MACO and NOEL Calculation for Cleaning Validation

Manufacturing multiple drugs in the same facility is an efficient way to run the business. Yet, it involves risk of cross-contamination.

To avoid this, previous product traces must be completely removed before changing over the product.

This is called Product-to-Product cleaning.

Not only this but also a same product facility requires effective residue removal from batch to batch to avoid the carry over of impurities.

Well, cleaning validation is a scientific technique that ensures:

• Previous product traces have been removed
• Contamination-free operations
• Safe batch-to-batch transitions

Cleaning Validation

Establishing documented, scientific and risk-based evidence that provides a high degree of assurance that a typical cleaning method or procedure will consistently clean the equipment or a medical device in compliance with its predetermined specifications and quality attributes, taking the patient’s safety into consideration. In a nutshell, it is a technique of developing effective cleaning processes that’ll not harm the end-user.

When the product is manufactured, the equipment, systems or any product contact surfaces leaves behind the traces of the product, raw material, etc. If cleaning is performed then the cleaning agent too.

Now during the product change-over or for the next batch of the same product, these traces may cross-contaminate or contaminate the next product or batch of the same product respectively.

Hence, a cleaning validation program is conducted to ensure that the cleaning process effectively removes such traces and there are no chances of contamination anyway.

So, the cleaning validation is a regulatory requirement to ensure:

1. Product safety
2. Patient safety

Without the Quality unit’s approval, the manufacturer’s generally don’t proceed for further manufacturing activities.

Therefore, after defining a cleaning validation program, it must be systematically integrated with QMS and QRM systems.

Because, it helps in:

• Understanding and identifying the risks to the patients, products, etc.
• Performing its impact assessment
• Outlining risk mitigation strategy
• Mitigating those risks at actual; and
• Assuring finished product safety, quality, and efficacy.

Contamination-

The product that is adulterated with the residue of the previous batch of the same product in particular equipment is called Contamination OR A new product that is adulterated with the residue of raw materials of the previous product is also called Contamination.

Cross-Contamination

A new product that is adulterated with the left behind traces of the previous product in particular equipment is called Cross-Contamination.

Successful cleaning validation is that which…

1. Assures patient safety through science- and risk-based studies conducted in the development of the cleaning processes.
2. Avoid product adulteration and achieve a contamination-free product.
3. Satisfies regulatory requirements.
4. Optimize processes and costs due to a systematic product change-over.

We needs to be focused on –

• Ways and types of cleaning processes
• How to develop a cleaning validation program, and
• The expectations of regulatory bodies.

When to Perform Cleaning Validation?

The following are the situations that triggers the requirement of a proper cleaning validation study.

• When establishing a fresh commercial process
• When reusing the existing facility for a different product every time
• Major changes in raw materials based on impact assessment
• Significant modifications in cleaning procedures
• Introduction of new equipment for the already established process
• Changes in cleaning agent
• Changes that might affect the CPPs and CQAs of already approved cleaning validation

Cleaning Classifications :

Cleaning processes based on industrial practices can be differentiated into three ways and two types.

Cleaning can be performed in 3 different ways using cleaning agents such as purified water, WFI (Water for Injection), or chemical solvents.

1. Cleaning In Place (CIP)

• CIP Skid to Clean the Equipment
• Automated CIP of the Equipment

1. Cleaning Out of Place (COP)

• Washers like Tray Washers, Dish Washers, etc.
• Sub-systems of equipment hard to approach in CIP

1. Manual Cleaning

• Components that are difficult to clean during COP and hence cleaned using tools such as Cleaning Brushes, Scrubbers, etc.
• Equipment is preferred for automated cleaning in place i.e., CIP.
• Components and sub-systems not being feasible for CIP are preferred for COP.
• Critical components or locations of the equipment that are hard to reach during CIP and COP are preferred for Manual Cleaning.

Types of Cleaning :

As per the common understanding among pharma professionals, there are two types of cleaning.

Batch To Batch

Cleaning of the process equipment in between two batches for ongoing manufacturing campaign of the same product.

Product to Product

Cleaning of the process equipment in between the two different products i.e. after finishing the previous product campaign and before initiating a new product campaign.

Critical Process Parameters (CPPs) for Cleaning

• Temperature
• Pressure
• Contact time
• Concentration of the cleaning agent
• Surface Roughness (More roughness means more difficult to clean)
• Flow rate
• Proper mixing RPMs
• Dirty Hold Time for Equipment
• Clean Hold Time for Equipment

Critical Quality Attributes (CQAs) For Cleaning

• Product residue
• Cleaning agent residue
• Required concentration of cleaning agent
• Microbial residue
• Drain ability
• Conductivity
• Number of rinses
• Time for cleaning

Pre-Requisites to Begin Cleaning Validation

Before commencing cleaning validation, the following pre-requisites should be met:

• Cleaning Validation Strategy and Protocols are approved and ready
• Equipment used for most of the products should be identified
• SOP for Equipment Cleaning has been established (draft)
• Sampling and Analytical Methods are validated
• A list of CPPs and CQAs is available
• Drug toxicity data is available
• Product contact surface area is calculated
• Training is given to the personnel involved in cleaning validation activity
• Drug characteristics are evaluated for cleaning difficulty

A risk-based approach would help identify the risks associated with:

• Concerned residue
• Priority of selecting sampling locations
• Determining and justifying product/equipment groups
• Defining acceptance criteria
• A sequence of protocol execution
• Product change-over SOPs

Cleaning Validation Protocol and Report

Instead of following any protocol template, it is important to understand its key technical aspects. A protocol should be prepared explaining technical cleaning validation activities that contain:

• Scope and Objective
• Cleaning SOP Draft
• Possible ways of contamination
• Sampling plan and rationale including prioritized sampling locations
• Worst-case conditions
• Selection of analytical methods and their validation
• Equipment Dirty and Clean Hold Time Study
• Acceptance criteria
• Technically sound deviation management strategy
• Data monitoring Annexures (if Applicable)

Successful execution of the protocol would be incomplete without a report. A report should be prepared to summarize the key achievements of the cleaning validation study, including a clear statement of acceptance/rejection.

Selection of Analytical Methods and Their Validation

Two fundamental types of analytical methods exist.

1. Specific
2. Non-Specific

The selection of these methods requires a science- and risk-based approach. Inappropriate evaluation and selecting any of these methods may invite regulatory objections.

For example, the FDA states that companies should determine the specificity of their analytical method. Most professionals misunderstand that the FDA expects to use only specific methods. On the contrary, this also means the selection of analytical methods requires proper evaluation.

However, the EU-GMP mentioned, “the analytical method must be specific for the target residue“. This may be interpreted in two ways.

Only specific methods are acceptable OR

Any of the methods must be specific to that particular target residue even with the non-specific method.

However, the second one sounds more logical and that’s the reason European drugmakers generally practice it.

Needless to say, there is much depth to it. Hence, a correct assessment of the method selection is very important to establish a scientifically meaningful cleaning process.

To bridge the gap, many pharmaceutical manufacturers follow the general practice i.e. a combination of both methods. Specific methods for primary cleaning validation while using non-specific methods for subsequent cleaning verification.

These methods can also be selected based on the stage of manufacturing. Initial stages such as bulk drugs or intermediates, excluding toxic APIs, may only use non-specific methods that may suffice the requirement. Whereas formulation and downstream processing may require both.

But the correct way is to conduct scientific studies and then choose the analytical method/s that accurately detects the compound of interest.

Analytical Method Validation

Upon successful selection of the analytical method, it is important to validate it for the intended use. All non-pharmacopeial analytical methods require validation.

However, if you want to use any of them even when a pharmacopeial method exists, you’ve to provide a rationale for doing so.

the following characteristics should be covered during the analytical method validation.

Specificity OR Selectivity

Ability to use analytical methods to accurately measure analytes and interferences such as cleaning agents.

Selectivity is checked with blank samples (without an analyte) examined in the anticipated time range of the peak that contains the analyte.

Accuracy(% Recovery)

Degree of agreement of the test results produced by the analytical method to the true value. Accuracy is generally established for a complete specified range of the procedure.

A known concentration of analyte standard spikes the sample matrix and measures the accuracy using the specified analytical method.

Precision

The degree of agreement between the individual test results for the repeatedly applied analytical procedure to multiple samplings.

It can be calculated by different methods like Statistical, Horwitz equation, etc.

Limit of Detection

The lowest concentration at which the instrument detects an analyte but does not necessarily quantify it. The noise to signal ratio should be 1:3.

Limit of Quantitation

The lowest concentration at which the instrument both detects and quantifies an analyte. The noise to signal ratio should be 1:10.

Linearity

The capability of the analytical method to obtain the outcome is directly proportional to the concentration of the analyte within a given range.

5 concentrations at a minimum are preferred from 50% to 150% across the working range and are injected with a mobile phase to produce a linear relationship.

Range

It is the concentration range and an interval between the upper and lower limit shown using precision, linearity, and accuracy.

Stability of Solution

The time duration of the sample for which it can be stored before the final analysis after extraction.

Ruggedness

It is a measure to determine the robustness and reliability of the analytical method to deliver linear, accurate, and precise results in all anticipated conditions.

Sampling Methods for Cleaning Validation

According to the FDA guidelines, there are 3 types of sampling. Out of that, 2 are commonly followed for cleaning validation.

Direct Surface Sampling (Swab Sampling)

Direct surface sampling (swab sampling) is the most preferred sampling method for hard-to-reach but reasonably accessible areas of the equipment.

A sterile swab made of cotton is attached to a compatible stick just like ear buds.

• The challenge is to use the solvent along with the swab comfortably without interfering with the analytical test. Swabs may contain certain adhesives that can alter the results.
• These swabs are stored and dipped in a buffer like a phosphate solution or as appropriate to soak the cotton. During sampling, swabs are removed and then applied gently on the equipment surface in one vertical and one horizontal direction without rubbing to and fro.
• Once done for at least 5 to 6 sampling locations, they are put back into the buffer solution and sent to the Quality Unit for evaluating and establishing an acceptable residue content per given surface area as CFU/cm² or as appropriate.

Though this method is more specific in terms of hard-to-reach areas, it has one major disadvantage i.e. the small surface area for a given sample.

Rinse Sampling

Unlike swab sampling, rinse sampling has the advantage of covering a large surface area of the equipment in a particular instance, including systems that are hard to disassemble frequently.

• The required amount of cleaning solvent with the help of a suitable spray ball preferably with 360° coverage used to rinse the equipment.
• Rinsed samples are then collected from sample points located near drain lines for physical and microbiological inspection.

One of the major disadvantages of rinse samples is when rinsed; the residue may incompletely solubilize in the rinse solvent like WFI or PW and remain clogged to the equipment surface. In this case, just checking downstream water for compendial requirements is illogical and hence unacceptable.

Instead, the system should be in place to identify the direct measurement of the residue in the rinse sample such as Infrared sensors or visual inspection, etc.

Indirect Testing or Monitoring Method

Although it is a sampling method or more specifically a monitoring method, it is also an indirect measurement that does not specifically provide us with exact quantification of the residue.

Hence not acceptable as a stand-alone sampling method during cleaning validation. Instead, it should either be used as complementary to the actual sampling method or should be routinely verified after the cleaning validation program.

It is more specific in cases like bulk drug manufacturing where sampling can be most easily performed through rinsing. The best examples are pH, conductivity, and TOC (Total Organic Carbon) measurements.

However, in special cases where other methods fail to detect any presence, this method can be scientifically justified.

Equipment’s Dirty Hold Time and Clean Hold Time :

Everything in the world comes with an expiry and cleaning is not an exception to this. Therefore, the timeframe for the cleaning validity and dirty conditions becomes crucial. But what do they mean?

• The equipment’s idle time between the end of the last batch and the start of the cleaning process is called Dirty Hold Time.
• The equipment’s idle time between the end of the cleaning process and the start of manufacturing is called Clean Hold Time.

Dirty Hold Time

Dirty hold times are the most crucial aspect of a cleaning validation program as they directly impact the efficiency of the cleaning process.

When equipment is left uncleaned for longer durations, the residue attached to the surface may become rigid and dry over a period of time, ultimately challenging the cleaning process.

Establishing dirty hold times for a particular production process depends on the nature of the product, associated processing materials, and cycle times.

The general practice is to conduct 3 consecutive runs of cleaning procedure considering maximum dirty hold time as per the requirement (in most cases around 72 hrs.).

These runs should demonstrate the cleaning procedure effective in removing the residue at the considered maximum dirty hold times. Obviously, through bio-burden testing.

According to one of the FDA’s 483 observations, cleaning validation and dirty hold times should be established for dedicated as well as non-dedicated equipment. This should also include hard-to-clean equipment to obtain overall confidence in cleaning validation.

Clean Hold Time

Just like dirty hold times, the FDA also expects to define clean hold times during the cleaning validation program.

Clean Hold time study generally includes a sampling of clean equipment at a regular time interval of around 6 to 8 hrs. till the equipment completes 24 hrs.

After 24 hrs., the sampling is done once per day. Sampling is performed immediately after cleaning and thereafter at specified intervals.

It is better to have a data recording sheet that captures the necessary information when the samples are sent to the QC lab for bio-burden testing (microbiological proliferation).

Worst-Case Conditions in Cleaning Validation Program

Conditions that may critically impact cleaning validation efforts or even cause a failure should be identified with the most specific challenges.

Following are the challenges considered for establishing the worst-case conditions.

• Drugs with the lowest solubility in their cleaning agent
• Swab locations that are difficult to clean
• Lower therapeutic doses for effective cleaning measurement
• Equipment catering for the largest number of products
• Drugs with higher toxicity

Establishing Cleaning Limits and Requirements

When it comes to cleaning validation, defining the incomplete acceptance criteria compromises your cleaning efforts. The two concepts commonly talked about are NOEL and MACO.

Maximum Allowable Carryover (MACO) tells you mathematically how much of your previous product will carry over to the next product.

Whereas, the No Observed Effect Level (NOEL) tells the drug quantity that has no observable effect on human health when provided with a 50% Lethal Dose.

1. a) Calculating NOEL

NOEL can be calculated as:

Where LD50 – Lethal Dose is at 50% reduction in mg or kg and NOEL is generally measured in “mg”.

However, this approach of risk identification is pointless to carry forward. According to one of the European Medicines Agency (EMA) Question and Answer documents, the use of LD50 to determine Health-Based Exposure Limits (HBEL) for drug products is an inadequate point of departure.

1. b) MACO Based on Therapeutic Daily Dose and Safety Criteria

Based on the above calculated NOEL values, the MACO values can be calculated as:

As per this criterion, no more than 0.1% normal therapeutic dose of the previous product shall appear in the maximum daily dose of the next product.

Here, the min. batch size is considered for the next product.

1. c) MACO Based on 10 PPM Criteria

As per this, no more than 10 ppm of the previous product shall appear in the next product.

MACO can be calculated as:

1. d) Calculating MACO Using Toxicity Data

This approach is generally considered during the early stages of drug manufacturing such as Intermediates or APIs. Additionally, this technique is used to calculate MACO for cases that don’t have information regarding the therapeutic dose.

HBEL is another concept that is gaining popularity within different regulatory bodies due to its risk-based approach (This section is under review and will be updated with a thorough explanation).

MACO and NOEL calculations have great importance in the pharmaceutical industries. NOEL refers to “No observed effect level” and MACO is a “Maximum allowable carryover”. NOEL is used to determine MACO during cleaning validation.

NOEL Calculation

NEOL is calculated by using LD₅₀ and avg. Adult dose.

NEOL= LD₅₀ x Avg. adult dose/ 2000

LD₅₀ is a lethal dose (it may vary), considered an adult’s avg. Weight is 70kg, and 2000 is content. For example, if an LD₅₀ of any drug product is 250mg/kg. then NOEL calculation is

NEOL= 250×70/2000= 8.75mg.

MACO Calculation :

For a multiproduct facility where equipment is shared, there is always a risk from cross-contamination. The correct calculation of the cleaning validation limits from maximum allowable carryover (MACO) of a marker compound to the next product is vital for the integrity and success of the cleaning validation program. However, the process yielding those limits often involves cumbersome, error-prone manual calculations. Herein, we describe an innovative yet simple tool that uses a combination of spreadsheet software and a statistical platform to fully automate science- and risk-based MACO calculations in pharmaceutical cleaning validation.

Before the cleaning validation limit is assessed and applied, an analytical method with adequate sensitivity, specificity, and recovery should be developed and validated. The sampling of the cleaned surface with a suitable swab material or rinse solvent is an important next step to calculate the cleaning validation limit. Generally, predefined areas (usually 10 cm × 10 cm) are swabbed or rinse samples are collected with a known volume of solvent. The formulas used to calculate the swab or rinse limit for each MACO are as follows:

Swab limit (mg/swab)=MACO(mg)Surface area(cm2)×Swab area(cm2)

Rinse limit (mg/rinse)=MACO(mg)Rinse volume(mL)

For each method of calculation, the lowest MACO and cleaning limit are obtained and proposed as acceptance criteria for cleaning validation. For most cases, the selection of the limit is straightforward and based on patient safety; however, there are other factors that could impact the selection, requiring further assessment. The technical and quality staff are responsible for the final decision with appropriate justification.

Pre- Requisite for MACO Calculation :

• The list of the products manufactured (and APIs used) in their respective sites;
• Physicochemical data on the APIs’ solubility, toxicity, potency, and cleanability to be used in calculating the MACO;
• The list of cleaning agents with their composition, acceptable daily intake (ADI), and safety data; and
• Relevant local and regional regulatory policies affecting cleaning validation.

MACO using four methods: health-based exposure limit (HBEL), therapeutic dose, toxicological, and 10-ppm approaches. Different safety factors were used, depending on the route of administration, and as accepted widely by the industry.

In the case study later in this article, the median lethal dose (LD₅₀) was used to calculate ADI. However, depending on the stipulations of the quality management system in the organization, ADI determined from animal toxicological studies (overt toxicity, biomarkers, exaggerated pharmacodynamic effects) to derive a safe starting dose in humans can also be used, if available. Irrespective of the approach used, the macro can easily be adapted to incorporate future changes.

If you don’t have the TDD of the products , you can calculate the MACO as per general limit criteria. O. 1% general limit for intermediates and 0.01% criteria for finished products. You can multiply the subsequent product batch size in mg and divided by 100 to get the MACO in mg.

Method 1:

If the MACO of the previous product is in the next batch. the calculation is as follows:

In this equation, product A is the previous product, and product B is the next product by taking into consideration of therapeutic dosage of the drug product in which the API is used.

Method 2:

The second method of calculation takes toxicological data into consideration. From the NOEL value, MACO is calculated as:

MACO= NOELxMBS/ SFxTDD

Whereas NOEL is 8.75mg.
MBS is the Minimum batch size for the next product
SF is the Safety factor.
TDD is the largest normal daily dose for the product

For example: if the total daily dose of a product is 350mg and the batch size is 100 kg, our NOEL is 8.75 mg. then MACO can be calculated as follow:

MACO= 8.75(mg)x100000000/1000×350(mg)= 250mg or 0.25 gm.

Acceptance criteria

This is the value of allowable residue of the previous product in the next product. Since the residue of the previous batch is contaminated in the next product, it is necessary to limit such carryover into the next product. The maximum limit that is permitted is called the MACO.

By using NOEL and MACO, we can find out the quantity of a drug that can not be carried out over to the next batch. As studies above 250mg /kg LD₅₀ should not be over 0.25gm in the next batch as per above the batch has 350mg daily dose and 100 kg batch size.

Regulatory Concerns

Auditors are not supposed to provide spoon-feeding. In fact, they don’t have much time to learn your complete cleaning philosophy.

They just try to realize the rationales behind your decisions and that the documents should speak for themselves. For example, choosing a method of analysis, cleaning agent, cleaning mechanism, etc.

The following are some of the regulatory audit observations and give us more clarity on their expectations.

• The company’s overall policy, intentions, and approach to validation, including the validation of production processes, cleaning procedures, analytical methods, in-process control test procedures, computerized systems, and persons responsible for design, review, approval, and documentation of each validation phase, should be documented – EU Guide To Good Manufacturing Practice Part II – Section 12.1 Validation Policy.
• Equipment used in the manufacture, processing, packing or holding of drug products is not of appropriate design to facilitate operations for its intended use and for its cleaning and maintenance. [21 CFR § 211.63] For example, CP-2 packaging line was modified in a manner that made it difficult for employees to remove the line cover. As a result, the line cover is not removed during line clearance operations and is only removed during preventative maintenance. Per firm personnel, unit dose strips can become caught in this area and are routinely found during maintenance – FDA warning letter 06-NWJ-14 (July 2006)
• The analytical methods should be challenged in combination with the sampling methods used, to show that the contaminants can be recovered from the equipment surface and to show the level of recovery as well as the consistency of recovery. This is necessary before any conclusions can be made based on the sample results. A negative result may also be the result of poor sampling techniques – EU Guide To Good Manufacturing Practice Annexure-15, Section 4.10.3.
• Validated analytical methods having sensitivity to detect residues or contaminants should be used. The detection limit for each analytical method should be sufficiently sensitive to detect the established acceptable level of the residue or contaminant – ICH Q7A.
• The specificity of test methods should be documented. For example, instructions for the identification and quantification of peaks when using integrators should be provided, and integrated peaks in the swab samples taken during cleaning validation run eluting close to the retention time of the standard peak should be identified or quantified during the validation exercise – FDA 483 Warning.
• Cleaning validation studies for multiple use equipment were inadequate in that the validation protocol did not identify the cleaning procedure, the total surface area was not considered during the validation study, recovery studies were not done to validate the swab sampling method or filtering of rinse samples, some rinse samples were not analyzed, dates of analyses were inaccurate, and analytical data on rinse samples were not checked by a second person – FDA 483 Warning.
• Pipework systems, valves, and vent filters should be properly designed to facilitate cleaning and sterilization – EU GMP Guide, Annexure 2, Premises and Equipment.
• Tanks, containers, pipework and pumps should be designed and installed so that they may be readily cleaned and if necessary sanitised. In particular, equipment design should include a minimum of dead-legs or sites where residues can accumulate and promote microbial proliferation – PIC/S GMP Guide PE 009-5, Guide to Good Manufacturing Practices For Medicinal Products, Annexure 9, Premises and Equipment.
• Washing and cleaning equipment should be chosen and used in order not to be a source of contamination – PIC/S GMP Guide PE 009-5, Guide to Good Manufacturing Practices For Medicinal Products, Chapter 3/3.37.
• The design of the equipment should be carefully examined. Critical areas (those hardest to clean) should be identified, particularly in large systems that employ semi-automatic or fully automatic clean-in-place (CIP) systems – EU GMP Guide, Annexure 15, Section 4.6.1.
• With regard to transfer lines, they are generally hard piped and easily cleaned and sanitized. In some cases manufacturers have used flexible hoses to transfer product. It is not unusual to see flexible hoses lying on the floor, thus significantly increasing the potential for contamination. Such contamination can occur by operators picking up or handling hoses, and possibly even placing them in transfer or batching tanks after they had been lying on the floor. It is also a good practice to store hoses in a way that allows them to drain rather than be coiled which may allow moisture to collect and be a potential source of microbial contamination. Observe manufacturing areas and operator practices, particularly when flexible hose connection are employed – FDA Guide to Inspections of Oral Solutions and Suspensions 1994.

Takeaway Message :

Considering both cleaning complexity and regulatory importance, defining and measuring your cleaning goals clearly is all that matters when it comes to establishing smart cleaning procedures.

Apart from the cleaning procedures, the selection of the analytical method and its validation carry equal importance.

• Upon successful completion of cleaning validation activities, firms should consolidate all the observations noted during cleaning validation.
• A report should be prepared to demonstrate that the predefined goals are met and summarized. Deviations and NCs should be summarized to show how they’re closed.
• Investigate your cleaning procedures to determine potential opportunities for improvement and consistency.
• The document should properly capture the activities with a clear statement of “Pass/Fail” for your cleaning validation activities.

To make the cleaning validation efforts robust, you’ll have to consider enough approaches from the development including risks, science, and statistics, to the fundamentals of contamination or cross-contamination.

for a general limit of 100 ppm: MACO = 0.01% of the minimum batch size (MBS), and for a general limit of 10 ppm: MACO = 0.001% of the minimum batch size (MBS).

MACO and NOEL Calculation are widely used for determining acceptance criteria, cleaning levels, Determination of the amount of residue present, and cleaning validation protocol.