Si Myra Tyson is a senior technical service associate for the Life Sciences Division of STERIS.

Si Myra Tyson

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.
