Cleaning Agent Residue Detection with UHPLC

April 26, 2013
The productivity potential of ultra-high-performance liquid chromatography is a “clean” cut case
Cleaning procedures are among the most critical tasks in the pharmaceutical and biopharmaceutical industries. Everyone understands proper cleaning of manufacturing equipment is required to guard against cross contamination and protect product purity. Just as important as the cleaning process itself is the ability to accurately determine the amount of residue present on the surface of the manufacturing equipment. It is essential to develop and qualify sensitive analytical methodologies to detect target residues on manufacturing equipment and other product contact surfaces. Although non-specific methods such as total organic carbon (TOC) or conductivity studies can provide general information about equipment cleanliness, many manufacturers prefer a specific method that can detect and quantify the active ingredient or cleaning agent, to ensure that they are adequately removed from surfaces. Various guidelines recommend thin layer chromatography, UV/Vis spectroscopy, gas chromatography (GC), and conventional high performance liquid chromatography (HPLC).1 Ultra high performance liquid chromatography (UHPLC) or UPLC offers the specificity of traditional HPLC with significantly shorter analysis times.
Methodology (HPLC vs. UHPLC)
HPLC has proven to be the predominant technology used in the pharmaceutical industry globally during the past few decades.2 The underlying principles of HPLC are governed by the van Deemter equation, which most users of chromatography methods are familiar. The equation describes the relationship between linear velocity (flow rate) and plate height (height equivalent to a theoretical plate (HETP), or column efficiency). Various techniques and changes to HPLC columns, including smaller columns, faster flow rates, and elevated temperatures, have been employed in an attempt to reduce the analysis time required by HPLC testing. However, using conventional resin particle sizes (3.5 – 5 µm), limitations are often reached and the user must sacrifice resolution for time.
To gain faster throughput and reduced turnaround times while maintaining adequate resolution in chromatography, smaller resin particles (sub-2.5 µm) have been created and used in UHPLC columns. This results in an increase in back pressure in the instrument. Typical back pressure for an HPLC is in the range of 100 – 300 bar (1450 – 4351 psi), while those encountered using UHPLC can be as high as 1000 bar (14,504 psi). The technology present in UHPLC enables users to maintain or increase the resolution and sensitivity of their liquid chromatography analysis, while reducing the amount of time required for each injection, and potential process analytical technology (PAT) applications. 
The effectiveness of UHPLC at measuring detergent residues was assessed by evaluating the following: an alkaline detergent, a combination of an alkaline detergent and peroxide based detergent additive, and an alkaline and acidic detergent designed for sequential use. 
Guidelines specify that after cleaning, surfaces must pass organoleptic testing (for example be visually clean), and residues from previous products or batches should not impact the strength, identity, appearance or quality of the next product produced.3 There are numerous specific and non-specific methods available. The methods used vary with the industry segment; for example, active pharmaceutical ingredient (API), excipient, dietary supplement and pharmaceutical manufacturers typically use HPLC, while biopharmaceutical companies may use conductivity, TOC and microbial assays.
Traditional HPLC offers an analytical methodology to pharma companies for analysis of final product, residual active ingredient(s), excipients and some degradation products.4,5,6,7,8 Due to the specificity of the analytical tool and widespread use within companies, the technology is frequently used for detection and quantification of residual cleaning detergents on the surface or in the rinse water.9,10,11,12 The speed and specificity of UHPLC, and its similarities to existing HPLC analysis have allowed companies to rapidly transition current validated HPLC methods to UHPLC methods. 
Technological advancements in manufacturing equipment have also allowed in-line, on-line or at-line sampling to monitor the cleaning process. The speed of UHPLC supports the use of line sampling for continuously monitoring the cleaning processes.
UHPLC is suited for specific detection and quantification of organic and inorganic residues during cleaning, and is also suitable for PAT applications for continuous monitoring of the process. PAT practices vary: Some companies monitor conductivity or TOC of the final rinse water to ensure pre-established values are achieved. Once the value has been reached, a signal is sent to the pump to stop the final rinse step. Others continuously monitor the critical parameters of time, temperature, action (pressure) and cleaning agent (conductivity) during the cleaning process such as the final rinse step or during all the rinse steps. The continuous monitoring allows operations staff to investigate irregularities, unmet specifications and/or non-compliance items in real-time; once a profile has been generated, changes to the existing product or process can be expedited.13 
Alkaline Detergent
Alkaline detergents are widely used in the pharmaceutical and biopharmaceutical industries because of the chemical properties attributed to caustic solutions. Caustic solutions are capable of ionizing acids, attacking oxides, and acting as nucleophiles that attack polar bonds in both organic and inorganic materials. Alkaline detergents are often formulated with additional ingredients such as chelants, surfactants and dispersants, to effectively clean a wide range of compounds encountered in pharmaceutical manufacturing. During the development, validation and routine use of an alkaline detergent in a cleaning process, manufacturers will frequently monitor levels of residual detergent on the equipment surfaces using HPLC. By using the technological advantages of UHPLC, manufacturers can reduce sample analysis time significantly. 
Based on the alkaline detergent retention times for each mode of analysis, one can see the time benefit of using the UHPLC method (Figure 1). The time for the peak to elute is reduced by 300%, and total run time by 500%, when using UHPLC instead of HPLC. To put this in terms of a typical analysis of 10 injections, (2 blanks, 6 replicates of a 10 ppm standard, and two samples), the total instrument time is reduced from 100 minutes for the HPLC analysis to 20  minutes for the UHPLC analysis. 
Alkaline Detergent Plus Detergent Additive
In certain cleaning processes, it may be necessary to use a detergent additive in order to adequately remove the process soils on the equipment surfaces. The additive may increase surfactancy, alkalinity (or acidity), or offer another mechanism (e.g., oxidation) to effect efficient removal of the process soils. An excellent example is the air-liquid interface on production bioreactors.14 Sharnez gives examples of effectively cleaning residues such as polyvinyl chloride, slip agents, and silicates which could not be cleaned by an alkaline cleaner alone. Additional examples included submerged parts, such as endcaps and baffles that could not effectively be cleaned using an alkaline cleaner alone. In some cases, the manufacturer may find it necessary to assay for, and quantify, any residual levels of each component; the detergent (whether alkaline or acid) and the additive. Due to the unique formulation and chromatographic properties of the detergent and additive, it can be difficult to test for both components simultaneously using a single HPLC method. The combination alkaline detergent — peroxide additive investigated in this study can be analyzed on a single HPLC analysis. The gradient elution required to achieve adequate separation requires approximately 10 minutes per injection, while the UHPLC method can achieve similar separation in a fraction of the time (approximately 1.1 minutes). The analysis time is reduced by about 900% using UHPLC vs HPLC; from 100 minutes down to 11 minutes for a typical analysis of 10 injections (Figure 2).
Alkaline Detergent Followed By An Acid Detergent
Many pharmaceutical and biopharmaceutical manufacturers need to use both an alkaline detergent and an acid-based detergent in their cleaning procedures. In a situation very similar to that of the detergent and detergent additive system described previously, it is often difficult to test for and accurately quantify low levels of residual detergent on the equipment surface using one HPLC method. Again, this is largely due to the unique formulations of alkaline and acid-based detergents, where each detergent has its own unique chromophores (and chromatographic profiles). In this example, the alkaline and acidic detergents share a common analyzable component that allows for simultaneous quantitation of both alkaline and acidic detergent residues. The analyzable method for each detergent (acid or alkaline) does not display any enhancement or interference with the other when run together (Figure 3).
Accurate quantitation of residues from cleaning agents is critical to a robust, validated cleaning process. As industry continues to refine the “risk-based” approach to manufacturing and cleaning, PAT continues to have an important role in developing, monitoring, and controlling processes. The significantly shorter analysis times offered by UHPLC allow manufacturers to view cleaning efficacy and residue level data within minutes of the operation. And, with the advent of UHPLC equipment that is designed to reside on the manufacturing floor, generating data specific to product or cleaning agents can help to meet one of PAT’s goals — variability is managed by the process.15
Another potential benefit of UHPLC technology is that the more efficient separation achieved with smaller resin may allow for methods to be developed for simultaneous analysis of multiple residues. Residues from cleaning agent and API, which previously required separate analyses, can be simultaneously assayed using one method. This can mean less time waiting for results and faster equipment turnaround, which can contribute to enhanced process efficiency and productivity.
Published in the April 2013 issue of Pharmaceutical Manufacturing magazine


[1] APIC Guidance on aspects of cleaning validation in API plants, 2000.

[2] Swartz, M.E., UPLC: An Introduction and Review; Journal of Liquid Chromatography & Related Technologies, 28 (2005) 1253-1263.

[3] Guide to Inspections Validation of Cleaning Processes; FDA, ORA, 1993 pp 1 – 6.

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[12] Zayas, J., et al. Cleaning validation 1: Development and validation of a chromatographic method for the detection of traces LpHse detergent. Journal of Pharmaceutical and Biomedical Analysis, 41 (2006) 589-593.

[13] FDA, Guidance for Industry, “PAT – A Framework for Innovative Pharmaceutical Development, Manufacturing and Quality Assurance” (Pharmaceutical cGMPs, September 2004).

[14] Rathmore, S.A., Case Study and Application of Process Analytical Technology (PAT) Toward Bioprocessing: II. Use of Ultra-performance liquid chromatography (UPLC) for Making Real-Time Pooling Decisions for Process Chromatogrphy., Biotechnology and Bioengineering, vol. 101, No. 6, December 15, 2008.

[15] Sharnez, R., et al. “Cleaning validation challenges for bioprocesses: strategies for eliminating tenacious residues”, Presentation at 2012 Parenteral Drug Association (PDA) Annual Meeting.

[16] Rathore, A.S., et al. “Case study and application of process analytical technology (PAT) towards bioprocessing: use of on-line high-performance liquid chromatography (HPLC) for making real-time pooling decisions for process chromatography.” Biotechnology and Bioengineering, (2008) Jun 1; 100(2):306-16. 

About the Author

Michael Gietl | Brook Meadows and Paul Lopolito