Dijana Hadziselimovic

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 

	
	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.

	Additional testing was performed with 1M chloride buffer solution, and an increased concentration and duration of  passivation treatment with a formulated citric/oxalic acid detergent to extend wet buffer storage time. The following conditions were evaluated.

		Decreased chloride concentration in Buffer A preparation solution from 2M to 1M.

		Evaluate passivation condition of 15% v/v formulated citric/oxalic acid detergent at 80

C for 60 or 90 min. prior to buffer exposure.

		Extend duration of buffer hold time to greater than 72 hrs post passivation condition.

	The additional testing found that the decrease in chloride concentration in Buffer A from 2M to 1M chloride as well as the increase in 15% v/v citric/oxalic acid detergent exposure from 60 to 90 min. extended the buffer storage condition exposure from 96 to 120 hours.

	Water is effective at cleaning Buffer A with 2M or 1M chloride. However, dirty hold time and solution storage time can affect the passive properties of the stainless steel surfaces when evaluated using the electrical pen test and cyclic polarization (not reported). The citric/oxalic acid detergent passivation conditions (time, temperature, and concentration) can affect the passive layer and the duration of buffer storage time. A passivation treatment of at least 5% v/v of a formulated citric/oxalic acid blend at 80 

C for 90 min. provided a passive surface when evaluated with the electrical pen, cyclic polarization, and X-ray photoelectron spectroscopy (XPS) or electron spectroscopy for chemical analysis (ESCA). The passivation of the stainless-steel coupon with 15% v/v citric/oxalic acid detergent maintained a passive surface using the electrical pen test for greater than 96 hours when exposed to the 1M chloride buffer.

	This information enabled the manufacturer to place timers on select buffer storage and product hold tanks to inform the operators when a passivation cycle is needed.

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