Some key methods for a more efficient cleaning validation process
Pharmaceutical manufacturing equipment must be properly cleaned to ensure the removal of product residue, cleaning chemical residue, and microbes prior to manufacturing. Because cleaning methods are developed and validated to prevent the risk of producing contaminated products by confirming that the cleaning process is sufficient, it’s important to establish method limits and select the proper cleaning techniques and detection methods.
Step One: Set Limits
One of the first steps in the development of an effective cleaning validation method is the determination of the necessary, non-regulated limits. Often, this can be the most daunting step for pharmaceutical manufacturing facilities throughout the cleaning validation process simply because of how specific each limit must be for every product produced. Pharmaceutical companies have several details to consider when setting their limits.
The regulation agencies approach cleaning validation limits with the idea that because of the vast variety of materials and equipment used to manufacture products throughout the industry, it would be impractical to try to set general limits.1 Most of them give examples of where limits should be set based on general criteria, however.
The Food and Drug Administration (FDA) Guide to Inspections Validation of Cleaning Processes, for example, states, "The firm’s rationale for the residue limits established should be logical based on the manufacturer’s knowledge of the materials involved and be practical, achievable, and verifiable." The FDA gives examples of analytical detection levels such as 10 parts per million (ppm), biological activity levels such as 1/1000 (0.1%) of the normal therapeutic dose, and organoleptic levels such as no visible residue.2 One can easily apply these examples to determine the amount of allowable carryover of product residues:
• No more than 10 ppm of any product should appear in another product;
• No more than 0.1% of the normal therapeutic dose of any product will appear in the maximum daily dose of the following product; and
• No amount of product residue should be visible on the surface of the equipment after the cleaning procedure has been performed.
Step Two: Inspect Visually
The FDA states, "When the cleaning process is used only between batches of the same product (or different lots of the same intermediate in a bulk process), the firm need only meet a criteria of ‘visibly clean’ for the equipment." One way to enhance visual detection is to set up spiking studies, in which coupons of the same type of surface that is going through the cleaning procedure are spiked with known amounts of residue. The coupons are then observed by trained personnel to determine at which level they appear clean. The coupon with the highest level of residue that appears to be clean by the personnel is considered the acceptance limit for that particular residue.3
The problem with visual inspection is that there are too many variables that can influence the results. The coupons must be observed in the exact same viewing conditions as the equipment in the field. Not all equipment can be inspected under conditions similar to a coupon sitting on a lab bench. The lighting, viewing angle, and even the distance of the observer from the surface should all be the same. If any of these circumstances is altered, the results may be skewed.
Issues may also arise when trying to simulate end-point residues on surfaces after the cleaning process. The question most commonly asked when viewing a spiked surface is, "Is the viewer seeing the actual residue stain, or are they just seeing the edge of the stain?"1 Most visual spiking studies include spiking a solution of dissolved product onto the surface and allowing it to dry. Sometimes a stream of nitrogen is used to speed up the drying process and to discourage product degradation. It is much easier to see the edge of a stain at low levels than it is to see the body of the stain itself. Under visual inspection, a stain of uniform thickness that covers the entire surface of a piece of equipment may be falsely mistaken for a clean surface. In this situation, a more sensitive means of detection would greatly improve recognition of a dirty surface.
Even though visual cleanliness is widely accepted as a means of evaluating product carryover, manufacturers should take their detection abilities one step further and develop a validated and quantitative method. This type of method is required when there is a change in the type of product being manufactured. The forms of detection generally use sample-based cleaning procedures involving collection of either swab or rinse samples. A quantitative method provides greater specificity, accuracy, and sensitivity than the visual approach.
Step 3: Swabbing Versus Rinsing
The selection of an appropriate extraction solution is an important step in establishing a swab or rinse procedure. Your decision should be based on the solubility of the cleaning detergent residue or pharmaceutical product residue in the selected solution. Various alcohols, water, buffers, or combinations of the three are common extraction solutions used for cleaning procedures. Once an extraction solution is chosen, equipment surfaces may be extracted using a swabbing or rinse method.
Rinsing is suitable for small surface areas where traditional swabbing procedures may be difficult. The specified area should be rinsed long enough to ensure complete coverage of the entire surface and sufficient removal of the target residue. Rinsing methods provide a more simplistic sampling approach because they avoid possible swab interference in the detection method or extraction issues in removing the residue from the swab surface.
Swabbing is ideal for hard-to-clean areas and can physically remove insoluble residues. Swabs are selected for their ability to recover the monitored residue from a given surface and their ability to release the residue to an extraction solution for analysis. The selected swab should not contribute excessive interference or background during analysis. Another consideration in swab selection is whether the area being swabbed is easily accessible or hard to reach. Swabs that are long with small heads are excellent for general purposes and hard-to-reach areas. Other swabs with larger heads are better suited for cleaning broad, flat areas.
Swabbing patterns can vary and are dependent on the surface or equipment being swabbed. Surface areas are defined and swabbed with the chosen solvent, which is usually the same as the extraction solution. Prior to swabbing, swabs are soaked for a few minutes in a vial of the extraction solution. Excess solution is removed from the swab head by gently pressing the head on the inside of the vial.
The prepared swabs are used to swab the appropriate area, which can be accomplished using various swabbing patterns. Common patterns use partially overlapping parallel strokes in one direction or back-and-forth strokes. Whichever you choose, it is important that you flip the swab head to the other side and repeat the same pattern at right angles to the first pattern (see Figure 1.A, p. 20). Another variation involves overlapping zigzag strokes in opposite directions, making sure that the swab head never leaves the surface being evaluated. An easy way to look at this is first, horizontally, and second, vertically (see Figure 1.B, p. 20).
The swab head is placed back into the vial after clipping the handle above the head with a clean cutting tool. One swab may be sufficient to remove residue, but a second or even a third swab can be used to repeat the swabbing pattern, increasing residue recovery.
Depending on the extraction solution, using a dry swab after the wet swab may be advantageous, helping to ensure that any remaining solution on the coupon is collected. Other swabbing patterns can be adapted for special surfaces or pieces of equipment.
Step 4: Choose Your Detection Methods
There are multiple detection options available for cleaning validation.
Ion Mobility Spectrometry (IMS)
IMS characterizes chemical substances based on their gas-phase ion mobilities, provides detection and quantitation of trace analytes, and offers atmospheric pressure chemical ionization (APCI), a soft ionization technique that produces molecular weight information.Benefits:
• Offers ultra-fast quantitative analysis (~30 seconds per sample);
• Has sub-nanogram sensitivity;
• Has the ability to analyze a broad range of compounds with no chromophore needed;
• Does not require mobile phases, columns, or vacuum for operation; and
• Designed for different ways of sample introduction, either by thermal desorption off a membrane (solid residue on swab giving a qualitative analysis or solution deposited on the membrane, which allows quantitative analysis) or by high performance injection, which allows for a gas chromotography-style temperature programmable split/splitless injection.Drawbacks:
• Compounds must be vaporizable and ionizable for IMS detection to be used;4
• Samples must be relatively clean;
• Ultra pure extraction solutions should be used; and
• Technique is not suitable for multiple component matrices.
Total Organic Carbon (TOC)
TOC analysis is specific to organic compounds and theoretically measures all the covalently bonded carbon in water.5Benefits:
• TOC detection is an acceptable way to detect residues of contaminants.Drawbacks:
• Considered a "worst case" analysis because TOC analysis incorporates all organic molecules in solution and represents surface area depending upon the sampling method (swab or rinse);5
• "In order for TOC to be functionally suitable, it should first be established that a substantial amount of the contaminating material(s) is organic and contains carbon that can be oxidized under TOC test conditions"5;
• Samples must be water soluble;
• Excellent water quality is needed for sensitivity; and
• Certain swab types may also interfere with TOC analysis, so swab selection is critical.
UV-Visible Spectrophotometry (UV-Vis)
UV-Vis is commonly used for detection of small molecule active pharmaceutical ingredients or detergent residues for swab and rinse samples.
• Not limited to water as the extraction solution;
• Provides quantitative results;
• Does not require a mobile phase or column;
• Offers fast spectral acquisition; and
• Allows for use of a larger swab selection, compared to TOC.
• Lacks peak separation; and
• Requires chromophore for specificity.
High Performance Liquid Chromatography (HPLC)
HPLC can be used for detection of small molecule active pharmaceutical ingredients (APIs) or detergent residues for both swab and rinse samples, allowing for separation of multiple components.
• Is not limited to water as the extraction solution;
• Offers peak separation via packed column;
• Provides identification of specific peaks of interest and quantitative results provided a suitable reference standard is used;
• Offers multiple detection options (UV, photodiode array, fluorescence, refractive index, evaporative light scattering, corona charged aerosol detection, etc.);
• Allows for the use of a large variety of swab types due to separation power; and
• HPLC in tandem with mass spectroscopy (MS) offers selectivity while separating the API from its degradates based off mass-to-charge ratio of the compound of interest.6
• May require more development and validation time in comparison to other forms of detection, depending on current information about the API and excipients being used in the formulation; and
• HPLC/MS analysis is more expensive.
GC and MS
Gas chromatography (GC) and GC/MS are mainly used for detection of detergent residue. These cleaning agents typically contain various solvents or compounds required to effectively clean equipment that may not be cleanable with typical detergents. Most solvent cleaners are volatile and will evaporate from equipment surfaces, but some residue may remain from less volatile compounds.
• Offers improved peak shape over HPLC due to capillary column usage; and
• Provides separation, identification, and quantitation of results when an acceptable reference standard is used.
• Samples require vaporization.
Step 5: Microbial Testing
A microbial cleaning validation can also be performed for equipment subsequent to or as part of the chemical cleaning validation. This is sometimes overlooked during the initial planning of the validation program, yet it provides additional data to support the effectiveness of the cleaning process by establishing the post-cleaning bioburden. As with chemical cleaning validations, the suitability and qualification of the recovery method should be considered when selecting methods.
Sample collection must be performed using an aseptic technique. The microorganisms captured on the swab or in the rinse can then be enumerated using direct plating, a pour plate technique, or membrane filtration.
For enumeration of swab samples, the swab is typically extracted in a buffered diluent or neutralizing broth (usually a 10 ml volume) to release organisms. A portion of the diluent can then be aseptically plated using a standard microbiological pour plate method by placing separate 1 ml aliquots into duplicate petri dishes.Tempered nutrient agar is added to these petri dishes, and the plates are swirled to mix.
After the agar solidifies, the plates are inverted and placed into an incubator at the desired temperature for a determined period of time to allow any microorganisms present to grow and become visible on the agar plates. An average count of the plates is obtained to determine the number of colonies (colony forming units or CFU) per milliliter of the original dilution.Plates having no growth are reported as <>
If more sensitive testing is required due to low bioburden requirements for the equipment or to enhance recovery of bioburden from very clean equipment, a membrane filtration method can be used. In this type of testing, the entire volume of diluent or an equipment rinse sample is filtered throughone membrane filter.The filter, usually a 0.45 micron filter, is then aseptically transferred to a prepared, solidified nutrient agar plate and incubated in the same manner as a standard pour plate.Visible colonies are counted at the end of the incubation period, and the CFU per swab or rinsed surface areais obtained. Filter plates having no visible growth are reported in the same manner as above, <>
The potential of any remaining chemical residues to inhibit microbial growth must be considered for a valid evaluation of the bioburden. For agents with inhibitory residuals, neutralization methods must be established using the maximum levels anticipated to be recovered in samples submitted for microbial testing.
The determination of appropriate residue limits and selection of appropriate cleaning and analytical techniques described are typically explored during the method development phase. n
Lingenfelter, Evans, and Atkins are senior chemists in the method development and validation group, and Clow is a manager of the pharmaceutical microbiology group at Lancaster Laboratories Inc. For more information, reach Lingenfelter at (717) 656-2300, ext. 1449, or at email@example.com.
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2. United States Food and Drug Administration. Guide to Inspections Validation of Cleaning Processes. FDA. Available at: http://www.fda.gov/ora/Inspect_ ref/igs/valid.html. Accessed May 6, 2009.
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