1. Validation Part 2: Cleaning validation
Supplementary Training Modules on Good Manufacturing Practices
Validation Part 2: Cleaning validation
Module 1, Part 2 focuses on Cleaning validation. The suggested time for this part is:
45-60 minutes
Although it is a formally designated and well-recognised regulatory requirement, cleaning validation continues to be a problem for many manufacturers and inspectors. It can be a complex undertaking, requiring an in-depth understanding of the process, materials and cleaning chemistries involved.
Additionally, the scientific basis for setting acceptable residual levels and the development of analytical procedures capable of detecting very low levels of target compounds needs to be understood.
(Note for the Trainer: the times noted are very approximate.)

2. Validation Objectives To review: General requirements
Validation protocol requirements
How to check limits
Analytical requirements
Sample methods
The objectives of Part 2 of Module 1 are to review in detail:
General requirements on cleaning validation, excluding specialized cleaning or inactivation that for example may be required for viral or mycoplasma removal in the biological manufacturing industry.
Validation protocol requirements: what needs to go in the protocol, and how it should be written.
How to check limits; the acceptance criteria need to be established before the experimental section.
Analytical requirements for cleaning; residues at lower levels and analytes can be many.
Sample methods; because the sampling efficiency is fundamental to cleaning validation.

3. Validation Why cleaning validation is so important (1)
Pharmaceuticals can be contaminated by potentially dangerous substances
Essential to establish adequate cleaning procedures
Why cleaning validation is so important:
Cleaning is like any other critical process that requires validation. However, it is not generally well understood or studied. The greater the risk of the product, the greater the drug potency or toxicity, the more effort on the validation of cleaning methods is required. Once established and validated, the cleaning process must be followed, adhered to, documented, recorded and maintained.
Pharmaceutical products can be contaminated by a variety of substances (some of them potentially dangerous) left over from previous batches by poor cleaning methods. They can also be contaminated by cleaning agents, micro-organisms and materials such as airborne particles, dust, lubricants and raw materials. It is essential that adequate cleaning procedures be established and validated.

4. Validation Why cleaning validation is so important (2)
“Particular attention should be accorded to the validation of … cleaning procedures” (WHO)
“Cleaning validation should be performed in order to confirm the effectiveness of a cleaning procedure” (PIC/S)
“The data should support a conclusion that residues have been reduced to an ‘acceptable’ level” (FDA)
Why cleaning validation is so important: (Contd.)
Three quotations are worth remembering:
“Particular attention should be accorded to the validation of … cleaning procedures”
“Cleaning validation should be performed in order to confirm the effectiveness of a cleaning procedure.”
“The data should support a conclusion that residues have been reduced to an ‘acceptable’ level.”
WHO Expert Committee on Specifications for Pharmaceutical Preparations. Thirty-second Report. Geneva, World Health Organization, 1992 (WHO Technical Report Series, No. 823). Annex 1, 5.1.
PIC/S GMP Guide, Annex 15 (cl.36)
FDA Guide to Inspections of Validation of Cleaning Processes July 1993

5. Validation Possible contaminants Product residues
Cleaning agent residues and breakdown
Airborne matter
Lubricants, ancillary material
Decomposition residues
Bacteria, mould and pyrogens
All contaminants need to be assessed as part of cleaning validation. It was once believed that the presence or absence of active materials was all that was needed to be tested for; but there are many other objectionable contaminants, as follows:
Product residues, both active and excipient
Cleaning agent residues
Airborne matter, such as dust and particulate
Lubricants, and ancillary material, such as disinfectants
Decomposition residues which include:
product residue breakdown products occasioned by e.g. use of strong acids and alkalis during the cleaning process, and
breakdown products of the detergents, acids and alkalis that may be part of the cleaning process.
Bacteria, mould and pyrogens. If the equipment is left wet for long periods before being cleaned it will encourage the growth of microorganisms. Conversely, if left to dry out, residues become extremely difficult to remove.

6. Validation Strategy on cleaning validation Product contact surfaces
After product changeover
Between batches in campaigns
Bracketing products for cleaning validation
Periodic re-evaluation and revalidation
The Manufacturer should have a strategy on cleaning validation covering:
Product contact surfaces.
Cleaning after product changeover, when one pharmaceutical formulation is being changed for another, completely different formulation.
Between batches in campaigns, when the same formula is being manufactured over a period of time, and on different days.
(Some participants may come from agencies where there must be extensive cleaning between all batches, even of the same formula. Be prepared to discuss the length of time for a campaign. It seems accepted that a campaign can last a working week but anything longer becomes difficult to control and define.)
Bracketing products for cleaning validation. This often arises where there are products containing substances with similar properties (such as solubility) or the same substance in different strengths. An acceptable strategy is to manufacture the more dilute form (not necessarily the lowest dose!) and then the most concentrated form. There are sometimes “families” of products which differ slightly as to actives or excipients.
(Be prepared to discuss this point with the participants.)
Periodic evaluation and revalidation.
(The trainer could ask: “How many batches are required to be assessed for cleaning validation, and for periodic revalidation”. “How often should a validated cleaning process be revalidated?”)

7. Validation Cleaning validation protocol (1) Should include :
Objective of the validation
Responsibility for performing and approving validation study
Description of equipment to be used
Cleaning validation protocol:
The protocol should be written before the experimental section of the work commences. It should be seen as the written description of the experiment, as is often taught in the first year of high school chemistry (Aim, Method, Equipment, Results, Conclusions). It should include:
Objective of the validation. What is the experiment trying to do?
Responsibility for performing the validation study: Who are the people and what are their responsibilities? It is extremely important that the person signing off the work, for example, has adequate qualifications and experience. In some situations, some inspection agencies have found the person to be not adequately qualified, trained or experienced and so all of the work was invalidated and the process deemed to be out of control.
Description of equipment to be used. This is where the list of equipment is needed which should include make, model and serial number or some other unique code.

8. Validation Cleaning validation protocol (2) Should include:
Interval between end of production and cleaning, and commencement of cleaning procedure
Cleaning procedures to be used
Any routine monitoring equipment used
Number of cleaning cycles performed consecutively
Sampling procedures used and rationale
Sampling locations (clearly defined)
Cleaning validation protocol: (Contd.)
Should include:
Interval between end of production and cleaning, and commencement of cleaning procedure. To take into account the maximum period that equipment will be left dirty before being cleaned. There may also be a need for the manufacturer to establish the time after cleaning and before use. This may be especially important for control of micro-organisms.
Cleaning procedures to be used for each product, each manufacturing system or each piece of equipment. Reference to the standard operating procedure is required here. Cleaning process must already be documented in an SOP. Note that some cleaning agents are incompatible with the drug. For example, removal of chlorhexidine residues (cationic) with an anionic surfactant will result in intractable, sticky residues.
Any routine monitoring equipment used. This includes conductivity meters, pH meters, and Total Organic Carbon analyzers.
The number of cleaning cycles to be performed consecutively.
Sampling procedures used and rationale for why a certain sampling method is used. A review of the two most commonly used methods, the swab or swatch sample, and the rinse fluid sample is given later in this module. The sample procedure should describe where the samples should be taken based upon a previous analysis of the equipment to identify the most difficult to clean places.
Clearly defined sampling locations.

9. Validation Record of cleaning validation Should include :
Data on recovery studies
Analytical methods including Limit of Detection and Limit of Quantitation
Acceptance criteria and rationale
When revalidation will be required
Must have management and QA involvement
Management commitment and QA involvement
Cleaning validation protocol: (Contd.)
Should include:
Data on recovery studies. The efficiency of the recovery of the sampling technique needs to be established.
Analytical methods, including Limit of Detection and Limit of Quantitation. Analytical methods should be referred to by a unique number, code or unique reference.
Acceptance criteria and rationale for setting the specific limits. The acceptance criteria should include a margin for error and for sampling efficiency.
When revalidation will be required.
The cleaning validation protocol must have management commitment, in order that money, time and resources are allocated to the validation work, as well as QA involvement.

10. Validation Results and reports
Cleaning record signed by operator, checked by production and reviewed by QA
Final Validation Reports, including conclusions
Results and reports:
The record of cleaning validation should be the raw data of the test results together with, for example, the cleaning record, which must signed by the operator, checked by production and reviewed by QA.
There must be a Final Validation Report including conclusions reached. The final outcome must be stated, such as: “All the acceptance criteria were met”.

11. Validation Personnel Manual cleaning methods are difficult to validate
Cannot validate people; can measure proficiency
Must have good training
Must have effective supervision
Personnel:
Manual cleaning methods are difficult to validate.
You cannot validate people but you can measure proficiency. People are not machines (they sometimes are much better than machines!). To compensate for any human weaknesses they should have good training (with appropriate records archived and which can be cross-referenced in the cleaning validation report) and they must have effective supervision.

12. Validation Microbiological aspects Include in validation strategy
Analyse risks of contamination
Consider equipment storage time
Equipment should be stored dry
Sterilization and pyrogen contamination
The microbiological review of cleaning is as important as the chemical cleaning since the equipment may appear to be clean but could be contaminated with micro-organisms, including pathogenic bacteria, during the cleaning process. Consequently the manufacturer must arrange to include the microbiological risk analysis in the validation strategy. This should mean advice from a competent, qualified person experienced in microbiology. He or she should consider unclean equipment storage time. Time frames and conditions for the storage of cleaned equipment should be established.
There should be some documented evidence that routine cleaning and storage of equipment does not allow microbial proliferation. Equipment should be stored dry and this should be reflected in the protocol and in the cleaning SOPs. Under no circumstances should stagnant water be allowed to remain in equipment after cleaning operations.
The control of the bioburden through adequate cleaning and storage of equipment is important to ensure that subsequent sterilization or sanitization procedures achieve adequate sterility assurance. This is also important for the control of pyrogens in sterile processing since equipment sterilisation processes may not achieve significant inactivation or removal of pyrogens.

13. Validation How to sample Swab/swatch Rinse fluid Placebo
The sample transport and storage conditions should be defined
The dirty item is cleaned according to the SOP and then it has to be checked for cleanliness according to the cleaning validation protocol. This means taking a sample of the surface and analysing it. How? Two common methods are the swab and the rinse sample. A less common method is the placebo batch. None is ideal, all have advantages and disadvantages. Check to see if the manufacturer uses them in combination.
The sample transport and storage conditions should be defined.
The swab and rinse methods are discussed on the following slides.

14. Validation Swab samples Direct sampling method Reproducibility
Extraction efficiency
Document swab locations
Disadvantages
inability to access some areas
assumes uniformity of contamination surface
must extrapolate sample area to whole surface
Swab samples:
This is a direct method of sampling, rather than indirect such as rinse sampling.
The swab is perhaps the most common method of checking. It involves taking an inert (usually cotton wool or similar) material on the end of a probe and rubbing it methodically across a surface. A “swatch” (a big piece of material) can sample a larger surface area. Advantages are that residues that are "dried out" or are insoluble can be sampled by physical removal, and areas that are hardest to clean and which are reasonably accessible can be evaluated.
It is expected the swab will pick up all the residues on the surface which can then be assayed. The reproducibility is suspect because of the human involvement. There must be a proper SOP and training on the technique. Sampling spiked surfaces is often used as a training method. Where possible, a template is used to ensure the same surface area is being swabbed. Similarly, the extraction efficiency must be checked because the swab may pick up the contaminant but may not necessarily release it to the extraction solution in the laboratory. The swab locations must be documented in the experimental section of the protocol.
There are a number of disadvantages of using swabs including:
Inability to access some areas; these are usually the most difficult to clean areas.
Assumes uniformity of contamination surface; invariably, contamination is not uniform.
Must extrapolate sample area to whole surface; it can be difficult to estimate the total surface area of the equipment and the calculation also requires that the swab location be carefully measured and recorded.
(Use flip chart and draw equipment and points to illustrate the above)

15. Validation Rinse samples Indirect method Combine with swabs
Useful for cleaning agent residues
pH, conductivity, TOC
Insufficient evidence of cleaning
Sample very large surface areas
Rinse samples:
This is an “indirect” sampling method. The solvent (usually water) used for the final rinsing of equipment has much useful information. It should not be discarded before all of the information can be gleaned from it.
The rinse sample should be used in combination with swabs; together they balance out the disadvantages each has on its own. The rinse sample is also useful for checking cleaning agent residues, e.g. detergents. Sometimes this can be with non-specific, simple tests such as pH, conductivity and, increasingly, Total Organic Carbon (TOC); which can also be put “on line” to measure routinely the cleaning endpoint.
However, consideration should be given to the fact that the residue or contaminant may be insoluble or may be physically occluded in the equipment.
Rinse samples on their own are insufficient evidence of cleaning and should be used in combination with other sampling methods, such as swabs. The manufacturer should be aware of the “dirty pot” syndrome. That is, the container may appear to rinse clean but it could still be contaminated. However, rinse samples allow sampling of a large surface area, of otherwise inaccessible systems or those that cannot be routinely disassembled.

16. Validation Analytical method (1) Validate analytical method
Must be sensitive assay procedure:
HPLC, GC, HPTLC
TOC pH
conductivity UV
ELISA
Analytical method:
The method must be a sensitive analytical procedure. Analytes of the order of parts per billion will be examined. A non-specific assay method is not a disadvantage for cleaning validation where total contaminants are being studied as opposed to just specific analytes. Examples of suitable methods are:
Chromatographic: HPLC, GC, High Performance Thin Layer Chromatography (HPTLC) are all very sensitive and also very specific. TLC may not be sensitive enough.
TOC: total organic carbon analysers are increasingly popular because they are very sensitive, but not specific. However, if the manufacturer puts a total residues limit of say 500ppb (parts per billion) then TOC is a very useful analytical tool. It should be used with pH and conductivity to be of value.
pH; very sensitive to hydrogen ions, so very good at checking even trace levels of acids and alkalis that may be used as part of the cleaning process.
Conductivity: a very sensitive method for total ions. (These last three, TOC, pH and conductivity, when used in combination, are proving very powerful cleaning validation assay methods.)
UV spectroscopy: Moderate specificity but not quantitative. Can have high sensitivity depending on analyte.
ELISA (Enzyme-linked immunosorbent assay): very sensitive and specific for biopharmaceuticals but very expensive, and labour intensive, with a long sample turn- around.

17. Validation Analytical method (2) Check:
Precision, linearity, selectivity
Limit of Detection (LOD)
Limit of Quantitation (LOQ)
Recovery, by spiking
Consistency of recovery
The laboratory must validate the analytical method before validation is started. Alternatively, there needs to be evidence that the analytical method is suitable for use.
The laboratory should check:
Precision, linearity, selectivity (the latter if specific analytes are being targeted). However, note that interference by another analyte will make the validation fail, rather than pass.
Limit of Detection (LOD).
Limit of Quantitation (LOQ).
Recovery, by spiking with the analyte; below 50% is considered unacceptable by some authorities, above 80% is good. The result is multiplied by the recovery factor to give the actual level of residue.
Consistency of recovery, or reproducibility of the method should be checked.
The last two measure the sensitivity of the procedure and are critical features of a good analytical method.

18. Validation Setting limits (1)
Regulatory authorities do not set limits for specific products
Logically based
Limits must be practical, achievable and verifiable
Allergenic and potent substances
Limit setting approach needed
Setting Limits:
It should be noted at the outset: “Regulatory authorities do not set limits for specific products”. It is up to the manufacturer to determine the level of carry over of any contaminant. (The US FDA says each manufacturer is responsible for setting its own limits.) Setting limits must be logically based. The limits must be practical, achievable and verifiable. The limit setting approach can be:
Product specific
Grouped into product families: for example all those products containing multiple
ingredients and one common, low-level ingredient, and choosing a worst case
product.
Collected into similar risk groups (e.g. very soluble products, similar potency, highly
toxic, or difficult to detect products).
Different safety factors for different dosage forms based on physiological
response. This method is essential for potent materials.
Setting limits on carryover.
Certain allergenic ingredients, (e.g. penicillins, cephalosporins) and highly potent material (e.g. anovulent steroids, potent steroids and cytotoxics) should not be detectable by best available analytical methods. In practice this may mean that dedicated manufacturing facilities are used for these products.
The methods of limit setting are discussed on the next slides.

19. Validation Setting limits (2)
Uniform distribution of contaminants not guaranteed
Decomposition products to be checked
Setting limits; cleaning criteria:
visually clean
10ppm in another product
0.1% of therapeutic dose
Setting limits: (Contd.)
Some points to note when setting limits:
As noted on a previous slide, uniform distribution of contaminants is not guaranteed.
Decomposition products should be checked. These can arise from interaction with the washing or cleaning procedures. If used, check reactions with alkalis, acids, lights, heat (polymerization of substances can make them extremely difficult to remove).
The three most common methods of setting cleaning criteria are:
visually clean
10ppm in another product
0.1% of therapeutic dose
Note that the PIC/S Guidelines on Validation calls for use of the most stringent
of three options: dose-based, 10 ppm default or the visually clean standard.
(Clause of PIC/S Recommendations on VMP, IQ, OQ, Non-sterile process
validation and Cleaning validation, August 2001.)

20. Validation Setting limits: “Visually clean” Always first criteria
Can be very sensitive but needs verification
Use between same product batches of same formulation
Illuminate surface
Spiking studies
Setting limits: (Contd.)
The human eye meets the criteria of a non-specific, sensitive analytical procedure. Of course, surfaces have to be visible! It is not a method for closed systems. “Visually clean”, on its own, is not an option for high potency, low dosage drugs.
“Visually clean” is always the first criterion. It can be very sensitive, and may allow detection of gross contamination concentrated in small areas that are not detected by other analytical methods. There are reports of consistent results of 4 micrograms per cm2.
(G.L. Fourman and M.V. Mullen, "Determining Cleaning Validation Acceptance Limits for Pharmaceutical Manufacturing Operations," Pharm.Technol. 17 (4), (1993))
A simple "visually clean" may be an appropriate acceptance criteria in some cases. Verification that the limit is acceptable is needed e.g. by spiking, and observing model surfaces, in a "blinded" manner.
Some inspection agencies accept 'visually clean' between batches of the same product of the same formulation. For example; the USA FDA's guidelines specify that when cleaning between lots of the same product, visually clean is sufficient and validation is not required. The checker must use a proper light source (white light, black light) oblique to the surface. Checking cleaned equipment using less than 400 lux is unsatisfactory.
The “visually clean” method is also useful for checking “non-product contact surfaces” such as walls, floors, doorknobs, ceilings, etc.
Spiking studies should determine the concentration at which most active
ingredients or contaminants are visible.

21. Validation Setting limits: “10ppm” Historical
In some poisons regulations
Pharmacopoeias limit test
Assumes residue to be harmful as heavy metal
Useful for materials for which no available toxicological data
Not for pharmacologically potent material
Setting limits: (Contd.)
The limit of 10ppm was used historically in some early food standards, as a limit for heavy
metals, etc. Consequently it is still a standard in some poisons regulations and is used in most
pharmacopoeias as the basis for limits on, for example: As, Pb, heavy metals for starting
materials. It:
assumes residue to be harmful as heavy metal;
is useful for materials for which no toxicological data is available such as detergents;
may be too generous for pharmacologically potent material.
Example: Maximum allowable amount of contaminant (MC) (10 ppm)
Product A
Product B (following product)
MC = R x S x U T
R: 10 ppm = 10 mg/Kg
S: Batch size of Product B (Kg)
T: Total surface area (inch2) where product A and B contact
U: Area of swab (usually 4 inch2)
e.g. Batch size of Product B = 13 Kg Total surface area = inch2
MC = 10 mg/Kg x 13 Kg x 4 inch2/swab = 116 mg/swab
4 500 inch2
(Tutor should use flip chart to illustrate the above.)

22. Validation Setting limits: not more than 0.1%
Proportion of MINIMUM daily dose of current product carried over into MAXIMUM daily dose of subsequent product
Need to identify worst case
Setting limits: (Contd.)
Toxicologists suggest that an acceptable level of a toxic material may be not more than 1/1000 of a toxic dose or 1/ /1000 of an amount which is not known to show any harmful biological effect in the most sensitive animal system known, e.g. no effect; the assumption that the proportion of the MINIMUM daily dose of the current product carried over into the MAXIMUM daily dose of a subsequent product should be not more than 0.1%.
Minimum daily dose = maximum daily dose x 1
1000
The manufacturer needs to identify worst case.
(Trainer should use flip chart to illustrate the above.)

23. Validation Other issues Clean-In-Place (CIP) systems Placebo studies
Detergent residues; composition should be known
Scrubbing by hand
Other issues:
Clean-In-Place (CIP) systems: Critical areas i.e. those hardest to clean should be identified, particularly in large systems that employ semi-automatic or fully automatic CIP systems.
Placebo studies: A placebo method relies on the manufacture of a placebo batch and then checking it for carry over of the previous product. It is an expensive and laborious process. It should be used in conjunction with rinse and/or swab samples. It is difficult to provide assurance that the contaminants will be dislodged from the equipment surface uniformly. Additionally, if the contaminant or residue is of large enough particle size, it may not be uniformly dispersed in the placebo. Lastly, the sensitivity of the assay may be greatly reduced by dilution of the contaminant.
Detergents residues: detergents should facilitate the cleaning process but are sometimes found to have the most persistent residues. For example, cationic detergents adhere very strongly to glass and are difficult to remove. Detergent composition should be known and removal demonstrated. Acceptable limits should be defined for detergent residues after cleaning. The possibility of detergent breakdown should also be considered when validating cleaning procedures. Detergents should be acceptable to QA/QC departments, and preferably be able to meet local food industry standards.
Scrubbing by hand: Note that manual cleaning methods are difficult to replicate.

24. Validation Questions for the GMP Inspector to ask
How is equipment cleaned?
Are different cleaning processes required?
How many times is a cleaning process repeated before acceptable results are obtained?
What is most appropriate solvent or detergent?
At what point does system become clean?
What does visually clean mean?
Questions for the GMP Inspector to ask:
How is equipment cleaned? Manual cleaning may be more difficult to validate than automated or Clean-In-Place procedures.
Are different cleaning processes required?
How many times is a cleaning process applied before an acceptable result is obtained? That is, will an inspection determine if the cleaning process meets specifications?
What is the most appropriate solvent or detergent?
At what point does the system become clean? With very good analytical procedures now available, it is possible to pick up insignificant amounts of residues. The regulators do not set limits but the manufacturer must. There is an extensive section on setting limits of contaminants earlier in this Part.
What does visually clean mean? “Visually Clean” can actually mean very clean indeed, provided the human eye has assistance e.g. from black and white light.

25. Validation Conclusion
The manufacturer needs a cleaning validation strategy
Assess each situation on its merits
Scientific rationale must be developed
equipment selection
contamination distribution
significance of the contaminant
“Visually clean” may be all that is required
Conclusion:
The cleaning validation programme should be based on detailed cleaning procedures, a good training programme, a validation protocol, validated chemical and microbiological methods, a change control programme, a final report and any auditing required to ensure compliance.
Assess each situation on its merits.
Scientific rationale must be developed
equipment selection
contamination distribution
significance of the contaminant
In some situations “visually clean” may be all that is required.