other side and rotate the coupon 90&#176;, clockwise or counterclockwise; thus, rotating the direction of swabbing by 90&#176;.</li>
	<li>Repeat the swabbing motion as detailed in steps 3.7 - 3.9. After the sampling of the surface, cut the swab head using scissors into the solvent vial.</li>
	<li>Repeat the swabbing process with a second swab while rotating the coupon 90&#176; toward the same direction chosen before.</li>
	<li>Sonicate the vial containing two swab heads for 5 min, then vortex for 10 s.</li>
	<li>Transfer the solution into an HPLC vial and label it as working solution.</li>
</ol>
4. Calculation of Recovery
<ol>
	<li>Sample recovery
	<ol>
		<li>Prepare the control solutions by mixing 200 &#181;L each of the coupon spiking solutions with 1,800 &#181;L of diluent.</li>
		<li>Run the chromatography system according to the conditions listed in Table 1.</li>
		<li>Calculate the recovery of the swab working solution based on the relative area under peak of the working solutions (AW) and the control solution (AC).</li>
		<li>Repeat the process on three coupons and calculate the average recovery along with relative standard deviation (Srel). 
		NOTE: All the analytical methods used here were validated according to the ICH Q2(R1) guidelines. The chromatography conditions are listed in the material section.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Validation of cleaning processes (7/93). Guide to inspections validation of cleaning processes. , (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Warning Letter: 320-12-08, Gulf Pharmaceuticals. , (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Warning Letter: 320-12-058, Novartis Internationsl AG. , (2012).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Warning Letter: 05-10, Teva Parenterals Medicines, Inc. , (2009).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Warning Letter: 320-14-09, Tianjin Zhongan Pharmaceutical Co., Ltd. , (2014).</li><li>Mantel, M., Wightman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+the+surface+chemistry+on+the+wettability+of+stainless+steel.">Influence of the surface chemistry on the wettability of stainless steel.</a> Surf Interface Anal. 21, 595-605 (1994).</li><li>Odaka, K., Ueda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependence+of+outgassing+rate+on+surface+oxide+layer+thickness+in+type+304+stainless+steel+before+and+after+surface+oxidation+in+air.">Dependence of outgassing rate on surface oxide layer thickness in type 304 stainless steel before and after surface oxidation in air.</a> Vacuum. 47, 689-692 (1996).</li><li>Kerber, S. J., Tverberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stainless+Steel+Surface+Analysis.">Stainless Steel Surface Analysis.</a> Adv. Mater. Processes. , 33-36 (2000).</li><li>Kusumaningrum, H. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Survival+of+foodborne+pathogens+on+stainless+steel+surfaces+and+cross-contamination+to+foods.">Survival of foodborne pathogens on stainless steel surfaces and cross-contamination to foods.</a> Int J Food Microbiol. 85, 227-236 (2003).</li><li>Liu, L., Pack, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleaning+verification+assays+for+highly+potent+compounds+by+high+performance+liquid+chromatography+mass+spectrometry:+Strategy,+validation,+and+long-term+performance.">Cleaning verification assays for highly potent compounds by high performance liquid chromatography mass spectrometry: Strategy, validation, and long-term performance.</a> J Pharmaceut. Biomed. 43, 1206-1212 (2007).</li><li>Lambropoulos, J., Spanos, G. A., Lazaridis, N. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+validation+of+an+HPLC+assay+for+fentanyl,+alfentanil,+and+sufentanil+in+swab+samples.">Development and validation of an HPLC assay for fentanyl, alfentanil, and sufentanil in swab samples.</a> J Pharmaceut. Biomed. 23, 421-428 (2000).</li><li>Chisti, Y., Moo-Young, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clean-in-place+systems+for+industrial+bioreactors:+Design,+validation+and+operation.">Clean-in-place systems for industrial bioreactors: Design, validation and operation.</a> J. Ind. Microbiol. 13, 201-207 (1994).</li><li>Haidar Ahmad, I. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleaning+verification:+Exploring+the+effect+of+the+cleanliness+of+stainless+steel+surface+on+sample+recovery.">Cleaning verification: Exploring the effect of the cleanliness of stainless steel surface on sample recovery.</a> J Pharmaceut. Biomed. 134, 108-115 (2017).</li></ol>

No financial interests or conflict of interests exist.

The major contributor to low and variable recoveries of API residues from stainless steel coupons was traced to the lack of a well-defined procedure for cleaning of the coupon surfaces. Cleaning the surface of the coupons resulted in consistent, accurate spiked recovery and reproducible results. With the demonstration of high recoveries from stainless steel coupons, the actual cleaning verification results obtained from the manufacturing equipment using validated method(s) should be accurate and precise, reflective of the residue level on the equipment with minimum risk of false negatives for product to product carryover that could jeopardize patient safety.
The initial approach followed by the analyst to troubleshoot the low and inconsistent recovery was by modifying the experimental conditions such as: percent of organic in the diluent, the spiking process, the type and strength of acid used in the spiking solution, etc. This approach did not solve the low and inconsistent recovery issue. Nonetheless, this aforementioned problem was completely solved when the stainless steel was adequately cleaned using clean-in-place solution. This achievement results in vastly increasing the chances of the successful pass of cleaning verification of the manufacturing equipment. It is important to note that the CIP solutions used here are limited to the pharmaceutical industry and thus suitable CIP solutions should be selected for other types of industries (processed food, dairy, cosmetics, etc.). The choice of CIP solutions and the cleaning process are critical steps for the success of this process. The protocol presented here will help analysts in the pharmaceutical industry as well as other industries to better design and execute successful cleaning verification. The work will also help minimizing batch-to-batch and/or product-to-product cross-contamination that may adversely affect the human health.

The cleanliness of non-dedicated equipment should be verified before its subsequent release for use in the manufacture of intermediates and active pharmaceutical ingredient (APIs), at product change over to prevent cross-contamination. Cleaning procedures should contain sufficient details to enable operators to clean each type of equipment in a reproducible and effective manner, and these procedures should be validated according to the U.S. Food and Drug Administration (FDA) requirements1. Numerous warning letters due to inadequate cleaning2,3,4, failure to validate the cleaning verification method, and failure to follow cleaning procedures5 have been issued by the FDA. 21 CFR &#167;211.67 outlines the requirements needed for successful cleaning verification.
It is the standard in industry that the validation of the analytical methods for cleaning verification is performed on stainless steel coupons with the same surface/finish as the manufacturing equipment. Stainless steel coupons (e.g., 50 cm2) are used to represent equipment surfaces for cleaning verification experiments in the laboratory. During the development and validation of these analytical methods, the sample of interest (i.e., the residues that should be recovered from the surface of the equipment) is spiked at the target residue onto the stainless coupon determined by the maximum allowable carry-over (MACO) limit. This level is determined based on acceptable exposure limit which is defined as the limit at which a patient can get exposed with no adverse health effects (no-observed-adverse-effect-level, NOAEL).
The analyst or manufacturing operator conducting the swabbing must follow a structured procedure to ensure that recoveries are reproducible regardless of who performs the swabbing. The procedure should explicitly detail the swab type, number of swabs used, the diluent, the amount of solvent used, the exact sweeping pattern, the number of strokes applied to the sampling surface, the amount of time spent swabbing/extracting the samples, the method of detection (ultra-violet, fluorescence, mass spectrometry, total organic carbon, etc.), the extraction technique of the material from the swab head, etc.
Besides all the above-mentioned factors that affect sample recovery, the surface of the coupon, and thus, the surface of the equipment also play a role. The surface of the coupon may be modified due to the deposition of a thin film of material on the surface or due to change in the oxidation state of one or more of the elements in stainless steel (e.g., Fe, Cr, and Ni)6,7,8. The regeneration of the surface of the stainless steel coupons back to its original state is vital for the success of the quantitative swapping process. Studies, in which the stainless steel coupons were not properly cleaned, showed variability in recovery including analyst to another, different drugs, or various spike levels9,10,11. The standard deviation in recovery of ten replicates on one coupon can be up to 14% and 26% on five coupons9. It is important to note that the relative standard deviation (Srel) values increased with increasing number of replicates or with increasing the number of coupons used (i.e., five coupons instead of spiking five times on the same coupon)11. In such cases, the variability cannot be interpreted as random fluctuation. Nonetheless, their results can be explained by our finding that the cleanliness of the surface of the coupon will affect the recovery. The results described in this paper illustrate a significant increase in recovery results and decrease in variability after properly cleaning the surface of the stainless steel coupons.
Clean-in-place (CIP) is an automated way of cleaning the surface of equipment that involves a minimal or no disassembling of the equipment. During the CIP cleaning process, a defined procedure of consecutive wash with a base followed by an acid is executed to remove organic and inorganic residues. Surfactants, chelating compounds, or complexing agents are usually added to the CIP solutions to enhance the efficiency of cleaning any product from the surface of the equipment. The efficiency of cleaning depends on several parameters including the choice and concentration of the CIP solutions (i.e., type and composition of base, acid, and surfactant), the cleaning time, temperature (typically 60 - 80 &#176;C), contamination type, and the presence of hard to clean parts12. Based on the type of drug product, CIP solutions 100 and 200 have been chosen to use for cleaning the stainless steel coupons used for cleaning verification, since it simulates the CIP process used for cleaning the manufacturing equipment.
This study reports the influence of different factors affecting the recovery of pharmaceutical residues from the surface of stainless steel coupons and recommends the best practices for analytical cleaning method development for small molecules, therapeutic proteins, and antibodies. The lack of a well-defined procedure that consistently cleaned coupon surface was identified as the major contributor to low and variable recoveries. High and reproducible recovery was obtained when the surface of the coupon was cleaned properly13.

<table><tbody><tr><td>stainless steel coupons</td><td>&amp;nbsp;GlobePharma (New Brunswick, NJ &lt;span class=&quot;font6&quot;&gt;).&amp;nbsp;&lt;/span&gt;</td><td>SS316-20RA-50cm2</td><td></td></tr><tr><td>Clean in place solutions (CIP100 and CIP200)&amp;nbsp;</td><td>were obtained from Steris Corporation (Mentor, OH)</td><td>1D10BG</td><td>Alkaline detergent and acid detergent, respectively</td></tr><tr><td>Positive displacement pipettes</td><td>Gilson (Middleton, WI).&amp;nbsp;</td><td></td><td></td></tr><tr><td>HPLC grade water</td><td>Millipore Milli-Q Advantage Water Purification System (Darmstadt, Germany)&amp;nbsp; or from Honeywell Burdick &amp;amp; Jackson (Muskegon, Michigan)</td><td>7732-18-5</td><td></td></tr><tr><td>HPLC grade Methanol</td><td>EMD</td><td>MX0475-1</td><td></td></tr><tr><td>glacial acetic acid&amp;nbsp;</td><td>EMD</td><td>MAX0073P5</td><td></td></tr><tr><td>HPLC grade Acetonitrile&amp;nbsp;</td><td>J.T. Baker (Avantor Performance Materials, Center Valley, PA)</td><td>75-05-8</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>J.T. Baker (Avantor Performance Materials, Center Valley, PA)</td><td>75-05-8</td><td></td></tr><tr><td>Chromatography column Zorbax Eclipse</td><td>&amp;nbsp;XDB-C18, 4.6 x 100 mm, 3.5 &amp;micro;m HPLC column</td><td>UNSPSC &amp;ndash; 41115709</td><td></td></tr><tr><td>Vanquish UHPLC system&amp;nbsp;</td><td>Thermo Fisher Scientific, Germering, Germany</td><td></td><td></td></tr><tr><td>Branson B8510 Ultrasonic cleaner&amp;nbsp;</td><td>Branson Ultrasonics (Danbury, CT, USA)</td><td>model (8510-D7H)</td><td></td></tr></tbody></table>

failure of cleaning verification in pharmaceutical industry due to uncleanliness of stainless steel surface

The aim of this work is to identify the parameters that affect the recovery of pharmaceutical residues from the surface of stainless steel coupons. A series of factors were assessed, including drug product spike levels, spiking procedure, drug-excipient ratios, analyst-to-analyst variability, intraday variability, and cleaning procedure of the coupons. The lack of a well-defined procedure that consistently cleaned the coupon surface was identified as the major contributor to low and variable recoveries. Assessment of cleaning the surface of the coupons with clean-in-place solutions (CIP) gave high recovery (&#62;90%) and reproducible results (Srel&#8804;4%) regardless of the conditions that were assessed previously. The approach was successfully applied for cleaning verification of small molecules (MW &#60;1,000 Da) as well as large biomolecules (MW up to 50,000 Da).

Representative results from the initial attempts for cleaning verification for drug A are summarized in Table 2. Before the coupons were cleaned according to the detailed procedure in the experimental section, inconsistent results were obtained at different spike levels, for various ratios of API/excipient, different analysts, and even for the same analyst at different days. The observed variability in the recoveries needed to be addressed, as some of the results failed the validation requirements (60% &#60; recovery &#60; 150%), such as the recovery for the 60% drug load at all cleaning limits.
The first type of variability observed in Table 2 is the variability in precision as can be seen from the high Srel associated with the majority of the recovery results (numbers listed in parentheses). Besides to the expected analyst-to-analyst variability (data shown in reference 13), day-to-day variability is also observed for one analyst with all other conditions not changed, as seen in the first two experiments in Table 2 (at 2.5% drug load). Inconsistent recoveries were observed at the different spike levels of 50%, 100%, and 150% of the cleaning limit (Srel up to 16% for the 60% drug load at 150% cleaning limit), regardless of the drug load or the analyst doing the experiment. In addition, there was variability even at different API/excipient ratios, at 2.5% and 60% drug load (Table 2) and at 50% drug load reported in reference 13). The low formulation gave the highest recovery on average, suggesting that the excipient was enhancing the recovery of the drug from the coupon. Quite possibly 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), a surfactant, shielded the organic drug compound from metal chelation interaction and enhanced the removal of the drug from the coupon surface when the drug/excipient ratio was lower.
Based on the types of variability discussed above, the initial approach to improve recovery was to redevelop the extraction method and the experimental conditions to obtain consistent and high recoveries. Adjusted parameters included: swabbing technique, the diluent (different solvents, various organic/aqueous ratios, different acids and acid concentration), the spiking solvent, the pH of the spike and the diluent, the spiking technique, and the extraction technique of drug from swab. The average recoveries on four different coupons along with relative standard deviation are shown in Figure 1 for some experiments. The main conclusion was that none of the aforementioned changes eliminated the previously observed variability in Table 2. Regardless of the experimental factor that was changed, the variability in recovery (Srel) from one coupon to another was evident and in some cases it was &#62;20%. Within experimental error, almost all of these experiments were not considered statistically different. The difference between the individual recovery at each coupon surface and the average recovery (&#916;Recovery) are shown in Figure 2. It is clear that the average recovery is different from one coupon surface to another. Therefore, the coupon surface is expected to be a major contributor to the observed variability.
There was a high probability that the previously observed variability in recovery was due to coupon-to-coupon variability. The cleaning verification experiments for 60% drug load were repeated six times, with each formulation spiked onto four coupons of identical material and surface finish, all from the same vendor. It was clear from the results shown in Figure 3 that the recovery for the 60% formulation was not very reproducible from one trial to another, with a general trend towards lower recoveries as the experiments progressed. Moreover, some differences were observed between coupons at this formulation (Figure 2). The observed variability suggested that the surface of the various coupons was not identical and interacted differently with the matrix.
The first approach to minimize the difference among the coupons was to thoroughly clean the surfaces of the coupons. Coupons used for obtaining recovery in Figure 3 were cleaned according to the procedure presented in the experimental section. The recovery results after cleaning the coupons are presented in Figure 3. It is clear that the recovery is virtually reproducible from one trial to another and that the difference in recovery between coupons is minimized.
Table 2 shows a comparison of the recovery results before and after cleaning the coupons under the same experimental conditions. The following conclusions can be drawn: 1) All the recoveries were high (90 - 100%); 2) The Srel values at each spiking level were acceptable and much smaller than the previously reported results on uncleaned coupons, 3) The variability in the recovery from one spike level to another was minimized, 4) The difference in formulation did not affect the recovery.
Coupons cleaned with CIP solutions were then used for cleaning verification of compounds B (another small molecule) C and D (large molecules, i.e., biologics) at different formulations and spike levels. The same conclusions drawn from experiments for drug A were applicable for drug B, C, and D (detailed results shown in reference 13). High recoveries were obtained across molecular size and physicochemical properties by applying a systematic cleaning approach for the coupons.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56175/56175fig1.jpg" /> 
Figure 1. Average Recovery Obtained from Four Coupons. Error bars represent relative standard deviation from four trials on four coupons. Experiment number: 1) normal spike, 2) 10% formic acid, 3) no formic acid, 4) no water in spike solution, 5) high placebo percentage (97%), 6) no placebo, 7) squeezing swab with spatula, 8) adding a centrifuging step, and 9) 0.1% HCL. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56175/56175fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56175/56175fig2.jpg" /> 
Figure 2. Difference in Recovery (&#916;Recovery) Obtained from Four Coupons. &#916;Recovery is the difference between the recovery on the coupon and the average recovery of the four coupons. Experiment number: 1) normal spike, 2) 10% formic acid, 3) no formic acid, 4) no water in spike solution, 5) high placebo percentage (97%), 6) no placebo, 7) squeezing swab with spatula, 8) adding a centrifuging step, and 9) 0.1% HCL. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56175/56175fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56175/56175fig3.jpg" /> 
Figure 3. Variability in Recovery of Drug A at 2.5 % Drug/Excipient Ratio Before and After Cleaning. Solid triangles correspond to recovery values before cleaning the coupons, while open symbols correspond to the recovery values after cleaning the coupons. Error bars represent relative standard deviation from four trials on four coupons. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56175/56175fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/56175/56175table1.jpg" /> 
Table 1: Chromatography Conditions.
<img alt="Table 2" src="/files/ftp_upload/56175/56175table2.jpg" /> 
Table 2.&#160;Spiked Recoveries for Different Drug Load Before and After Cleaning the Coupons According the Procedure in the Experimental Section.

Watch this Scientific Journal Video about Failure of Cleaning Verification in Pharmaceutical Industry Due to Uncleanliness of Stainless Steel Surface at JoVE.com

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

The lack of a well-defined procedure that consistently cleaned coupon surfaces was identified as the major contributor to low and variable recoveries in cleaning verification. This manuscript describes the correct protocol of cleaning stainless steel coupons.

The aim of this work is to identify the parameters that affect the recovery of pharmaceutical residues from the surface of stainless steel coupons. A ...

failure-cleaning-verification-pharmaceutical-industry-due-to

Nanyang Technological University

Research

JoVE Journal

Chemistry

8.1K Views.  Novartis Pharmaceuticals Corporation. Het ontbreken van een duidelijk omschreven procedure die consequent coupon oppervlakken gereinigd werd geïdentificeerd als de belangrijkste bijdrage aan lage en variabele terugvorderingen in het schoonmaken van de verificatie. Dit manuscript beschrijft het juiste protocol van de reiniging van roestvrij staal coupons.

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

Tracking Microbial Contamination in Retail Environments Using Fluorescent Powder - A Retail Delicatessen Environment Example

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne illness. Ready-to-eat (RTE) processed foods such as deli meats, cheese, and in some cases fresh produce, have been involved in foodborne disease outbreaks due to contamination with pathogens such as Listeria monocytogenes. With respect to L. monocytogenes, deli slicers are often the main source of cross contamination. The goal of this study was to use a fluorescent compound to simulate bacterial contamination and track this contamination in a retail setting. A mock deli kitchen was designed to simulate the retail environment. Deli meat was inoculated with the fluorescent compound and volunteers were recruited to complete a set of tasks similar to those expected of a food retail employee. The volunteers were instructed to slice, package, and store the meat in a deli refrigerator. The potential cross contamination was tracked in the mock retail environment by swabbing specific areas and measuring the optical density of the swabbed area with a spectrophotometer. The results indicated that the refrigerator (i.e. deli case) grip and various areas on the slicer had the highest risk for cross contamination. The results of this study may be used to develop more focused training material for retail employees. In addition, similar methodologies could also be used to track microbial contamination in food production environments (e.g.&#160;small farms), hospitals, nursing homes, cruise ships, and hotels.

The overall goal of this study was to demonstrate the potential cross contamination mechanism of foodborne pathogen Listeria monocytogenes in a retail deli setting. This methodology may be applied to a variety of different environments to track pathogen contamination. &#160;

Cross contamination of foodborne pathogens in the retail environment is a significant public health issue contributing to an increased risk for foodborne ...

Environment

Fabrication of Large-area Free-standing Ultrathin Polymer Films

This procedure describes a method for the fabrication of large-area and ultrathin free-standing polymer films. Typically, ultrathin films are prepared using either sacrificial layers, which may damage the film or affect its mechanical properties, or they are made on freshly cleaved mica, a substrate that is difficult to scale. Further, the size of ultrathin film is typically limited to a few square millimeters. In this method, we modify a surface with a polyelectrolyte that alters the strength of adhesion between polymer and deposition substrate. The polyelectrolyte can be shown to remain on the wafer using spectroscopy, and a treated wafer can be used to produce multiple films, indicating that at best minimal amounts of the polyelectrolyte are added to the film. The process has thus far been shown to be limited in scalability only by the size of the coating equipment, and is expected to be readily scalable to industrial processes. In this study, the protocol for making the solutions, preparing the deposition surface, and producing the films is described.

We describe a method for the fabrication of large-area (up to 13 cm diameter) and ultrathin (as thin as 8 nm) polymer films. Instead of using a sacrificial interlayer to delaminate the film from its substrate, we use a self-limiting surface treatment suitable for arbitrarily large areas.

This procedure describes a method for the fabrication of large-area and ultrathin free-standing polymer films.  Typically, ultrathin films are prepared ...

Biofilm Removal Using Carbon Dioxide Aerosols without Nitrogen Purge

Biofilms can cause serious concerns in many applications. Not only can they cause economic losses, but they can also present a public health hazard. Therefore, it is highly desirable to remove biofilms from surfaces. Many studies on CO2 aerosol cleaning have employed nitrogen purges to increase biofilm removal efficiency by reducing the moisture condensation generated during the cleaning. However, in this study, periodic jets of CO2 aerosols without nitrogen purges were used to remove Pseudomonas putida biofilms from polished stainless steel surfaces. CO2 aerosols are mixtures of solid and gaseous CO2 and are generated when high-pressure CO2 gas is adiabatically expanded through a nozzle. These high-speed aerosols were applied to a biofilm that had been grown for 24 hr. The removal efficiency ranged from 90.36% to 98.29% and was evaluated by measuring the fluorescence intensity of the biofilm as the treatment time was varied from 16 sec to 88 sec. We also performed experiments to compare the removal efficiencies with and without nitrogen purges; the measured biofilm removal efficiencies were not significantly different from each other (t-test, p &gt; 0.55). Therefore, this technique can be used to clean various bio-contaminated surfaces within one minute.

Biofilms on surfaces can be effectively and rapidly removed by using a periodic jet of carbon dioxide aerosols without a nitrogen purge.

Biofilms can cause serious concerns in many applications. Not only can they cause economic losses, but they can also present a public health hazard. ...

The Portable Chemical Sterilizer (PCS), D-FENS, and D-FEND ALL: Novel Chlorine Dioxide Decontamination Technologies for the Military

There is a stated Army need for a field-portable, non-steam sterilizer technology that can be used by Forward Surgical Teams, Dental Companies, Veterinary Service Support Detachments, Combat Support Hospitals, and Area Medical Laboratories to sterilize surgical instruments and to sterilize pathological specimens prior to disposal in operating rooms, emergency treatment areas, and intensive care units. The following ensemble of novel, &#8216;clean and green&#8217; chlorine dioxide technologies are versatile and flexible to adapt to meet a number of critical military needs for decontamination6,15. Specifically, the Portable Chemical Sterilizer (PCS) was invented to meet urgent battlefield needs and close critical capability gaps for energy-independence, lightweight portability, rapid mobility, and rugged durability in high intensity forward deployments3. As a revolutionary technological breakthrough in surgical sterilization technology, the PCS is a Modern Field Autoclave that relies on on-site, point-of-use, at-will generation of chlorine dioxide instead of steam. Two (2) PCS units sterilize 4 surgical trays in 1 hr, which is the equivalent throughput of one large steam autoclave (nicknamed &#8220;Bertha&#8221; in deployments because of its cumbersome size, bulky dimensions, and weight). However, the PCS operates using 100% less electricity (0 vs. 9 kW) and 98% less water (10 vs. 640 oz.), significantly reduces weight by 95% (20 vs. 450 lbs, a 4-man lift) and cube by 96% (2.1 vs. 60.2 ft3), and virtually eliminates the difficult challenges in forward deployments of repairs and maintaining reliable operation, lifting and transporting, and electrical power required for steam autoclaves.

The Portable Chemical Sterilizer PCS, D-FENS, and D-FEND ALL: Novel Chlorine Dioxide Decontamination Technologies for the Military

The Portable Chemical Sterilizer (PCS) is a revolutionary, energy-independent, almost waterless sterilization technology for Army medical units. The PCS generates chlorine dioxide from dry reagents mixed with water on-site, at-will, and at point-of-use (PoU) in a plastic suitcase. The Disinfectant-sprayer for Foods and ENvironmentally-friendly Sanitation (D-FENS) and the Disinfectant for ENvironmentally-friendly Decontamination, All-purpose (D-FEND ALL) produce aqueous chlorine dioxide in a collapsible spray bottle and other potential embodiments. These versatile decontamination technologies kill microbes in myriad diverse Dual-use applications for military and civilian consumers.

There is a stated Army need for a field-portable, non-steam sterilizer technology that can be used by Forward Surgical Teams, Dental Companies, Veterinary ...

Bioengineering

Light Enhanced Hydrofluoric Acid Passivation: A Sensitive Technique for Detecting Bulk Silicon Defects

A procedure to measure the bulk lifetime (&#62;100 &#181;sec) of silicon wafers by temporarily attaining a very high level of surface passivation when immersing the wafers in hydrofluoric acid (HF) is presented. By this procedure three critical steps are required to attain the bulk lifetime. Firstly, prior to immersing silicon wafers into HF, they are chemically cleaned and subsequently etched in 25% tetramethylammonium hydroxide. Secondly, the chemically treated wafers are then placed into a large plastic container filled with a mixture of HF and hydrochloric acid, and then centered over an inductive coil for photoconductance (PC) measurements. Thirdly, to inhibit surface recombination and measure the bulk lifetime, the wafers are illuminated at 0.2 suns for 1 min&#160;using a halogen lamp, the illumination is switched off, and a PC measurement is immediately taken. By this procedure, the characteristics of bulk silicon defects can be accurately determined. Furthermore, it is anticipated that a sensitive RT surface passivation technique will be imperative for examining bulk silicon defects when their concentration is low (&#60;1012 cm-3).

A RT liquid surface passivation technique to investigate the recombination activity of bulk silicon defects is described. For the technique to be successful, three critical steps are required: (i) chemical cleaning and etching of silicon, (ii) immersion of silicon in 15% hydrofluoric acid and (iii) illumination for 1 min.

A procedure to measure the bulk lifetime (&#62;100 &#181;sec) of silicon wafers by temporarily attaining a very high level of surface passivation when ...

Engineering

Preparation and High-temperature Anti-adhesion Behavior of a Slippery Surface on Stainless Steel

Anti-adhesion surfaces with high-temperature resistance have a wide application potential in electrosurgical instruments, engines, and pipelines. A typical anti-wetting superhydrophobic surface easily fails when exposed to a high-temperature liquid. Recently, Nepenthes-inspired slippery surfaces demonstrated a new way to solve the adhesion problem. A lubricant layer on the slippery surface can act as a barrier between the repelled materials and the surface structure. However, the slippery surfaces in previous studies rarely showed high-temperature resistance. Here, we describe a protocol for the preparation of slippery surfaces with high-temperature resistance. A photolithography-assisted method was used to fabricate pillar structures on stainless steel. By functionalizing the surface with saline, a slippery surface was prepared by adding silicone oil. The prepared slippery surface maintained the anti-wetting property for water, even when the surface was heated to 300 &#176;C. Also, the slippery surface exhibited great anti-adhesion effects on soft tissues at high temperatures. This type of slippery surface on stainless steel has applications in medical devices, mechanical equipment, etc.

Slippery surfaces provide a new way to solve the adhesion problem. This protocol describes how to fabricate slippery surfaces at high temperatures. The results demonstrate that the slippery surfaces showed anti-wetting for liquids and a remarkable anti-adhesion effect on soft tissues at high temperatures.

Anti-adhesion surfaces with high-temperature resistance have a wide application potential in electrosurgical instruments, engines, and pipelines. A ...

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation.

Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

Clostridium difficile is a pathogenic bacterium that is a strict anaerobe and causes antibiotic associated diarrhea (AAD). Here, methods for isolating, culturing and maintaining C. difficile vegetative cells and spores are described. These techniques necessitate an anaerobic chamber, which requires regular maintenance to ensure proper conditions for optimal C. difficile cultivation.

Clostridium  difficile is a Gram-positive, anaerobic, sporogenic bacterium  that is primarily responsible for antibiotic associated diarrhea (AAD) and is ...

Immunology and Infection

Micromechanical Tension Testing of Additively Manufactured 17-4 PH Stainless Steel Specimens

This study presents a methodology for the rapid fabrication and micro-tensile testing of additively manufactured (AM) 17-4PH stainless steels by combining photolithography, wet-etching, focused ion beam (FIB) milling, and modified nanoindentation. Detailed procedures for proper sample surface preparation, photo-resist placement, etchant preparation, and FIB sequencing are described herein to allow for high throughput (rapid) specimen fabrication from bulk AM 17-4PH stainless steel volumes. Additionally, procedures for the nano-indenter tip modification to allow tensile testing are presented and a representative micro specimen is fabricated and tested to failure in tension. Tensile-grip-to-specimen alignment and sample engagement were the main challenges of the micro-tensile testing; however, by reducing the indenter tip dimensions, alignment and engagement between the tensile grip and specimen were improved. Results from the representative micro-scale in situ SEM tensile test indicate a single slip plane specimen fracture (typical of a ductile single crystal failure), differing from macro-scale AM 17-4PH post-yield tensile behavior.

Presented here is a procedure for measuring fundamental material properties through micromechanical tension testing. Described are the methods for micro-tensile specimen fabrication (allowing rapid micro-specimen fabrication from bulk material volumes by combining photolithography, chemical etching, and focused ion beam milling), indenter tip modification, and micromechanical tension testing (including an example).

This study presents a methodology for the rapid fabrication and micro-tensile testing of additively manufactured (AM) 17-4PH stainless steels by combining ...

Determining Viral Disinfection Efficacy of Hot Water Laundering

This protocol provides an example of a laboratory process for conducting laundering studies that generate data on viral disinfection. While the protocol was developed for research during the coronavirus disease 2019 (COVID-19) pandemic, it is intended to be a framework, adaptable to other virus disinfection studies; it demonstrates the steps for preparing the test virus, inoculating the test material, assessing visual and integrity changes to the washed items due to the laundering process, and quantifying the reduction in viral load. Additionally, the protocol outlines the necessary quality control samples for ensuring the experiments are not biased by contamination and measurements/observations that should be recorded to track the material integrity of the personal protective equipment (PPE) items after multiple laundering cycles. The representative results presented with the protocol use the Phi6 bacteriophage inoculated onto cotton scrub, denim, and cotton face-covering materials and indicate that the hot water laundering and drying process achieved over a 3-log (99.9%) reduction in viral load for all samples (a 3-log reduction is the disinfectant performance metric in U.S. Environmental Protection Agency's Product Performance Test Guideline 810.2200). The reduction in viral load was uniform across different locations on the PPE items. The results of this viral disinfection efficacy testing protocol should help the scientific community explore the effectiveness of home laundering for other types of test viruses and laundering procedures.

In response to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, a laboratory protocol was developed to test the viral disinfection efficacy of hot water laundering of cloth face coverings, cotton scrubs, and denim pants. The Phi6 virus (bacteriophage) was used as the organism to test disinfection efficacy.

This protocol provides an example of a laboratory process for conducting laundering studies that generate data on viral disinfection. While the protocol ...

Microbial Control and Monitoring Strategies for Cleanroom Environments

Microbial Control and Monitoring Strategies for Cleanroom Environments and Cellular Therapies

A well-validated and holistic program that incorporates robust gowning, cleaning, environmental monitoring, and personnel monitoring measures is critical for minimizing the microbial bioburden in cellular therapy manufacturing suites and the corresponding testing laboratories to ensure that the facilities are operating in a state of control. Ensuring product safety via quality control measures, such as sterility testing, is a regulatory requirement for both minimally manipulated (section 361) and more than minimally manipulated (section 351) human cells, tissues, and cellular and tissue-based products (HCT/Ps). In this video, we provide a stepwise guide for how to develop and incorporate the best aseptic practices for operating in a cleanroom environment, including gowning, cleaning, staging of materials, environmental monitoring, process monitoring, and product sterility testing using direct inoculation, provided by the United States Pharmacopeia (USP&lt;71&gt;) and the National Institutes of Health (NIH) Alternative Sterility Testing Method. This protocol is intended as a reference guide for establishments expected to meet current good tissue practices (cGTP) and current good manufacturing practices (cGMP).

Author Spotlight: Microbial Control and Monitoring Strategies for Cleanroom Environments and Cellular Therapies  

The protocol summarizes the best practices to minimize microbial bioburden in a cleanroom environment and includes strategies such as environmental monitoring, process monitoring, and product sterility testing. It is relevant for manufacturing and testing facilities that are required to meet current good tissue practice standards and current good manufacturing practice standards.

A well-validated and holistic program that incorporates robust gowning, cleaning, environmental monitoring, and personnel monitoring measures is critical ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video