Paul Lopolito is a technical services specialist at STERIS, Mentor, OH.

Paul Lopolito

In the past several decades, advances have been made in the drug delivery vehicle for hydrophilic and lipophilic pharmaceutical actives (1). Drug delivery vehicles progressed from basic liposomes and emulsions to lipid nanoparticles (LNPs) and more advanced nanostructured lipid carriers (NLCs), which improved the therapeutic effect, reduced degradation, improved stability, controlled dosing, and minimized adverse toxicological effects of the target active (2,3). The particle size of these encapsulated LNPs and NLCs can range from 50–1000 nm and incorporate components such as lipids, surfactants, emulsifiers, co-surfactants, charge modifiers, preservatives, cryoprotectants, and others (

Table I. Typical components, processing techniques, and route of administration of lipid nanoparticles (LNPs) and nanostructured lipid carriers (NLCs) (4, 7).

Figure 1. Schematic of a cationic PEGylated mRNA lipid nanoparticle. (Figures courtesy of the authors)

Depending on the components and manufacturing process, the drug active may be within the core, lipid bilayers, surface, or combined locations (5). LNPs and NLCs delivery vehicles have been used for oral, pulmonary, nasal, topical, ocular, intravenous, and intramuscular drugs. Most recently, these LNPs and NLCs have been used in a wide range of vaccines, including hepatitis A, influenza, shingles, and the COVID-19 messenger RNA (mRNA) vaccines by Moderna/National Institute of Allergy and Infectious Disease (NIAID) and Pfizer/BioNTech (3).

The poor solubility and complexity of the LNPs with the active ingredient create cleaning challenges when cleaning with water, sodium hydroxide in water, or alcohol (

). Solubility of the LNP is improved with alcohol; however, this creates flammability, storage, handling, and disposal concerns. The investigation and design of a cleaning process using laboratory coupon studies incorporating the drug components, LNP or NLC delivery vehicle, manufacturing process conditions, and surface material align with testing a process’s life cycle design phase. Critical parameters to investigate may include different components, process conditions, surface materials, cleaning agent, temperature, time, mechanical action, water quality, and rinse parameters. The overall goal of a cleaning study is to design a repeatable cleaning procedure and maintain process equipment.

Table II. Lipid nanoparticle components, examples, and solubility in water.

This article explores the use of laboratory studies to recommend a cleaning process for COVID-19 mRNA vaccines. It provides a technique to easily design the cleaning process for other LNP and NLC delivery vehicles. The article also includes lab study results and a case study for cleaning LNPs currently used for the COVID-19 mRNA vaccine.

Developing a cleaning procedure for LNPs is the first step in a validation process to minimize or eliminate product contamination and ensure product reproducibility. The nature of the sample, processing time and temperature, dirty-hold-time (DHT), water quality, available temperature range, cleaning method capability, and preferred detergent of use are considered during the cleaning study (6). Studies are performed at a lab-scale using 304 stainless-steel coupons with a 2B finish to duplicate a large 316-L stainless-steel vessel used in production. The final product or components of the final blend are coated onto the surface and air-dried at ambient temperature for 24–72 hours, depending on the individual DHT requirement (

). At the end of the DHT, the coated coupon is placed in a prepared cleaning solution at the minimal temperature available using agitated immersion. Once the cleaning parameters are determined by agitated immersion, the same cleaning parameters are tested using spray wash, cascading flow, or manual scrub/wipe followed by rinse water. The cleaning parameters were evaluated by visual cleanliness, a gravimetric weight check of the pre-cleaned and post-cleaned stainless-steel surface, and a water break-free test.

In review of the manufacturing process, the cleaning and compatibility of various substrates should be considered and possibly evaluated when designing a cleaning procedure. In one example, platinum-cured silicon was tested for substrate compatibility (

. At a minimum, this process requires the substrate to be exposed to the recommended detergent at a specific concentration for a designated period. If no significant changes are observed to the substrate, it is usually considered compatible.

Figure 2. L–R: (a) Lipid mixture, (b) messenger RNA (mRNA)-encapsulated lipid nanoparticles in ethanol/citrate buffer, (c) mRNA encapsulated lipid nanoparticles in 10% trehalose, and (d) clean coupon. All residues (a-c) were applied to 304 stainless-steel surfaces and baked at 50 °C for 48 hours to simulate the process condition, followed by dirty hold time (DHT) conditioning at 30 °C for 48 hours. This residue was slightly corrosive to 304 stainless-steel coupons with the development of a small micro-pit on the surface. The process should be repeated with passivated 316-L stainless steel coupons to simulate the effect on production equipment. Early warning signs of corrosivity of process conditions within cleaning studies can be investigated using a predictive model to define a preventative maintenance plan or justify the inclusion of routine maintenance steps with a formulated acid detergent (8). Stainless-steel maintenance studies performed early in the cleaning design phase can greatly reduce unscheduled maintenance events and investigations. (Figures courtesy of the authors)

Table III. Compatibility of platinum-cured silicone in water for injection (WFI) in comparison to formulated alkaline detergent after 24 hours of submersion at 70 °C. The temperature was maintained throughout the testing cycle.

Based on the processing conditions and DHT, the cleaning recommendations ranged from a solution of 1–5% v/v formulated alkaline detergent at 45 °C–80 °C for a contact cleaning time of 15–45 minutes per agitated cleaning, spray wash cleaning, and cascading flow cleaning. These test conditions were successful as reported in 

, based on visually clean, water-break free, and gravimetric analysis (6). In comparison, a solution of 1N sodium hydroxide (NaOH) at 60 °C, cleaned for 60 minutes, was not effective in completely removing the LNP residue.

Table IV. Dirty hold time (DHT) and cleaning parameters for cleaning lipid nanoparticles (LNPs). NaOH is sodium hydroxide.

Progress in mRNA technologies and LNP-based delivery systems has allowed the development of mRNA COVID-19 vaccines at unprecedented speed, demonstrating the clinical potential of LNP–mRNA formulations and providing a powerful tool against the coronavirus pandemic (1). Along with the mRNA vaccine development, the goal was to develop optimum cleaning parameters, so production was safe and reproducible.

A large pharmaceutical company that developed a mRNA vaccine sent their new product to the laboratory for testing. All coupons were coated and baked at 35 °C for two and 16 hours to simulate manufacturing DHT conditions. Cleaning parameters (time, temperature, action, and concentration) were identified through testing by agitated immersion. A 1% volume-by-volume (v/v) cleaning solution of potassium hydroxide formulated detergent at 45 °C for 15 minutes was able to clean the residue. Results were confirmed by spray wash, cascading flow, and manual cleaning. Sodium hydroxide, however, was unable to achieve acceptable results.

After the successful cleaning trial with potassium hydroxide formulated detergent, the entire manufacturing area, including filling and packaging lines, adopted the same cleaning regimen.

Modifying the delivery vehicle of hydrophilic or lipophilic drugs from basic liposomes to complex LNPs and NLCs has improved these drugs’ therapeutic effect, stability, and toxicological profile. Water-insoluble lipids, practically insoluble charge modifiers, and hydrophilic or lipophilic active ingredients create difficulties with water or caustic (hydroxide in water) cleaning processes. Using flammable solvents, such as ethanol and isopropyl alcohol, improves lipids’ solubility and creates storage, handling, and disposal hazards, due to the volatile carbon emissions and flammability. Early investigations into the cleaning process of LNP delivery vehicles with small molecule drugs, and recently with mRNA vaccines, have demonstrated that a formulated alkaline cleaning agent can successfully clean both the active ingredient and the LNP or NLC delivery vehicle to an acceptable limit.

N. Naseri, H. Valizadeh, and P. Zakeri-Milani, 

D. Chatzikleanthous, D.T. O’Hagan, and R. Adamo, 

Indian Journal of Pharmaceutical Sciences,

E. Rivera, D. Hadziselimovic, and P. Lopolito, 

Paul Lopolito is a senior technical services manager, Dijana Hadziselimovic is a technical services laboratory specialist, and Si Myra Tyson is a senior technical service associate, all for the Life Sciences Division of STERIS.

When referring to this article, please cite it as D. Hadziselimovic, Si Myra Tyson, and P. Lopolito, “Considerations for Cleaning Lipid Nanoparticles,” 

Articles

This article explores the concerns with cleaning pharmaceutical products utilizing LNP delivery vehicles and 
provides a general cleaning recommendation based on laboratory and field testing.


Considerations for Cleaning Lipid Nanoparticles 

Regulated companies that use automated cleaning applications have always struggled with correlating rinse sample quality or visual inspection to surface cleanliness. The process typically involves performing 100% spray coverage testing, rinse recovery testing, specific or non-specific analytical method qualification, surface sampling and recovery testing of the sampling technique, and visual inspection performed during the cleaning validation. Each element of the justification needs to be reviewed and evaluated during the cleaning process design stage to defend a lean approach in continuously monitoring the cleaning process. A risk-based approach, supplemented with laboratory studies and information from published literature, can be leveraged in the justification to reduce testing during cleaning qualification and continuous monitoring stages without impacting quality. Laboratory results from total organic carbon (TOC) and conductivity rinse sample for an alkaline detergent along with determining visible residue limits using multiple concentrations, light intensity, viewing angles, inspectors, and distances have been used by multiple biopharmaceutical companies. This approach has helped correlate rinse sample quality or visual inspection and justify a lean approach to continuously monitoring surface cleanliness.

	FDA’s 1998 cleaning validation guidance document focuses on general aspects and qualification of the cleaning process (1). The design and post-validation monitoring aspects are factored into the validation process, but are not specified in the regulatory guide nor in the industry practices. Today, this is referred to as the old or traditional approach to validation. Other countries used a similar approach to provide guidance to the industry of the regulatory expectations regarding cleaning and cleaning validation (2–3). 

	In 2011, FDA issued a revised process validation guidance document that introduced a concept known today as the product lifecycle model (4). This model separates the various steps and activities of validation into three stages: process design, process qualification, and continuous process verification. Since then, the lifecycle model has become the “gold standard” for all types of processes including cleaning validation; it provides a better understanding of the design and monitoring of the cleaning process (see 

) and, consequently, it ensures a more robust cleaning validation program.

	Along with the process lifecycle approach, other guides have been issued by FDA and the International Council for Harmonization (ICH), which include the concepts of quality by design (QbD), risk management, and process analytical technology (PAT) (5, 6). The purpose of these guidance documents is to promote enhanced understanding of products and processes, to build quality into manufacturing, and to provide the basis for continuous improvement of products and processes. 

 shows the correlation between these documents and the lifecycle model. Consequently, the lifecycle model places more attention on understanding the process and its design, and continued monitoring of the operation to ensure the expected results. By comparison, the old validation approach placed most efforts on qualifying the process rather than understanding it.

	Cleaning in the GMP industry is a critical process intended to prevent or, in more modern terms, reduce risk of contaminating the subsequent product with undesirable residues that may impact patient safety. For that reason, it is important to make the right connection between rinse sampling and/or visual inspection to surface cleanliness. Reducing the risk requires good understanding of the cleaning process. Some aspects of the lifecycle model must be considered to ensure that surface cleanliness not only is feasible and consistent but also relates to patient safety. 

 depicts a workflow that helps bridge the gap between sampling to surface cleanliness, which will be discussed in more detail.

	Review engineering design and coverage testing

	The design of the process equipment must be considered to ensure that the cleaning recommendations can be successfully scaled-up from laboratory-scale experiments. For automated clean-out-of-place (COP) and clean-in-place (CIP) applications, it is important to review coverage and flow velocity in vessels and piping, drainability of the wash and rinse solutions, surface finish, and materials of construction (7–9).   

	Spray devices should cover all the surfaces that they are intended to clean. These types of devices can be fixed or dynamic. Riboflavin (vitamin B

 and an ultraviolet (UV) light ensures that the spray device gives full coverage during the wash and rinse steps. For vertical tanks, the flow rate of a static spray device should be about 2.5–3 gallons per minute per feet of internal tank diameter (10). Vessel inserts such as agitators, probes, and baffles may be challenging to clean in place and hence, coverage testing becomes more crucial to designing for a successful cleaning. 

	Flow rates in piping are important for proper coverage of solutions, as is providing turbulent flow. The flow velocity in piping should be five feet per second (approximately 1.5 m/s) to prevent air entrapment at vertical elbows. Dead legs should be minimized, and the length-to-diameter ratio of less than or equal to 1.5 is preferred. Dead legs should also be oriented at an angle to allow for full coverage and drainage during washing and rinsing. 

	The drainage of wash and rinse solution is also important. Pooling in vessels and piping may lead to microbial blooms, biofilm, and carryover of process and cleaning agent residues. The mixing of the drain solution (heel) can reduce residue deposition on the side walls. Horizontal tanks, equipment surfaces, and piping should be sloped at 1/8–1/16 inches per square foot (3.6–1.8 cm per square meter) to allow full drainage.

	The preferred materials of construction in the pharmaceutical industry are 304 and 316L stainless steel. Other materials of construction are used throughout the industry. The selection of materials should be based on both the manufacturing process and on the cleaning procedure required for reducing the amount of residue to safe levels. Surface roughness as well as other materials of constructions in use should be included in the laboratory studies to ensure that such levels do not adversely impact cleanability and recovery of residues. 

	Cleaning agent and analytical method selection

	Cleaning agents should be selected based on laboratory studies that simulate the process soil, soil condition, and cleaning method used, as well as performing a supplier qualification and technical support review. Further consideration should be given to formulation, toxicity, analytical method for residuals, rinsability of components, stability of closed and open containers, and assurance that the product will be made consistently over the life of the product, which is often in decades for cleaning agents intended for GMP applications. Several cleaning agent options are available, including water, solvents, commodity chemicals, and formulated cleaning agents (11). Formulated cleaning agents are blends of the latter components that help improve cleaning performance by incorporating several cleaning mechanisms in solution. 

lists different components used in designing cleaning agents (12). 

	A variety of analytical methods are used to detect active ingredients, degradation or byproducts, bioburden, endotoxin level, and cleaning agent residues. The selection may be based on multiple factors including the analyte of interest, analytical resources available, rinsability, and carry-over risk.

	The rinsability of a cleaning agent is crucial in demonstrating that the components within the formulation rinse freely from the surface with water. If the components within the formulation rinse at similar rates, then either non-specific methods, such as conductivity or TOC, or specific methods, such as ultra high-performance liquid chromatography (UHPLC), can be used (13, 14). If the components do not rinse at similar rates, then it is important to identify and set residual limits based on the last-to- rinse component. Refer to 

to compare rinsing results with various analytical methods for monitoring components within a formulated cleaning agent (13). 

	Cleaning agents used for product contact surfaces must have toxicity information available, which is important in determining safe levels of cleaning agent residues. The toxicity of components is important, especially if they are the last to rinse from the surface or if they display mutagenic, reproductive, carcinogenic, developmental, or chronic toxicity. In the case of cleaning agents, the sum of components may have increased or decreased toxicity compared to individual components, and toxicity of the cleaning agent should be available from the suppliers.  

	The ability to sample the surface for cleanliness is the preferred technique to demonstrate the equipment is ready to manufacture the next batch of product, a new product, or to proceed with the next manufacturing process step. Surface testing, however, requires a significant amount of time to: prepare the equipment for sampling; prepare and source sampling tools; sample the surfaces; transport samples for testing; test samples; and review results of testing.

	In addition to the amount of time required for sampling and testing, there is allocation of labor for sampling and testing, often scheduling between multiple departments, and more importantly lost time that could be used for manufacturing or sampling and testing critical steps within the manufacturing process. The selection of sampling sites and test methods and performing surface sampling recovery will help in significantly reducing or even eliminating routine surface sampling to demonstrate surface cleanliness.

	The European Union GMP guideline, Annex 15 section 10.11, cites that cleaning validation protocols should specify or reference the locations to be sampled, specify the rationale for the selection of these locations, and define the acceptance criteria (15). A documented sampling and testing plan must be prepared describing: location of samples (and rationale), number of samples per location, sampling test methods used, wetting agent, and surface area sampled. 

	The use of diagrams, such as the one illustrated in 

, provides the operator with a better picture of the sampling areas to focus on (16). They also aid in understanding the rationale behind the sampling site selection. 

, for example, shows the interior of a vessel with multiple accessories that reduce the coverage provided through the spray device (top circumference) in a CIP system. The shaded area in the vessel represents the normal process air-to-liquid interface, which is often an area where solids settle onto the surface and form a scum-like ring on the walls. 

	Sampling sites should be defined based upon the geometry and design of the equipment, materials of construction, hard-to-clean areas, and locations that may have a higher risk to product contamination (i.e., risk assessment), or they may be based on historical reference from similar equipment on site (i.e., benchmark). The riboflavin testing used for spray device qualification in CIP systems also helps in identifying areas that must be sampled due to limited coverage. 

Risk-based surface sampling locations and testing performed

	A risk assessment should be performed to minimize the number of sampling sites. 

 is an example of a list of sampling sites and assigned risk factors. 

	A risk assessment should also be performed to minimize the number of tests performed, such as microbial, detergent, or active ingredient testing. For example, a TOC swab may be able to demonstrate removal of the large molecule API as well as the cleaning agent used. A single UHPLC swab may be able to demonstrate the removal of multiple detergents that are used in separate wash steps of the cleaning procedure (17). Applying a scientific, risk-based rationale can drastically reduce the number of samples taken as well as testing performed.

	Surface sampling recovery studies using swabs, wipes, or direct sampling techniques should be performed as part of the design stage to ensure that testing of the surface can meet predefined acceptance criteria. Surface sampling should define the swab or wipe used, surface location sampled, surface area sampled, material of construction of the surface, number and condition of the swab(s) or wipe(s), sample storage and stability conditions, swab or wipe diluent, and sampling accessories; qualified analysts should perform the testing. The surface sampling recovery study should also consider, if applicable, the swab or wipe wetting solution and various spiked amounts on the surface. The spiked amount and design of the surface sampling recovery test should challenge the sampling and analytical procedure. 

	The analysis of swab recovery from various active pharmaceutical ingredients as well as cleaning agent residues from a single site using various materials of constructions (including metals, plastics, and elastomers) supported that stainless steel could be used as a representative surface for swab recovery (18). Forsyth, et. al. pooled recovery factors from 16 sites, 29 different materials, and hundreds of samples. The reported recovery factor from a formulated alkaline cleaning agent, CIP 100 detergent, from 316 stainless steel was 93% (18).

	Visual inspection is important to ensure that the equipment looks sufficiently clean to proceed with analytical testing. There are limitations, similar to surface sampling, with routine visual inspections, such as large equipment or lengths of piping, as well as the time and resources required for proper visual inspection. Visual inspection can be defined as the process of using the unaided eye as the sensing mechanism from which judgments can be made about the condition of a unit to be inspected. A visual non-uniformity on equipment surfaces may present as an interruption of the normal pattern or grain either by residue or discoloration in amount as perceived by the unaided eye. This non-uniformity could be on the surface, such as visible residue, particulate matter, pooling of liquid, or rouge (oxides) or within the surface, such as scratches, corrosion, or etching. 

	During the design and qualification stages of the lifecycle model, a correlation between visual inspection, surface sampling, and rinse sampling should be performed, so that a visual inspection or rinse sample can be scientifically justified to determine that the equipment, piping, or parts are cleaned. Visual inspection as the method to assess for cleanliness is ideal for small parts or open process equipment that are easy to inspect. However, “visually clean” as the only acceptance criterion will require a justifiable, scientific rationale that will need to be defended when inspected (15). Several published articles have evaluated the use of visual inspection procedures and the role of visible cleanliness in control of critical variables, as well as establishing visible residue limits of select analytes. 

	In 1993, Fourman and Mullen specified a visual limit of small- molecule active ingredients of 1 to 4 mcg/cm

 (19), and this article was referenced in the FDA guide to cleaning validation (1) as well as Parenteral Drug Association (PDA) technical report 29 (20). Forsyth et al. have published several articles that include testing and defining critical variables as well as presenting various case studies. The case studies include spiking a 1 cm

 surface with residue at various concentrations and on different substrates. Once the residue is dried, it can then be inspected visually  at different distances, angles, light intensities, with the use of mirrors, and by different analysts (21, 22). By defining the operators’ qualifications, visual inspection tools and conditions, procedures, and training and retraining activities, a company can quantify and validate the visual inspection procedure. The 

describes a VRL case study for a biopharmaceutical facility.

					Sidebar: visible residue limit case study

					A large biopharmaceutical manufacturer was designing a new facility for the final compounding and filling of their product. This standard parenteral facility consists of an automated parts washer, steam sterilizer, automated vial washer, depyrogenation tunnel, filling machine, closure machine to insert stoppers, capping machine, and product labeler. This product was being filled by a contract manufacturing organization (CMO), which was using only water for cleaning.

					During the design phase of the cleaning program, coupon testing was performed by coating the finished product onto stainless-steel coupons and evaluating the cleaning with water and a formulated alkaline cleaning agent to remove the finished product. Water-only cleaning at ambient to 80 oC was not sufficient to yield a visual clean and water-break free surface. Cleaning the coupons with water was successful in meeting the gravimetric testing of around 1–2 mcg/cm2. In support of a visual inspection program of the items being washed in the parts washer, a visible residue limit (VRL) study was performed on the final drug product as well as on a formulated alkaline cleaning agent on stainless-steel and glass surfaces.

					The VRL study procedure consisted of precleaning and drying 304 stainless-steel coupons with a 2B finish as well as borosilicate-glass coupons. The roughness of the 304 stainless coupons was comparable to the 316L stainless-steel specifications for the filling parts. The formulated acid and alkaline cleaning agents as well as the finished product were compatible with 304 stainless steel, so the use of these coupons as a test substrate versus 316L was justified. The test samples were serially diluted, and 1 mcl of diluted sample and 20 mcl of low TOC de-ionized (DI) water were applied over a 1-cm2 area. The sample was then allowed to air-dry at ambient temperature for at least 16 hours.

					After drying, the coupons were inspected by two analysts, in duplicate, at one of the following distances (0.45 m, 1.0 m, and 1.5 m); lighting conditions (250 lux, 500 lux, and 1000 lux); viewing angles (30o, 45o, and 90o); and with and without a viewing mirror.  A light meter (Cooke Corporation Cal-Light 400 lux), digital protractor, and 1–5 mcl syringe (Hamilton) were used in the study. The acceptable results for both analysts on two coupons per set for the formulated alkaline cleaning agent are noted in 

. The borosilicate glass surface was used in the construction of the viewing endcap on the final formulation compounding tank, so the higher VRL observed was not critical.

					The visual inspection procedure as well as the inspector qualification study consisted of precleaning and visual inspection of the coupons before spiking. An analyst receives a yearly eye exam with 20/20 vision (glasses or contact lenses are acceptable), wears safety glasses during visual inspection, and is informed that some coupons may not contain a sample. This procedure is used to try to eliminate phantom residues or false positives.

					The coupons were placed flat on a table in front of a fume hood in groups of three, and the inspector stood back approximately 0.85 m and inspected the coupons at a 50o angle. The groups of three coupons were mixed in order as seen in 

. The lighting in the room was between 500 and 1000 lux.

					An acceptable result is that the inspector identifies all coupons with a loading of 10 mcg/cm2 and 12 out of 15 of the remaining coupons.

	Rinse sampling is commonly used to evaluate surface cleanliness of closed production equipment, hoses, and piping commonly cleaned by CIP systems (23–25). The advantages of rinse sampling are that the entire surface can be sampled, with no disassembly of equipment and no direct sampling of the surface, and that rinse sample analysis via conductivity, TOC, UV, and other methods in-line or on-line can be adapted to PAT technologies. The disadvantages of rinse sampling are that the analyte measured may not be soluble in the rinse solution, rinsing may not pick up the residue due to poor coverage during rinsing, and the analyte may be too diluted in the rinse solution volume.

	Rinse recovery studies can be used in addition to assessing the solubility of the active ingredient in the rinse solution. The studies are performed by adding a specified concentration, around the acceptable limit, of the residue on the surface. The selection of the residue, conditioning of the residue, surface material, roughness of the surface, rinse solution, volume of rinse solution per surface area, rinse solution temperature, and flow rate should all be considered in setting up a rinse solution recovery study. 

	Two rinse recovery studies are discussed as examples. The first rinse recovery case study includes a linearity between concentration and conductivity of a formulated alkaline cleaning agent 

 as well as the recovery factor from stainless steel, polytetrafluoroethylene (PTFE), rubber, and glass surfaces 

. The second rinse recovery case study includes a linearity between concentration and TOC of a formulated alkaline cleaning agent 

 as well as the recovery factor from stainless steel, PTFE, rubber, and glass surfaces 

Three lots of the formulated alkaline cleaning agent were diluted at various concentrations between 1 and 100 ppm by volume using DI water and tested at ambient temperature. A conductivity meter (Radiometer/Copenhagen CDM83) was used, and the conductivity probe was standardized following the manufacturer’s recommendations with 50 and 100 mcS/cm standards prior to testing. Temperature compensation was not used for this testing. The conductivity to concentration curve is reported in 

	Three lots of the formulated alkaline cleaning agent were diluted at various concentrations between 0.1 and 5 ppm C by volume using DI water and tested at ambient temperature. A laboratory TOC analyzer (Sievers 900) was used for testing, and the TOC analyzer was standardized following the manufacturer’s recommendations with reference standards for sucrose and benzoquinone. The TOC to concentration curve is reported in 

	Rinse recovery testing was performed by applying 1 +/-0.1 g of the prepared 0.24% w/w formulated alkaline cleaning agent to about 100 cm

 area of one side of the stainless steel, PTFE, glass, and rubber coupons using an analytical balance (Mettler Toledo XS). Solution was applied in drops to provide a uniform coverage. Samples were air-dried on the coupons in a horizontal position. After drying, 300 mL of DI water at ambient temperature was poured from a squeeze bottle over the coupon so that the flow cascades down the face of the coupon for approximately 30 seconds. The collected rinse water was agitated for 30 seconds to ensure uniformity, then 39 mL was poured into a labeled TOC vial, sealed, and measured for TOC (Sievers 900 laboratory TOC analyzer). The TOC recovery results are reported in 

 The remaining sample (39 mL) was measured for conductivity (Fisher Scientific AB30 conductivity meter). The conductivity recovery results are reported in 

	Surface cleanliness is crucial in ensuring that process residue, cleaning agent residue, and bioburden do not adversely affect the safety, quality, and potency of the drug manufactured. Surface cleanliness can be determined through surface sampling, rinse sampling, and visual inspection. The preferred method is surface sampling through swabbing, wiping, or direct measurement. Surface sampling can add production delays, increased sampling costs, and often increased safety risk to the analyst. If the engineering of the equipment, coverage testing of the equipment, and cleaning agent and analytical method selection have been well vetted during the design and qualification stages of the lifecycle approach, then rinse sampling or visual inspection can be successfully used to demonstrate surface cleanliness. Attention to detail and application of a risk-based approach during the design stage can provide justification for using either rinse sampling or visual inspection in determining surface cleanliness.

	The authors are grateful to Amanda Deal for excellent technical assistance in performing the VRL study as well as the first and second rinse recovery case studies.

Guide to Inspections Validation of Cleaning Processes

Guide-0028 Cleaning Validation Guidelines

PE-006-3 Validation Master Plan Installation and Operational Qualification Non-Sterile Process Validation Cleaning Validation 

Process Validation: General Principles and Practices

Guide for Industry. PAT-A Framework for innovative pharmaceutical development, manufacturing, and quality assurance

Harmonised Tripartite Guideline-Q9 Quality Risk Management

Basic equipment-design concepts to enable cleaning in place: Part I

Pharm. Tech. Equipment and Processing Report

 (June 15, 2011), accessed Oct. 10, 2017.
	8. G. Verghese and P. Lopolito, “Cleaning Engineering and Equipment Design,” in 

Cleaning and Cleaning Validation Volume I

, P. Pluta Ed. (DHI Publishing and the Parenteral Drug Association, 2009) pp 123-150.
	9. J. Voss, 

Cleaning and Cleaning Validation: A Biotechnology Perspective 

pp 1-39 (PDA, Bethesda, MD, 1996).
	10. ASME, 

p. 52 (2012).
	11. G. Verghese and N. Kaiser, “Cleaning Agents and Cleaning Chemistry, Chapter 7” in 

, P. Pluta Ed. (Davis Healthcare International and Parenteral Drug Association, 2009) pp 103-121.
	12. P. Lopolito, “Critical Cleaning for Pharmaceutical Applications, Chapter 17” in 

Handbook for Critical Cleaning Applications, Processes and Controls, Second Edition

, B. Kanesgsberg and E. Kanesgsberg, Eds. (CRC Press, Taylor & Francis Group, 2011).
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6 (1) 424-436 (1999).
	14. H. J. Kaiser and B. Ritts, “Validation of Analytical Methods Used in Cleaning Validation,” 

 10 (3) (May 2004).
	15. European Commission, 

Good Manufacturing Practice Medicinal Products for Human and Veterinary Use-Annex 15, Qualification and Validation

 (2015).
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Contamination Control in Healthcare Product Manufacturing, Vol 3.

 R. Madsen and J. Moldenhauer, Eds. (DHI Publishing, PDA Books, 2014).
	17. M. Gietl, B. Meadows, and P. Lopolito, “Cleaning Agent Residue Detection with UHPLC” 

 (April, 2013), www.pharmamanufacturing.com/articles/2013/1304_SolutionsTroubleshooting/, accessed Oct. 10, 2017.
	18. R. L. Forsyth, J.C. O’Neill, and J.L. Hartman, 

  31 (10) 103-116 (2007).
	19. G. L. Fourman and M.V. Mullen, 

Technical Report No. 29 (Revised 2012) Points to consider for cleaning validation.

 (Bethesda, MD, 2012).
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 28 (10) 58-72 (2004).
	22. D. A. LeBlanc, 

 56 (1) 31-36 (2002).
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12 (5) 66-74 (1988).
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	December 2017
	Vol. 41, No. 12
	Pages: 28–37

	When referring to this article, please cite it as E. Rivera and P. Lopolito, "Evaluating Surface Cleanliness Using a Risk-Based Approach," 

	Paul Lopolito is a senior technical services manager and Elizabeth Rivera is a technical services manager, both at the Life Sciences Division of STERIS Corporation in Mentor, Ohio.

Rinse sample analysis or visual inspection are risk-based approaches that can be correlated to surface cleanliness to replace surface sampling in a biopharmaceutical equipment cleaning process.

Evaluating Surface Cleanliness Using a Risk-Based Approach

	
	Stainless steel is usually the preferred substrate for good manufacturing practice (GMP) applications, and it constitutes the majority of GMP product-contact surface areas. The austenitic stainless-steel series (e.g., 304L and 316L) has been popular in pharmaceutical applications because of its high stain and corrosion resistance and affordability. Although stainless steel is named “stainless

, it is really “stain resistant”. This property comes from its ability to naturally form an oxide-rich, passive layer on the surface.

	Passivation is a process in which a passive layer forms on the stainless-steel surface; this layer occurs naturally in the presence of oxygen when the surface has been cleaned of exogenous matter (1–3). The passive layer on the stainless-steel surface becomes the primary means of protection to prevent corrosion. Stainless steel can corrode, however, when the chromium-to-iron ratio has been significantly reduced, resulting in the oxidation and subsequent release of iron oxides that form deposits on surfaces.

	Rouge is the commonly used term for the visible corrosion product of stainless steel; it can be composed of several forms of iron oxides, with ferric oxide being the predominant form (4–7). Rouging is typically found in water generation systems, process tanks, and pipeline systems that are routinely exposed to corrosive solutions. Rouge seems to be a common problem. Regulatory agencies such as FDA have cited in at least one warning letter that corrosion is unacceptable in direct-contact pharmaceutical systems (8). The reasoning is that rouge on product-contact surfaces can create an environment for process residues and microbes to tenaciously adhere to the rouged area and become more difficult to clean and sanitize (9-11). Residues and microbes might also reside within the rouge layers, where the routine cleaner and sanitizer may not be able to penetrate.

	Considering the risk associated with rouged surfaces, manufacturers would benefit from focusing more attention on treatments that prevent rouge from happening. Some companies take a reactive approach and wait until rouge has been detected or has impacted production before taking corrective action. Process attributes such as elevated temperature, extreme pH solutions, or surface damage (e.g., from poor quality welding) can corrode stainless-steel surfaces (4-7). If a process or surface condition is expected to lead to corrosion at some point during the life of the equipment, then an effort should be made to investigate and prevent that corrosion from occurring.

	Unlike preventive maintenance, which is done to mitigate the cause of the potential problem or undesirable situation, corrective maintenance is done to correct a problem or fault once it has been detected. With a stainless-steel preventive maintenance procedure, for example, an operator knows exactly what needs to be done at a predefined schedule. On the contrary, in the case of a corrective maintenance, the critical parameters and overall procedure would depend on what is found. Corrective maintenance often requires evaluation of the severity of the rouge problem before any treatment can be recommended. This evaluation is referred to as a risk assessment, and includes a review of the potential impact to the patient, product, personnel, and equipment (12). Once the rouge is formed, there may be unknown variables associated with it and correcting the problem typically takes much longer than a preventive task. Many published references about stainless-steel corrosion are focused on corrective maintenance (3–11, 13–15).

	When assessing the risk of the corrosion or rouge to the quality of the next product or batch, one should ask the questions about risk and mitigation actions summarized in 

	For more information about passivation treatments, laboratory tests to evaluate the effectiveness of passivation treatments, and tests to determine critical cleaning parameters for passivation, read the complete article by E. Rivera, P. Lopolito, and D. Hadziselimovic, 

"A Risk-Based Approach to Stainless Steel Equipment Maintenance," 

A967–13,Standard Specification for Chemical Treatments for Stainless Steel Parts 

Standard Practice for Cleaning, Descaling, and Passivation of Stainless Steel Parts, Equipment, and Systems 

		ASME, “Nonmandatory Appendix E: Passivation Procedure Qualification,” in 

		M.M. Gonzales, “Stainless Steel Tubing in the Biotechnology Industry, Biotechnology/Pharmaceutical Facilities Design,” (April 30, 2001) 

www.ispe.org/san-francisco/stainless-steel-tubing-biotechnology-industry.pdf

		J.C. Tverberg and J.A. Ledden, “Rouging of Stainless Steel in WFI and High Purity Water Systems,” Proceedings of Tube 2000, (Dusseldorf, 2000).	

		P. Lopolito, “Addressing Rouge in Biopharmaceutical Manufacturing Equipment,” 

Pharm.Tech. Equipment and Processing Report

www.pharmtech.com/addressing-rouge-biopharmaceutical-manufacturing-systems

		GMP Trends, “GMP Trends #886,” (Dec. 15, 2013).	

Rouge & Biofilm: Compounding Cleaning Challenges

www.pharmaceuticalonline.com/doc/rouge-biofilm-compounding-cleaning-challenges-0001

		A. Deal, D. Klein, and P. Lopolito, “Strategies for biofilm remediation,” 

www.cleanroomtechnology.com/technical/article_page/Strategies_for_biofilm_remediation/110760

		A. Deal et al., “Use of the CDC Biofilm Reactor to Test Cleaning and Disinfection on Rouged Stainless Steel,” 

 (March 20, 2015) www.reinraum.de/news_en.html?id=3179.	

		International Conference on Harmonization (ICH) Q9 


	Elizabeth Rivera is a technical services manager; Paul Lopolito is a senior technical services manager; and Dijana Hadziselimovic is a senior technical services associate, all at the Life Sciences Division of STERIS Corporation in Mentor, Ohio. 

Process conditions can corrode stainless-steel surfaces, necessitating corrective and preventive maintenance.

Preventive and Corrective Maintenance for Rouge in Stainless-Steel Equipment

Stainless steel is usually the preferred substrate for good manufacturing practice (GMP) applications, and it constitutes the majority of GMP product-contact surface areas. The austenitic stainless-steel series (e.g., 304L and 316L) has been popular in pharmaceutical applications because of its high stain and corrosion resistance and affordability. Although stainless steel is named “stainless

	There is no globally accepted test to guarantee that a stainless-steel surface has been adequately passivated. Generally, when stainless-steel equipment has been exposed to a passivation treatment, some documentation should be generated describing the passivation procedure with emphasis on the critical parameters. A test matrix has been suggested to be used as a guide for acceptance criteria and to confirm that a surface is passivated (3). The overall scope of these test methods is to either verify that the surface has been passivated by removal of exogenous matter (e.g., free iron or hydrophobic films) or by direct measurement of the quality of the passive layer. 

 summarizes the advantages and disadvantages of common passivation test methods (2–3). Representative coupons of similar material and finish to the production equipment can be used to validate a passivation protocol.

	Passivation treatments are dependent on the chromium content, surface finish, and machinability characteristics of the grades in each family of stainless steel. The chemical treatment should help restore the inert oxide layer in a consistent, faster-forming passive layer than found naturally. Passivation treatment using citric acid blends and phosphoric acid blends are highly effective for a large number of stainless-steel families. The conventional nitric acid passivation methods and others are summarized in 

. The choice of passivation chemistry and method depends on the acceptance criteria imposed by the end-user as well as corporate, local, and national waste regulations. Typically, a passivation process includes the following steps:

		Alkaline cleaning (either single pass or recirculated) to remove all contaminants, oils, and foreign material.

		Acid treatment (either single pass, recirculated, or immersed) to dissolve any free iron and sulfides and to expedite the formation of the oxide layer

		Visual inspection and tests, as appropriate.

	Laboratory test model and verification of test conditions

	Laboratory testing has been successful in determining critical cleaning parameters for passivation of stainless-steel surfaces. A formulated phosphoric/citric acid detergent and a formulated citric/oxalic acid detergent were used to treat 316L stainless-steel coupons in the passive layer evaluation experiments described as follows.  

	The following procedure was employed for the experiment:

		316L stainless-steel coupons (1”x3”) were cleaned in a laboratory washer.

		Cleaned coupons were treated with concentrated hydrochloric acid for 30 seconds to remove the passive layer.

		All the coupons were tested with an electrical pen technique to make sure that all coupons were not passive.

		Coupons were rinsed with ambient water for injection and subsequently exposed to the different cleaning agents, concentration, time, and temperature.

		After exposure, the coupons were rinsed with deionized water and left to dry for 60 min.

		Coupons were tested with an electrical pen technique to determine whether the treatments resulted in a passive condition.

		The electrical pen technique indicates a passive range between (-200 mv) – (-400 mv) and registers pass/fail status via an indicator light (see 

		Final confirmation testing was performed using copper sulfate and salt spray cabinet techniques (see 

		The parameters tested were: cleaner (formulated phosphoric/citric acid detergent or formulated citric/oxalic acid detergent); concentration (5, 7, 8, 10, and 15% v/v); temperature (45, 60, 70, and 80

C); and time (20, 30, 45, 60, 90, and 120 min.).

		Testing included the electrical pen, copper sulfate, and salt spray cabinet methods to verify the passive layer on stainless-steel coupons.

The experiments showed that the following parameters resulted in passivated coupons:

		8% v/v formulated phosphoric/citric acid detergent at 80 °C for 120 min.

		10% v/v formulated phosphoric/citric acid detergent at 70 °C for 30 min.

		10% v/v formulated phosphoric/citric acid detergent at 60 °C for 60 min.

		10% v/v formulated phosphoric/citric acid detergent at 80 °C for 30 min.

		10% v/v formulated citric/oxalic acid detergent at 70 °C for 30 min.

		10% v/v formulated citric/oxalic acid detergent at 80 °C for 20 min.

		15% v/v formulated citric/oxalic acid detergent at 60 °C for 120 min.

	A large biotech manufacturer consistently observed rouge in select 316L stainless-steel buffer preparation and storage tanks. Based on solubility profiles of the buffer components, purified water should have been effective at removing the buffer residue. Persistent rouge, however, impacted the use of the equipment. Upon visual inspection of the tanks, obtaining a third-party service provider specializing in stainless steel cleaning and maintenance was required. This process resulted in increased maintenance costs and decreased production time due to equipment downtime. The biotech manufacturer investigated their cleaning and maintenance procedure to develop a scientific, risk-based approach to equipment preventive maintenance.

	The objectives for the investigation were as follows:

		Confirm condition of exposure that resulted in loss of passive layer

		Confirm that passivation treatment protects the surface from buffer exposure.

	The testing of condition 1 was performedby applying 1ML of buffer onto a precleaned, 304 stainless-steel coupon with a 2B finish. The treated coupon was dried at ambient temperature for a prespecified dirty hold time. The coupon was then cleaned with deionized water for 5 min. at 65 °C or 80 °C. The coupon was determined to be cleaned if it met the characteristics of: visually clean, water-break free, and no weight change (by gravimetric testing).

	The testing of condition 2 was to evaluate the effect of buffer condition on a passive 316L stainless steel coupon using periodic checks of the passive layer with an electrical pen technique and copper sulfate test kit. Final confirmation was performed with salt spray cabinet test. If the passive layer was damaged, additional testing was performed using frequent rinses of low concentration of a formulated acid detergent to maintain the passive layer on the 316L stainless steel surfaces. Testing was performed in replicates. Control coupons were passivated with 10 % v/v formulated acid detergent at 80 °C for 60 min. prior to exposure to the buffer.

Deionized water was effective in cleaning surfaces with dried buffer A (60 mM sodium phosphate, 2 M Chloride, pH 5.6) after 5 min. at either 65 °C or 80 °C. Coating the coupon with Buffer A and drying it overnight resulted in a non-passive surface. Treatment using concentrations of 10–15% v/v formulated citric/oxalic acid detergent at times of 20–60 min. at 80 °C or at times of 40–60 min. at 65 °C were effective at passivating the buffer exposed surfaces and maintained a passive condition for at least a 24-hour buffer exposure. Specifically, surfaces maintained their passive state under the following buffer exposure time:

		Up to 48 hours after passivation with 10% v/v formulated citric/oxalic acid detergent at 65 °C for 40 min.

		Up to 72 hours after passivation with 15% v/v formulated citric/oxalic acid detergent at 65 °C for 60 min.

		Up to 24 hours after passivation with 10 % v/v formulated citric/oxalic acid detergent at 80 °C for 20 min.

Laboratory tests can determine critical cleaning parameters for passivation treatments used to prevent rouge on GMP stainless-steel equipment.