Donald Singer is senior microbiology technical consultant at Ecolab Life Sciences.

Donald Singer

Pharmaceutical products are required to provide a therapeutic response in patients, with a minimum of adverse side effects. They must also be safe for patients to take. Ensuring quality and safety requires that they be free of microorganisms that could harm patients or affect product quality.

 The presence of microbial contaminants poses a serious threat to raw material, drug substance, and drug product quality and patient safety. Bioburden levels must be strictly controlled in order to prevent serious product quality problems and adverse patient reactions.

FDA has spelled out requirements in its CGMP code (1) and in more recent risk-based guidance documents (2). The 

Chapters <62>, Microbiological Examination of Non-Sterile Products and <1111> Microbiological Examination of Non-Sterile Products Acceptance Criteria (3,4).

However, subsequent USP guidance (USP <1115>) focused less on in-process and post-production microbial testing and more on facility, equipment, and process design and operation. In this article, two members of USP’s expert committee on microbiology , suggest ways in which USP <62> and <1111> might be improved, to more closely reflect today’s manufacturing realities and subsequent guidance. Before making formal suggestions for change, they are seeking industry opinion. Please contact them to discuss these issues, share your thoughts, or for more information.

FDA’s CGMPs specifically requires that written procedures be established and followed to prevent “objectionable micro-organisms” from affecting patient safety and drug substance and product quality (5), The agency also expects that appropriate tests be run on each individual lot of product, as well as on containers and closures, to check for potential microbial contamination (6).

Considered properly, the regulations expect procedures and testing to be designed so that they minimize any adverse consequences associated with the potential presence of ‘objectionable’ microorganisms. They do not and should not mandate the absence of all such microorganisms because that would be impossible to achieve for a non-sterile product (7).

Sampling and analysis cannot ensure the absence of objectionable microorganisms. Quite simply, nothing can, and the means to provide patient safety lies in other aspects of manufacturing a non-sterile drug product (e.g., facility, equipment, and process design and operation), along with expert assessment of the potential impact of microbiological contaminants, as advanced by USP’s Chapter <1115> (

Bioburden Control of Nonsterile Drug Substances and Products

 first included recommendations on microbial control for non-sterile products in chapter <62>, first published in 

29 (5) in 2003 (9). However, the expectations outlined in that chapter had previously been revised and harmonized with requirements from other global pharmacopeia (10,11). The results were 

Chapters <62> and <1111>, published in 2006, and effective 2009, which broadened CGMP expectations by requiring the use of microbial tests designed to ensure the absence of various ‘objectionable’ strains in non-sterile dosage forms and APIs.

These standards place undue emphasis on the testing of finished materials, diverting attention from the means (i.e., the equipment, systems, and facility design and operational practices) required to protect products and patients from microbial contamination. They also formalized expectations that specific strains of microorganisms be considered for each type of drug administration, which implied that other microbes were of less or no concern.

addressed some of these shortcomings by developing a draft informational chapter on environmental monitoring for non-sterile manufacturing, which endeavored to shift the focus from product testing to facility, system, and equipment design and operation, which, collectively, provide the foundation for microbial contamination prevention and control.

Bioburden Control of Non-sterile Drug Substances and Products

, which was finalized in 2015 andoutlines the concepts and principles necessary to protect products from adventitious microorganisms (12).

This informational chapter helps clarify how the regulatory expectations (stated in 21 

 211.113 a) are to be realized. While microbial sampling and testing play an important role in microbial control, the chapter suggests, they cannot assure the quality of the materials being produced, whether they are APIs. raw materials, or finished pharmaceuticals.

More recently, USP revised the content of USP <1211> 

 following the principles outlined in <1115>. During the development of this chapter, the limitations of microbial testing as evidence of microbial control were discussed. It was generally acknowledged that testing, and particularly sterility testing, was an ancillary component of microbial control. Consequently, the microbial prohibitions outlined in <1111> are even less useful because sterilization is not used to prepare non-sterile products, so they can never be rendered completely free of microbial contamination.

 <62> and <1111> be revised to achieve the following goals:

Increase awareness that microbial control for non-sterile products is not absolute and must be provided operationally (i.e., mandating the absence of any microorganisms is an unrealistic expectation).

Replace the emphasis on ‘absence’ expected in these chapters with a fuller understanding that microbial control is actually provided by design of process and facility, along with personnel practices and procedures other than microbial sampling and testing.

Broaden the scope of concern for microbial control to include other microorganisms, rather than limiting it to the specified species identified in these chapters.This is problematic because the use of specified species to aid in in microbial control is not well understood, except by academically trained microbiologists.

Shift the emphasis on microbial control for non-sterile products from a laboratory (testing) focus to a manufacturing control focus.

Additional revisions to <1111> would reinforce its message that microbial control of non-sterile products is provided by appropriate process/facility design and operational practices.

The authors suggest that the wording of the opening paragraph of USP <62> be changed to the following:

The tests described hereafter will allow determination of the absence of, or limited occurrence of, specified microorganisms in those monograph products where their presence is specifically considered potentially detrimental to patient health that may be detected under the conditions described.

The tests are designed primarily to determine whether a substance or preparation complies with an established monograph specification for microbiological quality. When used for such purposes, follow the instructions given below, including the number of samples to be taken, and interpret the results as stated below. If a product monograph does not indicate a requirement, or there is no monograph for the product, this test is NOT required.

Alternative microbiological procedures, including automated methods, may be used, provided that their equivalence to the Pharmacopeial method has been demonstrated.

They also suggest revising USP <1111> to remove the specific mention of individual microbial strains, as seen in Colum 4 (enumerating “Specified Microorganism(s)” in 

In addition, they suggest incorporating the following passages into the Chapter text:

The presence of high concentrations of microorganisms in nonsterile preparations may have the potential to reduce or even inactivate the therapeutic activity of the product and has a potential to adversely affect the health of the patient. Manufacturers have therefore to ensure a low bioburden of finished dosage forms by implementing current guidelines on Good Manufacturing Practice during the manufacture, storage, and distribution of pharmaceutical preparations.

Microbial examination of nonsterile products is performed according to the methods given in the texts on Microbiological Examination of Nonsterile Products: Microbial Enumeration Tests <61> and Microbiological Examination of Nonsterile Products: Tests for Specified Microorganisms <62>.

Acceptance criteria for nonsterile pharmaceutical products based upon the total aerobic microbial count (TAMC) and the total combined yeasts and molds count (TYMC) are given in Tables 1 and 2. Acceptance criteria are based on individual results or on the average of replicate counts when replicate counts are performed (e.g., direct plating methods).

When an acceptance criterion for microbiological quality is prescribed, it is interpreted as follows:

— 101 cfu: maximum acceptable count = 20;
— 102 cfu: maximum acceptable count = 200;
— 103 cfu: maximum acceptable count = 2000; and so forth.

If it has been shown that none of the prescribed tests will allow valid enumeration of microorganisms at the level prescribed, a validated method with a limit of detection as close as possible to the indicated acceptance criterion is used.

, recovery of isolates should be followed by an assessment for their relevant pathogenicity or significance for concern in the respective administered dosage form.Significance relates to ensuring patient safety and the maintenance of product quality and efficacy. The significance of microorganisms recovered should be evaluated in terms of the following:

Use of the product, since hazard varies according to the route of administration (e.g., whether via eye, nose, or respiratory tract).

Nature of the product. Questions would include: Does the product support growth? Does it have adequate antimicrobial preservation?

Intended recipient, since risk may differ for different patient populations (e.g., neonates vs. infants, adults, and the debilitated.

Use of immunosuppressive agents and corticosteroids.

Presence of disease, wounds, and organ damage.

Where warranted, a risk-based assessment of relevant factors should be conducted by personnel with specialized training in microbiology and in the interpretation of microbiological data. For raw materials, the assessment takes account of the processing to which the product is subjected, the current technology of testing, and the availability of materials of the desired quality.

In short, these are relatively limited revisions, but could have important implications for industry and make these important USP chapters more useful and reflective of industry practice.

, Chapter <62> Microbial Examination of Nonsterile Products: Tests for Specified Miocroorganisms, Sixth Interim Revision Announcement (USP, 2016).

, March – April 2001).
7. K. Ginsbury, “Proposed USP Chapter on Nonsterile Bioburden Long Overdue, But Clarification Still Needed,”

, PharmTech Sterile Manufacturing Supplement, 2009.

James Agalloco, is principal of Agalloco & Associates, 

; (908) 874-7558*
and Donald Singer is senior microbiology technical consultant, North America, Ecolab Life Sciences.


* to whom all correspondence should be addressed.

Articles

Subsequent guidance and manufacturing realities make portions of USP Chapters <62 >and <111> significantly less relevant. The authors suggest formal chapter revisions. 

Refining Microbiological Control for Non-Sterile Products

In aseptic manufacturing, the application of cleaning and disinfection agents reduces contamination to an acceptable level for the grade of cleanroom and prevents cross-contamination from surfaces that are part of or adjacent to good manufacturing practice (GMP) manufacturing operations. 

	There is systemic complacency within the industry regarding the cleaning and disinfection products used and the associated program. A common response within industry when asked about the rationale for a given cleaning regime is: “We have always done it that way.” When a review of the associated environmental monitoring trends demonstrates a degree of control, everyone is satisfied.

	Historically, cleaning has been the “residual contaminant” removal step and disinfection has followed, often at times leaving the disinfectant on the surfaces indefinitely. Legacy cleanroom environments were often following the old adage that “visible disinfectant residues on the surface are a preventative measure.” But this rationale may be changing. The authors have seen a prolonged and increased concern from regulators over residues left from disinfectants post-application. 

	Current industry thinking, along with recent compliance mandates (1), is that any residual chemical is a potential chemical or particulate contaminant to a process and possibly to product. This renewed focus has led a change from legacy thinking to a consideration of how to assess and address disinfectant residues, including an evaluation of what properties other than efficacy, such as residue profile, should be considered for cleaning and disinfectant products.

  The presence of a disinfectant residue represents a risk to product quality as either a physical or chemical contamination risk, which is why products such as alcohols are commonly used in close proximity to open product, as they leave little or no residues. For aseptically produced and low-bioburden products, contamination control is intertwined within the quality system, facility design, and process validation. The US Pharmacopeial Convention (USP) says (2): 

“The removal of residual disinfectants should be monitored for effectiveness as a precaution against the possibility of product contamination.”

	Cleanrooms are typically designed to facilitate contamination control for pharmaceutical products at the point in the manufacturing process where internal controls are most important. The highest risk products, sterile injectables, require product contact surfaces to be contamination-free and are commonly subjected to validated sterilization processes. Adjacent surfaces on production machinery and containment equipment (e.g., isolators or restricted access barriers) can be decontaminated, with the assurance that residual agents are removed, to prevent possible cross-contamination during the filling process where product is minimally exposed. 

	Biopharmaceutical cleanrooms can range in use from bulk manufacturing, containing large tanks/vessels and complex purification equipment, to fill/finish operations for sterile injectables. The concern with residual disinfectant cross-contamination into product processing is a moderate risk in bulk manufacturing areas, unless product processes are directly adjacent to a decontaminated surface, such as a fermentation tank with any size hatch that can open. Filling operations are similar in high risk to the sterile injectable.

	Cleanrooms used for cell therapy manufacturing may have more containment features to protect the live cells and inherently short manufacturing times, yet there are still many surfaces that need to be part of the contamination control strategy. These surfaces are disinfected routinely with expectations that no residue exists that can be carried by operators (on their gloves or gowns) or mobile equipment from one activity to another in the manufacturing suite(s). Appropriate choice of disinfectants is also crucial when using single-use bags containing live cells throughout the life cycle of a cell therapy.

	Despite the expectation that no disinfectant residue exists in these critical and adjacent manufacturing areas of any type of pharmaceutical operation, broad-spectrum disinfectants and sporicidal agents used in these same areas often leave residue, which may or may not be visibly apparent. 

	The new draft of Annex 1 contains a specific regulatory guidance statement about disinfectants, which highlights that cleaning programs should be effective at removing disinfectant residues. This expectation is in line with the developing expectations of industry and with historical regulatory observations. Annex 1, section 4.36 says (1): 

“The disinfection of cleanrooms is particularly important. They should be cleaned and disinfected thoroughly in accordance with a written programme. For disinfection to be effective, prior cleaning to remove surface contamination should be performed … Cleaning programs should effectively remove disinfectant residues.” 

  Regulatory compliance is always a business expectation because of its impact on final products and the patients who are the primary customers. Regulatory inspections follow the legal requirements stated in practical terms as current good manufacturing practices (CGMPs). Inspectors have authority to interpret CGMPs when they evaluate a manufacturing operation. 

	Evaluation of cleaning and disinfection practices are part of the CGMP inspection. Most evaluations of the cleaning and disinfection program are based on visual observation. Inspectors will not only indicate and question visual observation of residues from product, but they also will note disinfectant-type residues. Since cleaning validation usually relates to product contact surfaces, most non-product contact surfaces do not have a quantitative analysis of residues. Thus, the visual observation of residue by an inspector is often generalized and unqualified as to its identification as a residue from manufacturing products (actives or excipients), cleaning, or disinfection. Notation of color and location is often the extent of an observation, which leaves the identification of the residue and the subsequent corrective action with the manufacturer. In most situations, residue of any kind becomes an observation/finding from the inspection with a requirement to identify and mitigate/prevent future occurrence. 

  Another aspect to consider is the health and safety risk disinfectants may have on cleanroom operators and cleaning technicians. All disinfectants are by their very nature toxic to living organisms; however, these chemicals are an effective and safe tool when handled appropriately with adequate safety measures in place. Training on proper storage, mixing, handling, and application procedures is essential. 

	The interaction between some disinfectants can lead to undesirable risks to cleanroom operations. If the cleaning and disinfection program does not address disinfectant residues prior to applying different chemistries, there is a potential for chemical interactions between the chemistries in use (3). For example, a chlorine-based disinfectant applied after a phenolic-based disinfectant may result in the release of toxic chlorine gas. Additionally, these chemical reactions may also interfere with the disinfectant’s efficacy. The presence of disinfectant residues may also reduce the biocidal activity of disinfectants subsequently applied to the surface. This may result in frequent environmental excursions or increased recovery of microorganisms that the cleaning and disinfection regime should have been effective at managing throughout the facility. 

	Disinfectant residues can also interact with one another, causing sticky or slippery floors. Both outcomes pose a risk of slips, trips, and falls to cleanroom operators. In addition, the presence of sticky floors may also lead to the accumulation of debris on the surfaces, posing a gross contamination risk. It is imperative that pharmaceutical manufacturers review their cleaning and disinfection program to ensure that a residue removal step is incorporated when changing or rotating between different disinfectants, such as prior to sporicides. 

  Cleanroom operators are sure to have seen evidence of disinfectant residues, such as an oily sheen on stainless steel or the chalky white powder on the floor coving. Other effects from the use of disinfectants on cleanroom surfaces can be rouging on stainless steel or the reduction of the epoxy floor sheen. 

	In cleanrooms, disinfectant residues are often monitored, or measured, visually. These residues, if not managed preventatively, can cause degradation to the facility over time, which can lead to costly reconstruction or require deep cleaning measures. 

	A significant source of facility degradation tends to be rotational sporicides. Due to effective use levels of sporicidal formulations, these chemicals tend to be corrosive in nature and can quickly age a facility if not appropriately managed. It is crucial that the residues of these types of chemicals are removed from the surface after the appropriate contact time to avoid degradation over time. However, it should be noted that residue should not only be removed from cleanroom surfaces where the residue is visibly apparent, such as on stainless steel and glass, but also on surfaces where the residue may not be visibly apparent, such as non-reflective surfaces. 

. The disinfection residue removal process is likely to require additional time and resources in the form of increased cost of labor and supplies to remove residues, which in turn leads to reduced production time and productivity. 

	Disinfectant residues need to be removed from the cleanroom environment, but most disinfectants have some degree of residues. It begs the question: How can cleanroom operations meet the regulatory requirements without impacting their production schedule and targets?

	Several aspects of the cleaning and disinfection program can be reviewed to reduce the impact and/or risk posed by disinfectant residues, such as introducing a residue removal regime, reviewing application techniques, and choosing low-residue formulations. 

Insufficient or non-existent residue removal.

  All disinfectants, with the exception of some isopropanol and hydrogen peroxide formulations, leave some amount of residue on the surface, which will require routine residue removal. 

	Cleanroom disinfectants are typically aqueous-based formulations and are therefore readily dissolvable in water. Thus, the best solvent to remove disinfectant residues is water. Normally, this is water for injection or purified grade water, depending on the location and risk to the cleanroom. However, water poses another risk to the cleanroom: origin or potential for microbiological growth. To address this concern, 70% alcohols are commonly used in critical environments. While 70% alcohols are disinfectants, they are also used to reduce the build-up of other disinfectant residues. 

	The effectiveness of the residue removal step should be assessed for each disinfectant used in the site’s cleaning and disinfection program. The frequency of the residue removal will depend on the means of application and the disinfectant formulation. 

  When applying disinfectant to cleanroom surfaces, the end-user should be cautious of oversaturating a surface. Over-application can be the result of many common challenges within the cleanroom. One challenge is achieving the validated contact time. A heavy application of disinfectant may seem like a way to achieve the validated contact time. However, by oversaturating a surface, more disinfectant is being applied, resulting in more disinfectant residues building-up over time. 

	Another common challenge is improper use of the cleaning and disinfectant tools. The user should seek cleaning and disinfecting tools that apply the disinfectant in a controlled manner, such as an effective wringer and a defined saturation level for wipes. If controls are not in place, variability in application can result among operators, which in turn can impact effectiveness of the cleaning and disinfection program. Over application can also impact the frequency of the residue removal program. 

	Manufacturing sites should also ensure that their cleaning and disinfection personnel are adequately trained to apply disinfectants in a cleanroom setting. This includes training on the saturation level of mops and wipes, as well as how controlled application is important on surfaces. Multiple coatings and overlapping the same surface do not only serve as a potential cross-contamination issue, but also contribute to disinfectant residue build-up. 

  It is important to be aware of and understand the formulation of a disinfectant when applied to a particular surface for consideration about potential residues. Good practice utilizes high quality pharmaceutical-grade water (e.g., water for injection) for dilution of disinfectant concentrate. Following a manufacturer’s label for the volume of water to use when diluting disinfectant concentrate is critical, and any variation can influence the occurrence of residue and performance. Disinfectant stability may be related to incorporated inactive ingredients in the formulation, yet also may influence residue occurrence. Compatibility of the disinfectant on a particular surface is another parameter.

	Consider the intended purpose that the disinfectant was formulated to serve. Some cleaning and disinfection products intended for GMP environments may have been originally formulated for clinical or hospital settings that are more commonly highly soiled environments. Although these products are available for use within a cleanroom environment, these products will be formulated to address greater soiling and contamination than would be anticipated in the average pharmaceutical cleanroom. Therefore, it is likely that these types of disinfectants will contain a higher degree of surfactants and actives than are necessary to control and maintain a classified cleanroom; all contribute to the residue profile of the product. 

	Disinfectant residues pose various risks to the cleanroom environment, which is why the industry and regulatory groups have a renewed focus on the effective removal of their residues. End-users can combat disinfectant residues in many ways, such as implementing a routine residue removal program, instituting "low-residue" disinfectant formulations, and focusing on operator training to control application.

EudraLex, Volume 4, EU Guidelines to Good Manufacturing Practice Medicinal Products for Human and Veterinary Use,

 Annex 1, Manufacture of Sterile Medicinal Products, Draft (2020).
	2. USP, 

 General Chapter <1072>, “Disinfectants and Antiseptics,” 

The CDC Handbook: A Guide to Cleaning and Disinfecting Cleanrooms,

 T. Sandle Ed. (Grosvenor House Publishing Ltd, London, England, 2012). 

	Madison Hoal is global technical consultant, and Donald Singer is senior microbiology technical consultant, both at Ecolab Life Sciences.


	Vol. 44, No. 6
	June 2020
	Pages: 28–31, 60

	When referring to this article, please cite it as M. Hoal and D. Singer, "Why Do Disinfectant Residues Matter?" 

Consider how to assess risks and understand possible sources of disinfectant residues in pharmaceutical manufacturing.