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

Si Myra Tyson

Stainless steel pipe fittings, clamps, and connectors arranged in industrial. Precision manufacturing, mechanical assembly, and hygienic processing systems for food and pharmaceutical industries. | Image Source: ©Artinun -stock.adobe.com

Type III or Class III rouge (magnetite) appears as a black oxide in water systems and production equipment exposed to sanitization, pasteurization, or steam sterilization conditions. Remediation of type III rouge often requires electropolishing or mechanical polishing of the stainless-steel surface, resulting in significant labour cost, safety risk to operators with confined space entry, downtime of the equipment or system, and a reduction in thickness of the stainless steel. Raman spectroscopy is a non-invasive analytical method that can quickly identify the presence of magnetite (Fe

) in rouge. Based on the identification of magnetite, a detergent additive can be combined with a formulated citric/oxalic or a phosphoric/citric acid blend to transform insoluble magnetite to a more soluble form of iron oxide. This approach to type III rouge remediation utilizing a detergent blend, spray device, and recirculating pump reduces downtime, safety risk, material loss, and overall cost of maintenance.

A common surface in production equipment and piping is 300 series stainless steel. This surface is compatible with most pharmaceutical and biopharmaceutical processes; however, certain conditions can lead to discoloration and/or rouge on the surface. Rouge is a corrosion product on or in the surface of the iron alloy. Rouge is generally classified as Type I–III or Class I–III.

 Type I rouge (migratory rouge) is an iron oxide that is loosely attached to the surface and can be wiped off the surface. The origin of this rouge is often from an upstream source. Type II rouge (in situ rouge) is difficult to wipe off and often contains underlying damage to the surface. Examples of Type II rouge include galvanic, intergranular, pitting and stress corrosion. Common acid blends, such as citric/oxalic or phosphoric/citric solutions, can be effective at removing Type I and II rouge.

 These treatments do not restore original surface roughness. Electro- or mechanical polishing may be required to correct surface damage due to Type II rouge from pitting corrosion. Type III rouge is often associated with high temperature and purity water systems. Type III rouge is challenging to remediate due to the presence of magnetite (Fe

). Magnetite removal often requires aggressive chemicals or electro- or mechanical polishing. An acid detergent with phosphonate can be used alone or in combination with a citric/oxalic or phosphoric/citric acid blend to improve the solubility of magnetite and the removal of Type III rouge.

Understanding when and which acid detergent (or detergent additive) to use in rouge remediation can be difficult. The selection of the acid detergent should consider effectiveness to remove the rouge, substrate compatibility, rinse profile, stability and shelf life, analyzability, toxicity, disposal constraints, personnel safety, toxicity, foaming characteristics, microbial effectiveness, quality of manufacture, long-term availability, quality of manufacture and overall cost.

 Raman spectroscopy can be a powerful tool in identifying the oxide compound(s) present and provide direction to successful remediation. Alternative spectroscopy and microscopy techniques may be applicable for surface characterization and corrosion monitoring, such as X-ray diffraction (XRD) and scanning electrochemical microscopy (SECM).

 Raman uses inelastic light scattering, where the incident light (from a high-intensity laser light source) transfers energy to molecular vibrations, and the scattered light collected at the detector provides structural information relating to the molecule. Each molecule/compound will have a unique spectrum. Magnetite displays a primary peak with Raman spectroscopy between 662 and 670 cm

 A secondary peak is often observed at 532 and 550 cm

. A common roadblock in successful rouge remediation is the presence of magnetite. A quick identification of magnetite in a rouge surface using Raman spectroscopy can be helpful in selecting the right chemical treatment and cleaning parameters.

This article reviews the use of Raman spectroscopy in a series of case studies to quickly identify the iron oxide compound(s), including magnetite and hematite, on the surface of representative endcaps or piping spool pieces. The article also provides a strategy using a detergent additive to remove magnetite without performing costly electro- or mechanical polishing.

A Raman microscope with a 532 nm laser (DXR3, Thermo Scientific) was used to analyze selected sample areas. Areas that visually exhibited the greatest amount of residue were selected and focused on using the confocal microscope prior to analysis. The samples were analyzed as is (without cleaning, modification, etc.) using the following conditions in 

After analysis of the selected sample areas, a spectral library search (Wiley Science Solutions KnowItAll Spectral Library) was performed for each Raman spectrum.

For reference and comparison throughout the following case studies, the Raman spectra of hematite (Fe

Hematite exhibits peaks at approximately 220, 290, 400, 600, and 1300 cm

Figure 2. Raman Spectrum of Magnetite (Fe

A large multinational biopharmaceutical drug manufacturer indicated that visible rouge was unable to be removed from a tank using 10% v/v citric/oxalic acid detergent at 70 to 80 

C. Citric/oxalic and phosphoric/citric acid blends are commonly recommended for rouge removal.

 The nature of the rouge was investigated using Raman spectroscopy to identify the oxide composition and determine the reason for the difficulty in the chemical removal. Raman spectroscopy is an analytical tool that can be used in the laboratory and modified for use on the manufacturing floor.



. The following swab samples of the rouge were analyzed using Raman spectroscopy (See 

TA-9620 Pre-Derouge Swab Bottom Visible Ring

TA-9640 Rouge Observation Top Spot Near Sight Glass

Figure 3. Raman Spectra of Swab Samples. Figure courtesy of the authors.

. A spectral library search (Wiley KnowItAll Spectral Library) was performed for each Raman spectrum. All the residue samples exhibited a best match to a mixture of iron oxide compounds—hematite (Fe

). A representative search result is shown in 

Figure 4. Raman Spectra of Iron Oxide Mixture Reference (Mixture of Fe

) and TA-9720 Bottom of the Tank Outlet. Figure courtesy of the authors.

. All the Raman spectra from the selected swabs were similar to each other and exhibited peaks around 200, 300, 400, 700, and 1300 cm

, which can be attributed to an iron oxide mixture of Fe

. The general recommendation based on the swab analysis by Raman spectroscopy was to increase the concentration of the citric/oxalic agent detergent to 15% v/v and add a detergent additive at 15% v/v with recirculation through a device at 60 to 80

C for 1 to 3 hours to solubilize and remove the rouge containing magnetite (Fe

A large multinational biopharmaceutical blood product drug manufacturer submitted two end caps (one smaller and one larger) for Raman spectroscopy analysis. Both end caps exhibited brown discoloration consistent with rouge (

Figure 5. Small End Cap. Figure courtesy of the authors.

Figure 6. Large End Cap. Figure courtesy of the authors.

With 10x magnification, the small end cap exhibited orange, blue and purple/brown discolored areas. Areas of each colour were selected for Raman analysis to determine if there were any differences in the Raman spectra. Representative areas of analysis for the three different colours observed on the end cap’s surface are marked by a red square in the images below (

Figure 7. Small End Cap – Orange Area (10x mag). Figure courtesy of the authors.

Figure 8. Small End Cap – Blue Area (10x mag). Figure courtesy of the authors.

Figure 9. Small End Cap – Purple/Brown Area (10x mag). Figure courtesy of the authors. 

The three different colored areas of the end cap were analyzed, and the Raman spectra were found to be comparable to each other. A comparison of the spectra is shown in 

Figure 10. Raman Spectra of Analyzed Areas of Small End Cap. Figure courtesy of the authors.

. A magnified image of the residue present on the large end cap is shown in 

. The small area marked by a red square is representative of the area selected for Raman analysis.

Figure 11. Large End Cap – Brown Area (50x mag). Figure courtesy of the authors.

. The Raman spectrum of the brown area from the large end cap is shown in 

Figure 12. Raman Spectrum of Large End Cap. Figure courtesy of the authors.

. The three Raman spectra of the small end cap and the Raman spectrum of the large end cap were comparable to each other, with peaks at approximately 660, 600, 496, 408, 292, and 223 cm

.

When a spectral library search was performed (Wiley Spectroscopy KnowItAll Spectral Library), the residue from the end caps appeared to be a mixture of hematite (Fe

). A comparison of the Raman spectra of the residue from the small and large end caps to the hematite and magnetite Raman spectra is displayed in 

Figure 13. Raman Spectra of End Caps, Magnetite, and Hematite. Figure courtesy of the authors.



The end cap spectra matched well to the hematite spectrum; however, the peak around 665 cm

 had a greater intensity than the hematite reference spectrum, suggesting the presence of magnetite, which has a high intensity peak around 665 cm

 The residue was effectively removed from stainless steel by agitated immersion using a solution of 15% v/v formulated phosphoric/citric detergent plus 15% v/v detergent additive at 80 °C for 24 hours (

Figure 14. Derouge and Passivate End Cap Samples. Figure courtesy of the authors.

A large multinational biopharmaceutical drug product manufacturer submitted pipe samples with suspected rouge for Raman spectroscopy analysis. The pipe samples exhibited a brown discoloration consistent with rouge throughout its outer surface.

. Three different areas on the pipe were analyzed using Raman spectroscopy. The spectra of the three areas were found to be comparable to each other. A comparison is shown below in 

Figure 15. Raman Spectra of Analyzed Areas of Pipe. Figure courtesy of the authors.

. A spectral library search was performed (Wiley Spectroscopy KnowItAll Spectral Library), and the Raman spectrum of the brown discoloration from the pipe had a best match to the spectrum of a mixture of hematite (Fe

). A comparison of the Raman spectrum of the hematite and magnetite mixture and the Raman spectrum of the brown residue from the pipe is shown in 

Figure 16. Raman Spectra of Iron Oxide Mixture and Brown Residue From Pipe. Figure courtesy of the authors.

 The brown discoloration was effectively removed compared to untreated controls from stainless steel by agitated immersion using a solution of 15% v/v formulated phosphoric/citric acid detergent plus 15% v/v formulated detergent additive or a 15% v/v formulated citric/oxalic acid detergent plus 15% v/v formulated detergent additive at 80 °C for 4 hours. The surface was tested with an electrical pen test kit to confirm a passive surface following treatment. Testing with 15% v/v formulated phosphoric/citric acid or the formulated citric/oxalic acid detergent at 80 °C for 24 hours was not successful in removing the discoloration shown in 

 alongside untreated controls (left side) and successfully derouged and passivated pipe samples (right side).

Figure 17. Case Study 3 Heat Exchanger Pipe Samples, Left to Right:

Sample 3, Cleaned with 15% formulated phosphoric/citric acid detergent

Sample 4, Cleaned with 15% formulated citric/oxalic acid detergent

Sample 5, Cleaned with 15% formulated phosphoric/citric acid detergent plus 15% detergent additive

Sample 6, Cleaned with 15% formulated citric/oxalic acid detergent plus 15% detergent additive

A general requirement of pharmaceutical drug manufacturers is that equipment shall be constructed so that surfaces that contact components, in-process materials, or drug products shall not be reactive, additive or absorptive so as not to alter the strength, quality, integrity, identity and purity of the drug product.

 The presence of discoloration or rouge on direct and indirect product surfaces has been cited by regulatory authorities.

Guidelines for Good Manufacturing Practice for Medical Products for Human and Veterinary Use 

(Annex 1) state in section 2.5 that process equipment, utilities, raw materials and premises are potential sources for microbial and particulate contamination. Stainless steel maintenance and cleaning of direct, indirect, and non-direct surface must be considered within any contamination control strategy.

 A risk-based strategy to stainless-steel maintenance utilizing International Council for Harmonisation (ICH) Q9 

 has two primary principles: the evaluation of risk to quality from rouge should be based on scientific knowledge and ultimately linked to the protection of the patient and product, and the level of effort, formality and documentation should be commensurate with the level of risk.

 Stainless steel commonly used in pharmaceutical and biopharmaceutical manufacturing can be discoloured or rouged based on several factors, including the nature of the in-process solution, product, medias, buffers, reagents and water quality.

Through a series of case studies, the authors used Raman spectroscopy to identify the presence of magnetite on various process and utility system samples (swabs, end caps and spool pieces). Type III rouge (magnetite) presents considerable challenges to chemical remediation and stainless steel maintenance programs. Based on the identification of magnetite, a readily available detergent additive can be combined with a formulated citric/oxalic or phosphoric/citric acid detergent commonly used for derouging and passivating stainless steel. The two-detergent approach transforms the insoluble magnetite to a more soluble form of iron oxide for removal without polishing or a secondary passivation step. This science-based approach to type III rouge remediation incorporates a detergent additive, spray device and recirculating pump to reduce equipment downtime, operator safety risk, material loss from polishing and overall cost of maintenance and compliance to current good manufacturing practices.

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 A risk-based approach to stainless steel equipment maintenance. 

Emily Null, MS, scientist; Mildred R. Bernardo, lead scientist; Si Myra Tyson, MS, technical services lab specialist; Dijana Hadziselimovic, technical services laboratory manager; Paul Lopolito, technical services director; all with STERIS Corp.

Articles

Identification and Remediation of Type III or Class III (Magnetite) Rouge in Manufacturing Equipment and Water Systems

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,” 

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.
