Wednesday, 24 February 2010

Dry Vapour Disinfection Validation

Validation is achieved through the incubation of spore strips that have been exposed to the hydrogen peroxide vapour during the disinfection process. It is normal practice to place the spore strips in areas of the facility thought to be most inaccessible to the vapour (the strips must be retrievable without being compromised).

Hydrogen Peroxide Vapour

Validation spore strips & recovery media

The spore strips are stainless steel, oblong discs inoculated with Geobacillus stearothermophilius and are specifically for use in hydrogen peroxide vapour. Each strip or disc contains a standard spore population of 106. Geobacillus stearothermophilius is a commonly used indicator organism to verify the efficacy of sterilisation and disinfection procedures and is not harmful to humans.

Spore  Test StripTryptic soy broth is used as a recovery media and is placed alongside every spore strip placed in the facility target area prior to the disinfection process. At the end of the disinfection process the spore strips are aseptically transferred to the recovery media tubes and transported to an incubator.

Dry Vapour Disinfection Validation

Incubation & validation process

A control strip which has not been subjected to the dry vapour is placed in to recovery media and incubated, with the exposed strips from the facility, at 56°C. The tubes of media are checked for signs of growth, indicated by a change in colour, at the pre determined intervals for a period of 7 days. No colour change indicates that the disinfection process was sufficient to achieve the expected parameters and kill the test microorganisms.

The result of each spore strip used is recorded and a detailed report is provided after the 7 day period, however, no change after 1 – 2 days is usually indicative of a successful disinfection process.

Hydrogen peroxide vapour has been shown to bio-deactivate a wide range of micro-organisms including:

  • Endospore-forming bacteria
  • Vegetative bacteria
  • Atypical bacteria
  • Viruses
  • Fungi

During the delivery of the disinfection process Bio decontamination Ltd perform “real time humidity reporting” that produces an immediate indication of the success of the process. The data forms part of a comprehensive report provided to our clients.

Aspects of Validation of Aseptic Process and Sterilisation , (Process Simulations "Media Fill " , Filtration Efficacy , Sterilization of Equipment, Co


In this series of articles we are going to disscus aspects of Validation of Aseptic Process and Sterilisation routine qualifications and validation study recommendations .

Change control procedures are an important part of the quality systems established by any firm.
A change in facility, equipment, process, or test method should be evaluated through the written change control program, triggering an evaluation of the need for revalidation or requalification.

We have divided topic "Aspects of Validation of Aseptic Process and Sterilisation" in to three parts .

A. Process Simulations :

B. Filtration Efficacy. ( Filtration Efficacy will be discusses in this article )

C. Sterilization of Equipment, Containers, and Closures ,
( In next article we will be writing about this aspect "Sterilization of Equipment, Containers, and Closures ".

B. Filtration Efficacy.

Filtration is a common method of sterilizing drug product solutions. A sterilizing grade filter should be validated to reproducibly remove viable microorganisms from the process stream, producing a sterile effluent ( This article does not address virus removal ).
Currently, such filters usually have a rated pore size of 0.2 μm or smaller (0.22μ and 0.2μ are considered interchangeable nominal pore size ratings ).

Use of redundant sterilizing filters should be considered in many cases. Whatever filter or combination of filters is used, validation should include microbiological challenges to simulate worst-case production conditions for the material to be filtered and integrity test results of the filters used for the study. Product bioburden should be evaluated when selecting a suitable challenge microorganism to assess which microorganism represents the worst-case challenge to the filter. The microorganism Brevundimonas diminuta (ATCC 19146) when properly grown, harvested and used, is a common challenge microorganism for 0.2 μm rated filters because of its small size (0.3 μm mean diameter). The manufacturing process controls should be designed to minimize the bioburden of the unfiltered product. Bioburden of unsterilized bulk solutions should be determined to trend the characteristics of potentially contaminating organisms.

In certain cases, when justified as equivalent or better than use of B. diminuta, it may be appropriate to conduct bacterial retention studies with a bioburden isolate. The number of microorganisms in the challenge is important because a filter can contain a number of pores larger than the nominal rating, which has the potential to allow passage of microorganisms. The probability of such passage is considered to increase as the number of organisms (bioburden) in the material to be filtered increases. A challenge concentration of at least 107 organisms per cm2 of effective filtration area should generally be used, resulting in no passage of the challenge microorganism. The challenge concentration used for validation is intended to provide a margin of safety well beyond what would be expected in production.

Direct inoculation into the drug formulation is the preferred method because it provides an assessment of the effect of drug product on the filter matrix and on the challenge organism. However, directly inoculating B. diminuta into products with inherent bactericidal activity against this microbe, or into oil-based formulations, can lead to erroneous conclusions. When sufficiently justified, the effects of the product formulation on the membrane's integrity can be assessed using an appropriate alternate method. For example, a drug product could be filtered in a manner in which the worst-case combination of process specifications and conditions are simulated. This step could be followed by filtration of the challenge organism for a significant period of time, under the same conditions, using an appropriately modified product (e.g., lacking an antimicrobial preservative or other antimicrobial component) as the vehicle. Any divergence from a simulation using the actual product and conditions of processing should be justified.

Factors that can affect filter performance generally include (1) viscosity and surface tension of the material to be filtered, (2) pH, (3) compatibility of the material or formulation components with the filter itself, (4) pressures, (5) flow rates, (6) maximum use time, (7) temperature, (8) osmolality, (9) and the effects of hydraulic shock. When designing the validation protocol, it is important to address the effect of the extremes of processing factors on the filter capability to produce sterile effluent. Filter validation should be conducted using the worst-case conditions, such as maximum filter use time and pressure (Ref. 12). Filter validation experiments, including microbial challenges, need not be conducted in the actual manufacturing areas. However, it is essential that laboratory experiments simulate actual production conditions. The specific type of filter membrane used in commercial production should be evaluated in filter validation studies. There are advantages to using production filters in these bacterial retention validation studies. When the more complex filter validation tests go beyond the capabilities of the filter user, tests are often conducted by outside laboratories or by filter manufacturers. However, it is the responsibility of the filter user to review the validation data on the efficacy of the filter in producing a sterile effluent. The data should be applicable to the user's products and conditions of use because filter performance may differ significantly for various conditions and products.

After a filtration process is properly validated for a given product, process, and filter, it is important to ensure that identical filters (e.g., of identical polymer construction and pore size rating) are used in production runs. Sterilizing filters should be routinely discarded after processing of a single lot. However, in those instances when repeated use can be justified, the sterile filter validation should incorporate the maximum number of lots to be processed. Integrity testing of the filter(s) can be performed prior to processing, and should be routinely performed post-use. It is important that integrity testing be conducted after filtration to detect any filter leaks or perforations that might have occurred during the filtration. Forward flow and bubble point tests, when appropriately employed, are two integrity tests that can be used. A production filter’s integrity test specification should be consistent with data generated during bacterial retention validation studies.

Regulatory Aspects for This article .

21 CFR 211.63, 211.65, and 211.67 address, respectively, to the aspects of “Equipment design, size, and location,” “Equipment construction,” and “Equipment cleaning and maintenance.”

21 CFR 211.84(c) mentions, in part, that “Samples shall be collected in accordance with the following procedures: (3) Sterile equipment and aseptic sampling techniques shall be used when necessary.”

21 CFR 211.100(a) mentions , in part, that “There shall be written procedures for production and process control designed to assure that the drug products have the identity, strength, quality, and purity they purport or are represented to possess. Such procedures shall include all requirements in this subpart.”

21 CFR 211.113(b) mentions that “Appropriate written procedures, designed to prevent microbiological contamination of drug products purporting to be sterile, shall be established and followed. Such procedures shall include validation of any sterilization process.”

Saturday, 20 February 2010

Accuracy is one of several data elements required for assay validation. In a regulated pharmaceutical laboratory, determining accuracy can take on many diff e rent forms. This month’s installment of “Validation Viewpoint” focuses on ways to measure and documentn past “Validation Viewpoint” columns and other publications, we have discussed different methods of quantitation as a distinct and decided part of method validation (1–4). And while many factors are taken into account when validating a method, the determination of the method’s accuracy is an integral part of validating any quantitative method. Accuracy is defined as the closeness of the test results obtained by the method of i n t e rest to the true value, demonstrated a c ross the range of analyte levels expected to be found in samples during routine analyses (5). The determination of accuracy varies according to the method’s intended purpose, and whether the sample is a drug substance, the final drug product, or related impurities. In general, accuracy is determined for drug substances by comparison with standard reference materials; for drug products by the analysis of synthetic mixt ures spiked with known quantities of components; and for impurities, it is determined by the analysis of samples (either drug substance or drug product) spiked with known amounts of impurities. For drug substances and drug products, accuracy also can be inferred in some instances once precision, linearity, and specificity h a ve been established. accuracy for typical pharmaceutical formulations, including synthetic drugs and bioanalytical samples. For full article Click Here

Validation - The Essential Quality Assurance Tool For Pharma Industries

K. Dashora, D. Singh, Swarnlata Saraf and S. Saraf *.
Institute of Pharmacy, Pt.RavishankarShuklaUniversity, Raipur 492 010.
*Author for correspondence (e-mail:

Quality is always an imperative prerequisite when we consider any product. It becomes prime when it relates to life saving products like pharmaceuticals. Although it is mandatory from the government and regulatory bodies but it is also a fact that quality of a pharmaceutical product can not be adequately controlled solely by pharmacopoeial analysis of the final product. Today quality has to be built in to the product right from its inception and rigorous international environmental, safety and regulatory standards need to be followed. Validation had proven to be an important tool for quality management of pharmaceuticals. According to ISO 9000:2000

Validation is defined as "Confirmation, through the provision of objective evidence, that the requirements for a specific intended use or application have been fulfilled". In contrast with Verification, Validation rather focuses on the question whether a system can perform its desired functions. This review is an attempt to prove the it as essential tool for quality management in pharmaceutical industry.


Validation is a concept that has been evolving continuously since its first formal appearance in United States in 1978. The concept of validation has expanded through the years to encompass a wide range of activities from analytical methods used for the quality control of drug substances and products to computerized system for clinical trial, labeling or process control.1

Validation is the overall expression for a sequence of activities in order to demonstrate and document that a specific product can be reliably manufactured by the designed processes, usually, depending on the complexity of today’s pharmaceutical products, the manufacturer must ensure; "that products will be consistently of a quality appropriate to their intended use”. 2

To achieve this with confidence, only in process control and finished product testing alone are not sufficient to assure product quality; but all factors including the services which could affect product quality must be correctly designed, demonstrated to work effectively. Consistently and their performance is also regularly conformed so that consistent quality product is obtained. For example, no sampling plan for applying sterility tests to a specified proportion of discrete units selected from a sterilization load is capable of demonstrating with complete assurance that all of the untested units are infect sterile .

In recent year many manufacture houses have attempted to define their philosophy and strategy for self inspecting their plants for manufacturing ,processing and packing ,including holding of drugs .As much these manufacturers are interpreting the GMP guidelines as evaluated by Food and drug Authority and the schedule M after due modification in 1988.3

A philosophy of performing systematic inspection has worked and may be termed “Drug in- process inspection and validation”. The compliance to their working rules defines a validated manufacturing process as “one has been proven to do what it purport or it represented to do. The proof of validation is obtained through the collection and evaluation of data, preferably beginning from the process development phase and continuing through in to the product phase. Validation necessarily includes process qualification such as materials, equipment, system, building and personnel, but it also includes the control of the entire process for repeated batches or runs.

The word “validation” simply means assessment of validity or action of proving effectiveness. According to European community for medicinal products, validation is action of proving in accordance with the principals of good manufacturing practices, that any procedure, process, equipment, material, activity or system actually leads to expected results.

Validation is a proof that a process works and this must be done using scientific and statically principles. This is done to establish process capability and to confirm product acceptability.4 Validation determined process variables and the acceptable limits for these variables and accordingly sets up appropriate in process controls, which specifies alert and action levels.5


Conducting process validation is not only a regulatory requirement, but also makes a great deal of sense from engineering as well as a business point of view .It is evident that pharmaceutical companies that are well versed in conducting process validation have a competitive advantage over those who are not6. Process validation is required, in both general and specific terms, by the Current Good Manufacturing Practices regulations for finished pharmaceuticals, 21 CFR parts 210 and 2117,8 . A requirement for process validation is set forth in general terms in sections 211.100 written procedures; deviations–which states, in parts; “there shall be written procedures for production and process control designed to assure that the drug products have the identity , strength, quality, and purity they purport or are represented to posses”. Several sections of cGMP regulations states, validation requirement in more specific terms. Excerpts from some of the sections are:-section 211,100, sampling and testing of in –process materials and drug products.

a ) “------------- Control procedures shall be established to monitor the output and validate the performance of those manufacturing process that may be responsible for causing variability in the characteristics of in process material and drug products.”

Section 211, 113, control of microbiological contamination

b) “------------- Appropriate written procedures, design to prevent microbiological contamination of drug products purporting to be sterile, shall be established and followed. Such procedures shall include validation of any sterilization process.”

The requirement of process validation is implicit in the language of schedule M, Good manufacturing practices regulation which states “To achieve the objective, each licensee shall evolve methodology and procedure which should be documented and kept for reference and inspection”.

Process validation is required by the medical device GMP regulation,21 CFR part 820.Section 820.5 requires every finished device manufacturer to states: “……………….. Prepared and implement a quality assurance program that is appropriate to the specific device manufactured……………….”

Section 820.3 states: “………………….All activities necessary to verify confidence in the quality of the process used to manufacture a finished device…………”

A generally stated requirement for process validation is contained in section 820.100, states:

“Written manufacturing specification and processing procedure shall be established, implemented, and controlled to assure that device conforms to its original design or any approved changes in that design”. Validation is an essential element in the establishment and implementation of a process procedure, as well as in determining what process controls are required in order to assure conformance to specification.

Section 820.100(a) (1), states: “Control measures shall be established to assure that the designed basis for device, components and packaging is correctly translated in to approved specification” 9.

{mospagebreak title=Importance and Scope Of Validation}


The most compelling reasons to optimize and validate pharmaceutical productions and supporting processes are quality assurance and cost reduction .the basic principles of quality assurance has as their goal and the production of articles that are fit for there intended use.10 These principles are Quality, safety, and effectiveness must be designed and built in to the product, quality cannot be inspected or tested in the finished products and each step of the manufacturing process must be controlled to maximize the probability that the finished product meets all quality and design specification. The relationship of quality assurance and process validation goes well beyond the responsibility of any quality assurance functions, nevertheless it is fair to say that process validation is a quality assurance tool because it is establishes a quality standard for the specific process.

Quality control is the part of GMP, it is concerned with the sampling specification, testing and with organization documentation and release procedures.11,12 Where as assurance of quality is derived from careful attention to a number of factors including selection of quality materials, equipments, adequate product, process design ,selection of approved vendors, proper GMP inspections , employee training ,technical audit, critical evaluation of market complaints, in-process control of processes, and end product testing.13-20

Process validation should result in fewer product recalls and trouble shooting .process consistently under control requires less process support, will have less down time, fewer batch failures , and may operate more efficiently with greater output .In addition timely and appropriate validation improves quality assurance ,reduces cost by process optimization ,enables more effective and rapid trouble shooting ,shortens lead time leading to low inventories ,empowers all employees to control their processes and to improve them ,enables better system control ,maintains, and improves a high degree of assurance that specific process will consistently produce a product meetings its predetermined specifications and quality characteristics 21,22.


Following are the area were validation can be implied : Analytical test methods, Instrument calibration, Process utility services, Raw materials, Packaging materials, Facilities, Manufacturing, Product design, Cleaning and Operators. 23-26


The activities relating to validation studies are classified into three phases.

Phase 1:

Pre-validation phase or the qualification phase ,which covers all activities relating to product research and development, formulation, pilot batch studies, scale-up studies, transfer of technology to commercial scale batches, establishing stability conditions, storage and handling of in-process and finished dosage form, equipment qualification ,installation qualification, master production documents, operational qualification, process capability.

Phase 2:

Process validation phase (process qualification phase) designed to verify that all established of the critical process parameters are valid and that satisfactory products can be produced even under the worst case conditions.

Phase 3:

Validation maintenance phase requiring frequent review of all process related documents ,including validation audit report to assure that there have been no changes ,deviations, failures modification to production process, and that all SOP’s have been followed including change control procedures. At this stage the validation team also assures that there have been no changes /deviations that should have resulted in requalification and revalidation.


It would normally be expected that process validation be completed prior to the distribution of a finished product that is intended for sale (prospective validation). Where this is not possible, it may be necessary to validate processes during routine production (concurrent validation). Processes which have been in use for some time without any significant changes may also b validated according to an approved protocol (retrospective validation) 27,28.

Prospective validation:

In prospective validation, the validation protocol is executed before the process is put in to commercial use. During the product development phase the production process should be broken down into individual steps. Each step should be evaluated on the basis of experience or theoretical consideration to determine the critical parameters that may affect the quality of finished product. A series of experiment should be design to determine criticality of these factors. Each experiment should be planned and documented fully in an authorized protocol.

All equipment, production environment and the analytical testing methods to be used should have been fully validated. Master batch documents can be prepared only after the critical parameters of the process have been identified and machine settings, component specification and environment conditions have been determined. By using this defined process a series of batches should be produced. In theory, the number of the process runs carried out and observations made should be sufficient to allow the normal extent variation and trends to be established to provide sufficient data for evaluation. It is generally considered acceptable that three consecutive batches/runs with in the final agreed parameters, giving product of the desired quality would constitute a proper validation of the process. In practice, it may take some considerable time to accumulate these data.

Some considerations should be exercised when selecting the process validation strategy. Amongst these should be the use of different lots of active raw materials and major excipients, batches produced on different shifts, the use of different equipments and facilities dedicated of commercial production, operating range of critical process, and a thorough analysis of the process data in case of requalification and revalidation 29,30.

During the processing of the validation batches, extensive sampling and testing should be performed on the product at various stages, and should be documented comprehensively. Detail testing should also be done on the final product in its package.

Upon completion of the review, recommendation should be made on the extent of monitoring and the in-process control necessary for routine production. These should be incorporated into the batch manufacturing record and packaging record or appropriate standard operating procedures. Limits, frequencies and action to be taken in the even to the limits being exceeded should be specified.

Concurrent validation:

In using this approach there is the always the risk of having to modify process parameters or specifications over a period of time .this situation often leads to question regarding disposition of the batches that had already been released for the sale, subsequently known to have undesired quality characteristics.

Concurrent validation may be the practical approach under some circumstance. Example:

· When a previously validated process is being transferred to a third party contract manufacturer or to another manufacturing unit.

· Where the product is different strength of a previously validated product with the same ratio of active/inactive ingredients.

· When the number of lots evaluated under the retrospective validation were not sufficient to obtain a high degree assurance demonstrating that the process is fully under control.

· When the number of batches produced are limited.

It is important in these cases however, that the system and equipment to be used have been fully validated previously. The justification for conducting concurrent validation must be documented and the protocol must be approved by validation team. A report should be prepared and approved prior to the sale of each batch and a final report should be prepared and approved after the completion of all concurrent batches. It is generally considerable acceptable that a minimum of three consecutive batches within the finally agreed parameters giving the product the desired quality would constitute a proper validation of the process.

Retrospective validation:

In many establishments, processes that are stable and in routine use have not under gone a formally documented validation process. Historical data may be utilized to provide necessary documentary evidence that the processes are validated.

The steps involved in this type of validation still require the preparation of a protocol, the reporting of the results of the data review, leading to a conclusion and recommendation.

Retrospective validation is only acceptable for well established detailed process and will be inappropriate where there have been recent changes in the formation of the product, operating procedures, equipments and facility.

The source of data for retrospective validation should include amongst others, batch documents, process control charts ,maintenance log book, process capability studies, finished product test results, including trend analysis, and stability results.

For the purpose of retrospective validation studies, it is considered acceptable that data for a minimum ten consecutive batches produced be utilized. When the less than ten batches are available, it is considered that the data are not sufficient to demonstrate retrospective that the process is fully in control .In such cases the study should be supplemented with concurrent or prospective validation.

Some of the essential elements for retrospective validation are:

  • Batches manufactured for a defined period (minimum of last ten consecutive batches)
  • Number of lots released per year.
  • Batch size /strength /manufacturer /year /period.
  • Master manufacturing/packaging documents.
  • Current specification for active materials/finished products.
  • List of process deviation, corrective actions and changes to manufacturing documents.
  • Data for stability testing for several batches.
  • Trend analysis including those for quality related complaints.

Process Revalidation

Revalidation provides the evidence that change in a process and /or the process environments that are introduced do not adversely affect the process characteristics and product quality. Documentation requirement will be the same as for the initial validation of the process.

Revalidation becomes necessary in certain situations .Some of the changes that require revalidation are as follows.

  • Changes in raw materials properties such as density, viscosity, particle size distribution, moisture, etc. that may affect the process of product.
  • Changes in the sources of active raw material manufacturer.
  • Changes in packing material (primary container/closure system).
  • Changes in the process (such as mixing time, drying temperature, and batch size).
  • Changes in the equipment (e.g. addition of automatic detection system).Changes of equipment which involves the replacement of equipment on a “like for like ’’basis would not requires are validation except that this new equipment must be qualified.
  • Changes in the plant /facility.


All changes must be formally requested documented and accepted by the validation team .The likely impact/risk of the change on the product must assess and the need for the extent of revalidation should be determined .

Commitment of the company to control all change to premises, supporting utilities, system, materials, equipment and process used in the fabrication/packaging of pharmaceutical dosage forms essential to ensure a continued validation status of the system concerned.

The change control system should ensure that all notified or requested changes are satisfactory investigated, documented and authorized. Products made by process subjected to changes should not be released for sale without full awareness and consideration of the changes by the validation team. The team should decide if a. revalidation must be conducted prior to implementation of the proposed change.


The FDA’s guideline defines validation as:

“Establishing documented evidence, which provide a high degree of assurance that a specific process will consistently produce a product meeting with its pre-determined specifications and quality characteristics”.

The development of validation documentation is an essential part of any successful validation programmed or study. The documentation should be concise, unambiguous, detailed, and thorough31,32.

Table 1: Components of a good validation document.

Good validation documentation

A written historical perspective of what was manufactured, filled, cleaned, packaged, how it was done, and which controls were in place.

A way to minimize mistakes and variables.

Provide evidence that “something happened” how, when, and by whom.

Everything you wanted to know but were afraid to ask, written history of product, its components, equipment, and its process before product introduction to market.

Compliance to GMP requirement and ensure reproducibility

Validation takes place within following areas: New : Formula/product, process, procedures, manufacturing, packaging, Changes in : Processing procedures, manufacturing, packaging, cleaning, equipment, computer system and infrastructure and Failures : Revalidation.Within these area, the validation documentation requirements will depend on complexity of the process, project scope, GMP risk (it increases with the complexity of the system), and GAP analysis (define the strategies for achieving goals, identify the weakness).

Key Validation Documents

· Validation master plan (VMP).

·Validation protocols: Installation qualification (IQ), Operational qualification (OQ), Performance qualification (PQ), Computer systems, Facility/utility/equipment qualification protocols, process, packaging, and cleaning.

· Standard operating procedures.

·Optimization batch guidelines.

· Validation reports.

· Change control system 31,33,34.

Importance of the VMP:

The VMP describes clearly and concisely the company’s philosophy, expectations and approach to be followed. It identifies the systems and controls to be validated and the level of testing required. It covers all aspects of the project as equipment qualification, training, maintenance, and change control. It should be developed in the early stages of a project and allow a logical progression from plan to validation schedule. The VMP can also assist in monitoring and tracking the progress of the project by performing periodic audit reviews v/s the approved version of the VMP 31.

Contents of typical VMP:

Following are the contents of VMP : Introduction, Purpose, Scope, Overview /Description of system to be validated, Responsibilities, Validation methodology, Acceptance criteria, Validation report, Change control, Validation project milestone, Deliverables.

Benefits of VMP:

A VMP is created when the project is complex, include high risk ,and when more extensive and thoroughly verification and system review are required. If study is simple involving only one validation study /variables, a validation protocols may be used instead. The benefits of VMP includes, i) It provides the total pictures of the project. ii) It is a management tool for tracking progress. iii) Assignment of responsibility, which promote team work. iv) It identifies acceptance criteria before the start of validation.

The Validation protocol for process, packaging, and cleaning:

The Validation protocol for process, packaging, and cleaning studies is a written plan stating how validation will be conducted including purpose, scope overview/description of system to be validated, responsibilities, validation methodology, acceptance criteria, validation report, change control, required SOPs and decision points on what constitutes acceptable test results.

Format for validation protocol:

·Cover page (approvals)

·Scope of project (which process being validated)

·Objectives /backgrounds


· Installation qualification (IQ), Operational qualification (OQ), Performance qualification (PQ)

· Role and responsibilities.

· SOP’s requirement.

· Process monitoring.

· Sampling and testing.

· Process monitoring.

· Acceptance criteria /test methods.

· Deliverables.

· Documentation requirement

· Additional information-

§ A flow chart of the process

§ Sampling methods to be used

§ In process samples to be collected and details of collection

§ Testing to be conducted on samples collected

§ Sample size ,type of container, and swab techniques

§ Tools and precautions 31,33.

Equipment /Facility /Utility qualification protocols:

The qualification protocols are a very important document of the protocol process. The complexity of the equipment, facility, utility systems, involved and their relationship to the quality of the product dictate the scope ,details, and contents of the qualification protocol. The major component of the qualification protocols are 1,35.

  • Installation qualification (IQ):

Document that the equipment is properly installed according to the manufacturer and purchaser’s specifications. It covers equipment /system descriptions, which includes principle of operation, design requirements, equipment specifications piping, instruments diagrams, facility functional specifications, equipment utility requirements, and equipment safety features.

  • Operational qualification (OQ):

Document that the equipment operates within established limits and tolerances. It covers equipment operation procedures established and challenged equipment control functions, calibration requirements and schedules established, and maintenance requirements.

  • Performance qualification (PQ):

Document, which the equipment can operate reliably as intended for the process under routine, minimum, and maximum operating ranges.

  • Facility /Utility qualification: 31

It involves installation, operation and performance qualification of the building and the equipment. It covers -

  • Plant layout /construction. it includes material flow ,air locks, structure and finishing ,fire safety /alarm system ,manufacturing rooms ,and ware house.
  • Utilities and services. . it includes potable water, cooling water, drainage, plant system, purified water system, compressed air , heating ventilation air conditioning (HVAC) systems.


· Purpose.

· Scope.

· Equipment description includes master list of equipment requiring installation qualification/operational qualification.

· Role and responsibilities.

· Definitions.

· Qualification criteria for IQ methodology/execution, OQ methodology/execution, PQ methodology.

· Deviation reports.

· Acceptance criteria.

· SOP requirements.

· Executive summary report.

· Change control.

· Deliverables.

· Attachments: Raw data sheets, test results, preventive maintenance schedules, test incidence reports, and equipment calibration certifications.

Validation reports:

It summarizes the results, disposition, conclusions, and recommendations of the validation study relative to the protocol / VMP. The main components of the report are as follows 31:

· Cover page.

· Over view.

· Product name.

· Description of the process being validated.

· Location.

· Number of batches being validated.

· Validation study plan.

· Scope.

· Results.

· Discussion.

· Recommendations.

· Conclusion.

· Change control.


The quality assurance of pharmaceutical product involves a number of factors. The complexity of modern day medical products requires more than the routine end product testing, as the end product testing is not sufficient to assure quality of finished product.

The review highlights various aspects on process elements, regulatory requirements, and validation documentation that are considered by regulatory agencies. The particular requirement of process validation will vary according to the nature of the pharmaceutical product and type of process. The broad concepts stated in this review have general applicability and provide an acceptable framework for building a comprehensive approach for the validation.


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18. Iyer, R. S., Pharma Times., (1992), 24, (4), 11-17.

19. Lachman, L., In Hanna, S. A., and Kalrin, The Theory and Practice of Industrial Pharmacy, 3rd ed., 1987, 804-855.

20. Choudary, K. P. R., Sushma, K., The Eastern Pharmacist., (1998), XLI, (488), 51-54.

21. Kiffer, R.G., J.Pharm. Sci. Tech., (1995), 44, (5), 249.

22. Guidelines on General Principles of Process Validation, Food and Drug Administration, Maryland , 1984, 4-25

23. Harder, S. W., In: Encyclopedia of Pharmaceutical Technology, Swarbrick, K. j., Boylan, J. C. (Eds.) Marcel Dekker: New York, 1995, vol. 2, 447-456.

24. Zeller, A. O., Pharm. Tech., (1998), 17, (10), 70-80.

25. Amer, G. and Beane, R. g., Autoclave Qualification: Some Practical Advice,

26. Kaiser, H. J. and Minowitz, Analyzing Cleaning Validation Samples.

27. USA-FDA, Guidelines on General Principles of Process Validation, CEDR, FDA-USA, 1987.

28. Pharmaceutical Inspection Convention Draft Document, Recommendation on Validation Master Plan, Installation and Operational Qualification. Non Sterile Process Validation and Cleaning Validation, 1998.

29. Berry, I., Nash, r., (Eds.), 1993, 2nd ed., Marcel Dekker: New York, 247-257.

30. Essential Drugs and Medicines Policy,

31. Castilla, B. and Sena, F. J., Validation Documentation A Winning Approach,

32. Good Manufacturing Practices for Pharmaceutical Products, In: Who Expert Committee on Specifications for Pharmaceutical Preparations, 32nd Report, Geneva, WHO, (1992), 14-79.

33. Klenzaids GMP Academy, Express Pharma Pulse., (1998), Feb, 12, 15.

34. Klenzaids GMP Academy, Express Pharma Pulse., (1998), Jan., 29.

35. Klenzaids GMP Academy, Express Pharma Pulse., (1998), March, 5,13.

{mospagebreak title=About Authors}

About Authors

Mr. Kamlesh Dashora

Mr. Kamlesh Dashora has nearly 11 years of teaching, research and industrial experience. Mr. Dashora did his masters degree from Department. of Pharmacy, SGSITS, Indore, one of the premier institute of technical education in the central India. He has over 12 publications to his credit published in international and national journals. His research interest extends from Noble topical delivery systems, Delivery Systems for biologicals to regulatory affairs. Presently, he is working at Institute of pharmacy Pt. Ravishankar Shukla University, Raipur, (C.G.) INDIA

Mr. Deependra Singh

Mr. Deependra Singh has nearly 6 years of research and teaching experience. He is a hard working researcher . Mr . Singh did his masters degree at Dept. of Pharmacy, Dr. H. S. Gour University, SAGAR. he has over 16 publications to his credit published in international and national journals. he is an founder secretary of ipa local branch Chhattisgarh. His research interest extends from Noble topical delivery systems, Delivery Systems for biologicals to Plant tissue culture . Presently, he is working as a Lecturer at Institute of pharmacy Pt. Ravishankar Shukla University, Raipur, (C.G.)

Dr. (Mrs). Swarnlata Saraf

Dr. (Mrs). Swarnlata Saraf has nearly 14 years of research and teaching experience. She is a leading scientist and well-known in the field of herbal cosmatics . Dr. Saraf did her doctoral research at the Dept. of Pharmacy, Dr. H. S. Gour University, SAGAR. She has over 40 publications to her credit published in international and national journals. She is an active member of ipa ,apti and iste. Her research interest extends from Herbal Cosmetics to transdermal drug delivery (specially Iontiphoresis), New Drug Delivery Systems for biological therapeutic agents. She has Co-authored 1 books, in press. Presently, she is working as a Reader at Institute of pharmacy Pt. Ravishankar Shukla University, Raipur, (C.G.)

Prof.S. Saraf

Prof. S. Saraf has nearly 17 years of research and teaching experience at both U.G. and P.G. levels. He is a leading scientist and well-known academician . Prof. Saraf did his doctoral research at the Dept. of Pharmacy, Dr. H. S. Gour University, SAGAR. under the supervision of Prof. V. K. Dixit, a renowned Pharmacognosist. He has over 50 research publications to his credit published in international and national journals. He has delivered invited lectures and chaired many sessions in several National Conferences and Symposia in India. His research interest extends from Herbal Cosmetics to Herbal drug standardization Modern analytical techniques, New Drug Delivery Systems with biotechnology bias. He has authored 1 books, in press. Presently, he is Professor and Director Institute of pharmacy and Dean, Faculty of Technology, Pt. Ravishankar Shukla University , Raipur , (C.G.)

The Basic Facts of Cleaning Validation

Robin Fredric, QA-Validation Department, Novopharm Ltd, Canada. Email:

Cleaning validation is primarily applicable to the cleaning of process manufacturing equipment in the pharmaceutical industry. The focus of cleaning validation is those cleaned surfaces that, if inadequately cleaned, could potentially contaminate the product subsequently manufactured in that same equipment. This primarily covers product contact surfaces in the cleaned equipment. Cleaning validation is not performed only to satisfy regulatory authorities. The safety of patients is the primary objective, and product contamination presents serious liability issues for any pharmaceutical manufacturer or contract organization.

The history behind cleaning validation

The unhygienic conditions in Chicago’s meat- packing plants revealed in Upton Sinclair’s novel, “The Jungle”, allowed the government investigators and congress to enact the meat inspection law and the Pure Food and Drugs Act in 1906, the law forbade adulteration, misbranding adulteration, misbranding of foods, drinks, and drugs.
Thirty years later the drug tragedy “elixir of sulfanilamide” which killed over 100 people, greatly dramatized to broaden the existing legislation. On June, 25th 1938 Franklin D. Roosevelt signed the Federal Food, Drug, and Cosmetic Act, it required manufacturers to provide scientific proof of drug safety before it could be marketed.
All these events brought the current regulatory requirements for cleaning validation.


Cleaning can be defined as removal of residues and contaminants. The residues and contaminants can be the product themselves manufactured in the equipment or residues originating from the cleaning procedure (detergents / sanitizers) or degradation products resulting from the cleaning process itself.

The basic mechanisms involved in removing the residues and contaminants from the equipment are mechanical action, dissolution, detergency and chemical reaction.

1.Mechanical action – It refers to the removal of residues and contaminants through physical actions such as brushing, scrubbing and using pressurized water.

2.Dissolution – It involves dissolving the residues with a suitable solvent. The most common and practical solvent is water being non-toxic, economical, environment friendly and does not leave any residues. Alkaline and acidic solvents are sometimes preferred as it enhances the dissolution of the material, which are difficult to remove.

3.Detergency-Detergent acts in four ways as wetting agent, solubilizer, emulsifier and dispersant in removing the residues and contaminants from the equipment

4.Chemical reaction- Oxidation and hydrolysis reaction chemically breaks the organic residues and contaminant to make them readily removable from the equipment

What is cleaning validation ?

It is documented evidence with a high degree of assurance that one can consistently clean a system or a piece of equipment to predetermined and acceptable limits.

Why cleaning validation ?

To verify the effectiveness of cleaning procedures and to ensure no risks are associated with cross contamination of active ingredients or detergent/sanitizer.

When cleaning validation ?

· Initial qualification of a process/equipment
· Critical change in a cleaning procedure
· Critical change in formulation
· Significant change in equipment
· Change in a cleaning process
· Change in a cleaning agent.

Why we do validation for 3 times ?

Once an FDA was asked why do we do it 3 times?
His answer was - Because if it comes out right once it is an accident, twice coincident, three times validation.

Regulatory requirements:

FDA has required that the equipment to be cleaned prior to use (GMP regulation-Part 133.4) This is one of the basic GMP requirement and it is indicated in more than one section of 21CFR 211 (FDA, April 1998)
• Section 211.63 relates to the equipment design, size, location, and requires that equipment used in the manufacture, processing, packaging, holding of a drug product shall be of appropriate design, adequate size, and suitably located to facilitate operations for its intended use and for its cleaning and maintenance.
• Section 211.65 states that a) the construction of equipment which contact the in-process materials, or drug products shall not be reactive, additive or absorptive so as to alter the safety, identity, strength, quality or purity of the drug product beyond official or other establishment requirements.
b) Any substances required for operation, such as lubricants or coolants, shall not come into contact with components, drug product containers, closures, in-process materials, or drug products so as to alter the safety, identity, strength, quality or purity of the drug product beyond official or other establishment requirements.
• Section 211.67 further requires that the equipment and the utensils shall be cleaned, maintained and sanitized at appropriate intervals to prevent malfunctions or contamination that would alter the safety, identity, strength, quality or purity of the drug product in form of written procedure including all the parameters during cleaning.
• Section 211.180 and 211.182 relates to the record that should be kept for the maintenance, cleaning, sanitation and inspection of equipment.

The Common elements of Cleaning Validation

· Written cleaning procedures should be established. Attention should be addressed to dedicate certain equipment to specific products, such as fluid bed dryer bags and to residue originating from the cleaning detergent or solvent themselves.

· Procedure on how validation will be performed should be in place.

· Who is responsible for performing and approving the study.

· Acceptance criteria should be set.

· Procedure dealing with the subject of when revalidation study stating issues such as sampling procedure and analytical methods.

· Study should be conducted according to protocol.

· Approved report should state the validity of the cleaning process.

Cleaning procedure

The two common cleaning procedures are,

· Manual cleaning

· Automated cleaning procedures such as CIP (Cleaning In Place

Manual Cleaning Sequence
CIP Cleaning Sequence
Dismantle the parts of equipment to be cleaned
Pre-wash the parts in tap water
Pre-wash the parts with tap water
Wash the pre-washed parts with cleaning solution
Wash the pre-washed parts with cleaning solution
Blow out using compressed air
Rinse the parts in tap water
Rinse the parts with tap water
Rinse now with purified water
Final rinse using purified water
Dry the parts using hot air
Blow out using compressed air
Visual inspection is done to check whether the equipment is clean
Drying using hot and compressed air
Reassemble the parts finally

In all cases cleaning procedure must prove to be effective, consistent and reproducible.

FDA recommends (CIP) should be used to clean process equipment and storage vessels in order to reproduce exactly the same procedure each time (FDA, March 1998).

With manual procedure one must rely on the operator skills and thorough training of the operator is necessary to avoid variability in performance. However in some instances, it may be more practical to use only manual procedures.

Sampling methods for Cleaning Validation

There are three known sampling methods:

1.Swabbing (or direct surface sampling) method

2.Rinse sampling method

3.Placebo method.

Swabbing technique involves the use of a swabbing material, often saturated with solvent, to physically sample the surfaces.


· Dissolves and physically removes sample

· Adaptable to a wide variety of surfaces

· Economical and widely available

· May allow sampling of a defined area

· Applicable to active, microbial, and cleaning agent residues


·An invasive technique that may introduce fibres

·Results may be technique dependent

·Swab material and design may inhibit recovery and specificity of the method

·Evaluation of large, complex and hard to reach areas difficult (e.g., crevices, pipes, valves, large vessels)

·Subject to the vagaries of site selection

Rinse Sampling involves passing a known volume of solution over a large area and analyzing the recovery solution.


·Adaptable to on-line monitoring

· Easy to sample

· Non-intrusive

· Less technique dependent than swabs

· Applicable for actives, cleaning agents and excipients

· Allows sampling of a large surface area

· Allows sampling of unique (e.g., porus) surfaces


· Limited information about actual surface cleanliness in some cases

· May lower test sensitivity

· Residues may not be homogeneously distributed

· Inability to detect location of residues

· Rinse volume is critical to ensure accurate interpretation of results

· Sampling methodology must be defined since rinse sampling method and location can influence results

· May be difficult to accurately define and control the areas sampled, therefore usually used for rinsing an entire piece of equipment, such as a vessel

· Reduced physical sampling of the surface

Placebo sampling can be used to detect residues on equipment through the processing of a placebo batch subsequent to the cleaning process. It is appropriate for active residue, cleaning agent, particulates and microbial testing. Placebos are used primarily to demonstrate the lack of carryover to the next product. The placebo should mimic product attributes. The equipment characteristics also impact the choice of the placebo batch size.


· Placebo contacts the same surfaces as the product

· Applicable for hard-to-reach surfaces

· Requires no additional sampling steps


· Difficult to determine recovery (contaminants may not be evenly distributed in the placebo)

· Lowers analytical specificity and inhibits detectability

· Takes longer and adds expense since equipment must be cleaned after the placebo run

· Placebos must be appropriate for each potential product

· Residues may not be homogenously distributed

· No direct measurement of residues on product contact surfaces

The preferred sampling method and the one considered as the most acceptable be regulatory authorities is the swabbing method.

The Common analytical methods and their basic requirements

Specific and non-specific are the two analytical methods used widely to detect any compound. The choice of using a specific or non specific method can be difficult. If a drug active is highly toxic, a specific method is always recommended.

Chromatographic methods are preferred for cleaning validation studies because of their sensitivity, specificity, and ability to quantify.

Specific method:

It is a method that detects a unique compound in the presence of potential contaminants.

Some examples of specific methods are high performance liquid chromatography (HPLC), Ion chromatography, Atomic absorption, Capillary electrophoresis, and other chromatographic methods.

Non-specific method:

It detects any compound that produces a certain response.

Some examples of non specific methods are Total Organic Carbon (TOC), pH, Titration, and conductivity.

It is always wise to choose the simplest technique that can be used to reach the desired goal.

The basic requirement for the analytical method

The sensitivity of the method shall be appropriate to the calculated contamination limit.

The method shall be practical and rapid, and, as much as possible use instrumentation existing in the company.

The method shall be validated in accordance with ICH, USP, EP requirements.

The analytical development shall include a recovery study to challenge the sampling and testing methods.

An introduction to analytical method validation

Mr. Bhavadip B. Tanna

For Pharmaceutical drug analysis analytical method validation is very important parameter. So, there is a need for the study of validation parameters.

The analytical procedure refers to the way of performing the analysis. Analytical method validation is required for herbal procedure,new process and reaction,new molecules, active ingredients,residues, impurity profiling and component of interest in different matrices.An analytical methodology consists of the techniques,method , procedure and protocol.this methodology the required data for a given analytical problem, required sensitivity,required accuracy,required range of analysis and required precision to the analyst.It is required for assuring quality,achiving acceptance of products by the international agencies, mandatory requirment purposes for accreditation as per ISO 17025 guidelines,mandatory requirment for registration of any pharmaceutical product or pesticide formulation.The main objective is to demonstrate that the procedure is suitable for its intended purpose.the method validation studies for the developed methods for various parametersas per protocol and guidelines which are EN 45000 series of standards,ISO\ IEC Guide 25, Internation conference on harmonization (ICH) , US EPA, USP, published litreture.There are 4 types of analytical procedures which includes identification testes, impurity tests, limit tests and potency tests.Identification testes which normally compares that sample under evalution with a known reference sample standard with spectrographic or chromatographic methods. Impurity tests may be either quantitative or limit test and different validation requirements applied. For limit testes, specificity and detection limits only may required.For assay of actives or other key components of drug products potency tests is performed.

The validation parameters that should be considered during validation of analytical procedures are shown as:

§ Specificity: It conforms the ability of the methods to evalute the desired analyte in the presence of known other components like degradants, impurities, potential contamination and excipients.It is not possible to demonstrate that an analytical procedures is specific for a particular analyte. In such case a combination of two or more analytical procedure is recommended to achieve the necessary level of discrimination.lack of specificity of an individual analytical procedure may be compensated by other supporting analytical procedures or tests.

§Accuracy: The accuracy of analytical procedure expresses the closeness of agreement between the values which is accepted either as conventional true value or an accepted reference value in the value found which is sometimes called as trueness.It should be established across the specified range of procedure.

§ Precision: The procedure of an analytical procedure expresses the closeness of agreement (degree of scatter) between a series of measurements obtained from multiple sampling of the same homogeneous sample under the prescribed condition.

It may be considered at 3 levels:

~~Repeatability: It expresses the precision under the same operating conditions over a short interval of time .It is also known as inta-assay precision.

~~Inter mediate precision: It express within laboratory variations like different analysts, days and equipments.

~~Reproducibility: It expresses the precision between laboratories.There are variations like differences in room temperature and humidity, operating with different expertience and thoroughness,variation in material and instrument condition , equipment and consumable of different ages affecting a method’s reproducibility.

§ Limit of Detection (LOD): The detection of an individual analysis procedure is the lowest amount of analyte in a sample which can be detected but not necessarily quantify as an exact value. It may be determined by the analysis of sample with known concentrations of the analyte and by establishing the minimum level at which the analyte can be reliably detected.

Based on standard deviation of the response and the slope it is shown as:

DL= 3(SD/Slope)

§ Limit of quantification(LOQ) :The quantification limit of an individual analytical procedure is the lowest amount of analyte in a sample which can be quantitatively determine with suitable precision and accuracy .It is used particularly for the determination of impurities and degradation products. It is shown as:


§ Linearity : The linearity of an analytical procedure is its ability to obtain test results which are directly propotional to the concentration of analyte in the sample.Minimum 5 concentrations are recommended for the establshiment of linearity.

§ Range: The range of an analytical procedure is the interval between the upper and lower concentration of analyte in the sample for which it has been demonstrated that the analytical procedure has a suitable level of precision , accuracy and linearity.

§ Robustness : The robustness of analytical procedure is a measure of Its capacity to remain unaffected by small,but deliberate variations(e.g.stability of analytical solutions, extraction time ,etc.) in method parameters and provide an indication of its reliability during normal usage.

§ System Suitability Testing: It is an integral part of many analytical procedure.The tests are based in the concept that equipment electronics , analytical operations and sample to be analysed constitute an integral system that can be evaluated as such.


operating ranges should be defined for each method based on experience with similar methods. The availability of such operating ranges makes it easier to decide when a method should be revalidated.It is necessary whenever a method is changed and the new parameters is outside the operating range.For example , a revalidation is necessary if a high –performance liquid chromatography method has been developed and validated on a pump with a delay volume of 5ml and the new pump only has 0.5ml


The efficient development and validation of analytical methods are critical element in the development of pharmaceuticals. Success in this area can be attributed to several important factors,which in turn will contribute to regulatory compliance.A strong mmenotering and training program is an important factor for ensuring successful methods development and validation.


1. http://www.shriraminstitute .org

2. S.Iyer; “Guidelines on cGMP AND QUALITY OF PHARMACEUTICAL PRODCUCTS”; D. K. publications; First Edition ; January 2003 ; pg no. 145-157

3. Sidney H. Willing and James R. Stokar; “Good Manufacturing Practices for Total Quality Control”; Fourth Edition, Revised and expanded; MARCEL DEKKER INC.; 141-143

4. World Health Organization – Geneva “ Quality assurance of Pharmaceutical – A Compendium of guidelines and related materials”; Pharma Book Syndicate ; Volume -1 ; 119-123

5.Robert A. Nash and Alfred H. Wachter ; “ Pharmaceutical Process Validation; An International Third Edition ‘ Revised and expanded ; MARCEL DEKKER INC.; 507-522




About Authors:

Mr. Bhavadip B. Tanna

Bhavadip B. Tanna

Student, Final Year B.Pharm, Maliba Pharmacy College, Tarsadi, Surat – 394 350, Gujarat, India

Nilesh K. Patel

Nilesh K. Patel

M.Pharm.,Lecturer, Department of Quality Assurance, Maliba Pharmacy College, Tarsadi, Surat-394350, Gujarat, India
e-mail :

Mr. Bhavin P. Marolia

Mr. Bhavin P. Marolia

M.Pharm, Lecturer, Department of Quality Assurance, Maliba Pharmacy College, Tarsadi, Surat-394350, Gujarat, India
e-mail :

Dr. Dinesh R. Shah

Dr. Dinesh R. Shah

M.Pharm., Ph. D., Professor, Department of Quality Assurance, Maliba Pharmacy College, Tarsadi, Surat-394350, Gujarat, India

Validation: An Essentiality In The Pharmacy

Tarun Virmani

The development of a drug product is a lengthy process involving drug discovery, laboratory testing, animal studies, clinical trials and regulatory registration.

To further enhance the effectiveness and safety of the drug product after approval, many regulatory agencies such as the United States Food and Drug Administration (FDA) also require that the drug product be tested for its identity, strength, quality, purity and stability before it can be released for use. For this reason, pharmaceutical validation and process controls are important in spite of the problems that may be encountered1. Process controls include raw materials inspection, in-process controls and targets for final product. The purpose is to monitor the on-line and off-line performance of the manufacturing process and then validate it. Even after the manufacturing process is validated, current good manufacturing practice also requires that a well written procedure for process controls is established to monitor its performance2. This paper provides an overview of pharmaceutical validation and process controls in drug development. The validation concept can be applied to new drugs, new dosage forms and generic drug development.

Essentials of Pharmaceutical Validation

Validation is an integral part of quality assurance; it involves the systematic study of systems, facilities and processes aimed at determining whether they perform their intended functions adequately and consistently as specified. A validated process is one which has been demonstrated to provide a high degree of assurance that uniform batches will be produced that meet the required specifications and has therefore been formally approved. Validation in itself does not improve processes but confirms that the processes have been properly developed and are under control3. Adequate validation is beneficial to the manufacturer in many ways:

  • It deepens the understanding of processes; decreases the risk of preventing problems and thus assures the smooth running of the process.
  • It decreases the risk of defect costs.
  • It decreases the risk of regulatory noncompliance.
  • A fully validated process may require less in-process controls and end product testing.

Validation should thus be considered in the following situations:

  • Totally new process
  • New equipment
  • Process and equipment which have been altered to suit changing priorities and
  • Process where the end-product test is poor and an unreliable indicator of product quality.

When any new manufacturing formula or method of preparation is adopted, steps should be taken to demonstrate its suitability for routine processing. The defined process should be shown to yield a product consistent with the required quality. In this phase, the extent to which deviations from chosen parameters can influence product quality should also be evaluated. When certain processes or products have been validated during the development stage, it is not always necessary to revalidate the whole process or product if similar equipment is used or similar products have been produced, provided that the final product conforms to the in-process controls and final product specification. There should be a clear distinction between in-process control and validation. In production, tests are performed each time on a batch to batch basis using specifications and methods devised during the development phase. The objective is to monitor the process continuously4.

Major Phases in Validation

The activities relating to validation studies may be classified into three:

Phase 1:

This is the Pre-validation Qualification Phase which covers all activities relating to product research and development, formulation pilot batch studies, scale-up studies, transfer of technology to commercial scale batches, establishing stability conditions and storage, and handling of in-process and finished dosage forms, equipment qualification, installation qualification, master production document, operational qualification and process capacity.

Phase 2:

This is the Process Validation Phase. It is designed to verify that all established limits of the critical process parameter are valid and that satisfactory products can be produced even under the worst conditions.

Phase 3:

Known as the Validation Maintenance Phase, it requires frequent review of all process related documents, including validation of audit reports, to assure that there have been no changes, deviations, failures and modifications to the production process and that all standard operating procedures (SOPs), including change control procedures, have been followed. At this stage, the validation team comprising of individuals representing all major departments also assures that there have been no changes/deviations that should have resulted in requalification and revalidation. A careful design and validation of systems and process controls can establish a high degree of confidence that all lots or batches produced will meet their intended specifications. It is assumed that throughout manufacturing and control, operations are conducted in accordance with the principle of good manufacturing practice (GMP) both in general and in specific reference to sterile product manufacture5. The validation steps recommended in GMP guidelines can be summarized as follows:

  • As a pre-requisite, all studies should be conducted in accordance with a detailed, pre-established protocol or series of protocols, which in turn is subject to formal – change control procedures.
  • Both the personnel conducting the studies and those running the process being studied should be appropriately trained and qualified and be suitable and competent to perform the task assigned to them.
  • All data generated during the course of studies should be formally reviewed and certified as evaluated against pre-determined criteria.
  • Suitable testing facilities, equipment, instruments and methodology should be available.
  • Suitable clean room facilities should be available in both the ‘local’ and background environment. There should be assurance that the clean room environment as specified is secured through initial commissioning (qualification) and subsequently through the implementation of a programme of re-testing – in-process equipment should be properly installed, qualified and maintained.
  • When appropriate attention has been paid to the above, the process, if aseptic, may be validated by means of “process simulation” studies.
  • The process should be revalidated at intervals and
  • Comprehensive documentation should be available to define support and record the overall validation process6.

Process Validation

Process validation is the means of ensuring and providing documentary evidence that processes (within their specified design parameters) are capable of repeatedly and reliably producing a finished product of the required quality5. It would normally be expected that process validation be completed prior to the release of the finished product for sale (prospective validation). Where this is not possible, it may be necessary to validate processes during routine production (concurrent validation). Processes, which have been in use for some time without any significant changes, may also be validated according to an approved protocol (retrospective validation) 6-13.

Pre-requisites for Process Validation

Before process validation can be started, manufacturing equipment and control instruments as well as the formulation must be qualified. The information on a pharmaceutical product should be studied in detail and qualified at the development stage, i.e., before an application for marketing authorization is submitted. This involves studies on the compatibility of active ingredients and recipients, and of final drug product and packaging materials, stability studies, etc. Other aspects of manufacture must be validated including critical services (water, air, nitrogen, power supply, etc.) and supporting operations such as equipment cleaning and sanitation of premises. Proper

training and motivation of personnel are prerequisites to successful validation14-16.

Process validation decision

The following model may be useful in determining whether or not a process should be validated:

Process validation decision tree

Figure 1 - Process validation decision tree

The model shown describes a decision tree that a manufacturer can follow when deciding on whether a process needs to be validated. The process under consideration in this model is the simplest possible - many processes may be large and/or a complex set of sub-processes.

Each process should have a specification describing both the process parameters and the output desired. The manufacturer should consider whether the output can be verified by subsequent monitoring or measurement (A). If the answer is positive, then the consideration should be made as to whether or not verification alone is sufficient to eliminate unacceptable risk and is a cost effective solution (B). If yes, the output should be verified and the process should be appropriately controlled (C). If the output of the process is not verifiable then the decision should be to validate the process (D); alternatively, it may become apparent that the product or process should be redesigned to reduce variation and improve the product or process (E). Also, a change in a manufacturing process may result in the need for process validation even though the process formerly only required verification and control. The risk or cost may also be reduced by redesigning the product or process to a point where simple verification is an acceptable decision (E).

The Pharmaceutical Process Equipment

The key idea of validation is to provide a high level of documented evidence that the equipment and the process conform to a written standard. The level (or depth) is dictated by the complexity of the system or equipment. The validation package must provide the necessary information and test procedures required to provide that the system and process meet specified requirements. Validation of pharmaceutical process equipment involves the following:

  • Installation Qualification

This ensures that all major processing and packaging equipment, and ancillary systems are in conformity with installation specification, equipment manuals schematics and engineering drawing. It verifies that the equipment has been installed in accordance with manufacturers recommendation in a proper manner and placed in an environment suitable for its intended purpose.

  • Operational Qualification

This is done to provide a high degree of assurance that the equipment functions as intended. Operational qualification should be conducted in two stages:

    1. Component Operational Qualification, of which calibration can be considered a large part.
    1. System Operational Qualification to determine if the entire system operates as an integrated whole.
  • Process Performance Qualification

This verifies that the system is repeatable and is consistently producing a quality product. These exercises assure, through appropriate performance lists and related documentation, that equipment, ancillary systems and sub-systems have been commissioned correctly. The end results are that all future operations will be reliable and within prescribed operational limits. At various stages in a validation exercise there are needs for protocols, documentation, procedures, specifications and acceptance criteria for test results. All these need to be reviewed, checked and authorized. It would be expected that representatives from the professional disciplines, e.g., engineering, research and development, manufacturing, quality control and quality assurance are actively involved in these undertakings with the final authorization given by a validation team or the quality assurance representative17.


It is necessary, before approval of a new drug, that an accurate and reliable assessment for its effectiveness and safety for the intended indication and target patient population is demonstrated. Pharmaceutical validation which includes assay validation, cleaning validation, equipment validation as well as the overall process validation is crucial in stability analysis, animal studies and early phases of clinical development such as bioavailability/bioequivalence studies. After the drug is approved, pharmaceutical validation and process control are necessary to ensure that the drug product will meet/set pharmaceutical standards for identity, strength, quality, purity, stability, evaluation safety and efficacy. In general, pharmaceutical validation and process control provide a certain assurance of batch uniformity and integrity of the product manufactured.


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15. LeBlane DA. Establishing scientifically justified acceptance criteria for cleaning validation of finished drug product. Pharm Technol 1998; 23(10): 136-48.

16. WHO Expert Committee on Specifications for Pharmaceutical Preparations, 34th Report. WHO Technical Report Series no. 863, Annex 6, Geneva: WHO, 1966, pp 80-96.

17. Good Manufacturing Practices for Pharmaceutical Products, WHO/Pharm./93.562/Annex: Guidelines on Validation of Manufacturing Process. Geneva: WHO.

About Authors:

Tarun Virmani

Tarun Virmani

M.Pharm (Pharmaceutics), Lecturer, Department of Pharmaceutics, Rajiv Academy for Pharmacy, Mathura, Delhi Mathura bypass, Chhattikara 281006, India
Corresponding author –, Phone number - (+919412576525) , Fax number – 0565 -2530766

Kamla Pathak

Kamla Pathak

M.Pharm (Pharmaceutics),Ph.D.,Department of Pharmaceutics , Rajiv Academy for Pharmacy, Mathura Delhi Mathura bypass, Chhattikara 281006, India

Thursday, 11 February 2010

Validation of Microbiological Tests

The variety of microbiological tests makes it difficult, if not impossible, to prescribe a single, comprehensive method for validating all types of tests. By their very nature, microbiological tests possess properties that make them different from chemical tests. Consequently, the well-known procedures for validating chemical tests are not appropriate for many microbiological tests. Yet, it is necessary to validate microbiological tests if they are to be useful for controlling the quality of drug products and devices. Test-method validation provides assurance that a method is suitable for its intended use. Given this definition, any rational company would want to be sure that its methods are validated.

Some tests, such as bioburden or viral titer tests, are quantitative in nature while other tests, such as those for the presence of objectionable organisms, are qualitative. As with chemical tests, these differences necessitate different validation approaches. The purpose of a test also may change the procedures for running and validating it. As an example, consider a drug that will be orally administered. Normally, sterility is not a major issue, and the specification allows for a considerable number of organisms. However, if the drug will be administered to immunocompromised cancer or AIDS patients, the bioburden level must be reduced considerably, increasing the test sensitivity required in the validation study.

The nature of the test material itself changes how a test is run and the validation protocol. Consequently, testing for objectionable organisms is different when testing a diuretic for hypertension or an antibiotic for treating pneumonia. Also, a procedure that works perfectly well for checking the bioburden of granulated sugars may fail with sodium chloride. These differences make full coverage of the topic impossible within the context of this primer. This article will present the general considerations that apply to most microbiological tests. However, three excellent publications are available to analysts preparing validation study protocols for microbiological methods (see Suggested Reading).

Note also that certain microbiological tests are already associated with well defined validation procedures. For example, the endotoxin test and USP bacterial enumeration tests have clearly defined validation procedures. In addition, individual countries may have specific requirements that modify or change standard procedures. If a test is associated with a compendial or regulatory validation procedure, workers are advised to follow that procedure unless there are clear reasons for not doing so. In such cases, the reasons should be documented and filed with the test procedure.

Media The suitability of the medium used for cultivating organisms or cells obviously can have a major impact on the test results. Some organisms are extremely fastidious and require a precisely defined medium with several complex nutrients, while others grow in the presence of inorganic salt mixtures and simple carbon sources. It is commonly argued that delicate, fastidious organisms cannot survive manufacturing processes and should not be of concern, but organisms as delicate and fastidious as mycoplasmas can appear in final preparations of biologics.

In addition to the nutrient composition of the media, more general factors such as pH and ionic strength must be validated. While it is commonly believed that media in the range of pH 6.0 – 8.0 are suitable for sterility and bioburden studies, individual organisms may require a more restricted range. The same holds true for ionic strengths and osmolalities outside of the human physiological range. Shifting the pH range from 6.0 – 7.0 to 7.0– 8.0 and raising the ionic strength to 300 mOsm may select for a different set of organisms than those that would be present in the lower pH range at 150 mOsm.

Most validation schemes require the use of five or more "indicator organisms" to demonstrate the medium's ability to support growth. In addition to aerobic bacteria, anaerobic organisms, yeasts, and molds are usually included. This is an important step since a finding of "no growth detected" is meaningless if the medium was incapable of growing any organisms. This leads to two important points.

First, the indicator organisms are supposedly representative of the types of organisms that will be encountered during the testing, but this is not necessarily true. The indicator organisms are a subset of organisms that are known to grow on properly prepared media, but the organisms contaminating a manufacturing process may not belong to that subset. As a result the quality control laboratory may repeatedly face what appears to be a microbial contamination event despite monitoring cultures that show no growth. It is very important to know what organisms are normally present in the working environment and to include these environmental isolates in a validation program. There is little value in proving that a medium will support the growth of indicator organisms if the environment is full of organisms with very different cultivation requirements.

The second issue involves media handling. The qualification or validation study may require autoclaving the medium and then pouring culture plates as the autoclaved material cools. In laboratories with a low testing load, the excess material is often poured into large tubes or culture flasks to cool and solidify and then stored for future use, usually in a refrigerator. However, when future testing is done, the second heating of the medium may not be captured in the qualification or validation check and may not even be mentioned in the test procedure. If the agar is melted under gentle conditions and quickly poured, there may be no problem, but in some cases, technicians have placed the flasks in microwave ovens to heat the medium while taking a short break. With a powerful microwave oven it is easy to boil the medium for an unknown period of time. This can destroy nutrients or produce toxic or inhibitory substances. Consequently, in laboratories where this second heating is a common practice, this procedure must be captured in the validation and described exactly in the test procedures.

When preparing the validation protocol, the analyst should specify the recovery level expected for each of the indicator organisms. Generally, recovery of at least 80% of the inoculum or control is desirable. Recovery of less than 50% is usually unacceptable and should raise questions about the presence of inhibitory substances, especially when the testing is taking place in the presence of a raw material or product intermediate. It may be necessary to introduce — and validate the performance of — an agent that inactivates the inhibitor. It is important to set the specifications before the study is conducted and to hold to these specifications. If specifications are not pre-set and the test system cannot meet general acceptance specifications, it is very easy to set "acceptable" specifications that would otherwise have been unacceptable. The other problem is the "specification creep" that occurs when a recovery of 78% is found and the specification is 80%. A quality assurance or quality control worker who allows the 78% to pass will soon face the expectation that 75% should pass because it is "only slightly different from the other one." Over the course of a few years, an 80% specification can gradually turn into a 70%, then 65%, specification.

Environment The incubation temperature can have a major effect on the ability of an organism to grow in a given medium. It is well known that yeasts and molds require a different incubation temperature than bacteria in a sterility test. Similarly, cells in tissue culture are often extremely sensitive to small changes in temperature, not only for their growth but also in their susceptibility to being infected or lysed by viruses. The analyst may need to develop temperature curves to justify the incubation temperatures used for the test. It is also important to verify the incubator's ability to maintain the set temperature within the specified range. If a four-degree temperature variation can cause a significant change in the test results, the incubator's ability to hold a ±1° C range at all internal locations is critical. This may not be covered in a validation study, but it should be included in the incubator's qualification studies.

In addition to the usual range from 20 – 40° C, it may be necessary to demonstrate the ability to grow organisms at extreme temperatures. If it is necessary to monitor the presence of microbes in a hot or cold room, it will be necessary to demonstrate an ability to cultivate thermophiles or psychrophiles in addition to organisms that grow under more normal conditions. While the significance of these extremophiles may be open to question, their presence and the possibility that they may leave residues such as endotoxins must be considered.

The atmosphere in which the test system is immersed can have a major effect. Anaerobic organisms cannot grow in the presence of oxygen, and tissue cultures may require the presence of 5% CO2 to grow well. Certain facultative organisms will adjust their metabolic paths to cope with reduced levels of oxygen. This, in turn, can affect their growth rates. When media for general purposes, such as sterility tests, are being considered, it is normal to include one medium that provides anaerobic conditions. The detection of anaerobes is important as they include toxin-producing and other pathogenic bacteria.

Quantitative Issues One of the problems with quantitative microbiological tests is that as microbe counts become smaller, straight-forward linear behavior is less common than that which follows the Poisson distribution. This is because random distribution is not even distribution. Most quantitative tests for microorganisms require the plating of dilute liquid samples, and it is normal to prepare samples to ensure the dispersion of microbes and a random distribution of bacteria or viruses. When concentrations are high, the lack of even distribution is not a problem; simple linear averaging methods can compensate for the uneven distribution. Problems arise with smaller numbers of microbes.

Consider an example where there are exactly 100,000 organisms per mL. If 0.1 mL is taken and mixed with 0.9 mL of a diluent, it is highly unlikely that the new suspension will contain exactly 10,000 organisms; it would not be surprising to have anywhere from 9,800 – 10,200 organisms. Back-calculating the result produces a range from 98,000 – 102,000 organisms in the original sample, and, if there were enough replicates, the results could be averaged to obtain a number indistinguishable from 100,000. This is the result that would be expected based on linear thinking.

However, if there were only 10 organisms per mL, it is quite possible that a 0.1 mL aliquot would not contain any organisms at all. In fact, in this situation about one third of the aliquots will not contain a single organism. This could lead to the conclusion, on averaging, that the sample only contained 6.7 organisms per mL, which is a significant deviation from the true value.

A transition occurred from a high density that produces a fairly smooth, homogeneous distribution of organisms to a low density that results in organisms that are distributed with significant distances between them. Under these conditions, the suspension behaves according to the Poisson distribution and assumptions related to a normal distribution no longer hold. The Poisson distribution is an exponential function. The problem is that parameters such as the standard deviations may be logarithmic in nature, and when attempts are made to make these numbers "real" by taking the antilogarithms, the results may actually have no "real" meaning. This can cause great difficulties when attempting to validate quantitative microbial test procedures.

When it is necessary to deal with the Poisson distribution, it is wise to consult a statistician who is versed in the use of this distribution. It appears that the transition to the Poisson distribution occurs when approximately 100 colonies or plaques are counted. This is unfortunate because at this level many analysts will declare a colony or plaque count to be "too numerous to count" (TNTC) to avoid the tedium of these measurements. Therefore, most colony or plaque counting procedures actually operate under the Poisson distribution and calculations based on the normal distribution will be incorrect.

Revalidation The frequency of revalidation is a contentious question. There are many tests, such as the growth promotion test on culture media, that are essentially self-validating and are run frequently. It could be argued that if performance parameters (for example, percent recovery of indicator organisms) are monitored via control charting and no significant changes are seen, revalidation is unnecessary. However, control charting usually does not measure all the parameters included in validation studies. Consequently, it is wise to revalidate tests after any major change in constituents or procedures; in fact, revalidation may be needed to justify the changes. Changes in suppliers (especially of media components) and changes in the composition of test samples have resulted in major changes in microbiological tests. Finally, it is probably wise to revalidate procedures approximately every second year to protect against unseen or unreported changes. A media supplier may change its own suppliers or change its processing procedures without notifying customers. The supplier may have no idea of the impact these changes could have on the end use of their product. In addition, personnel changes in the laboratory and the maturing of analysts' techniques can have an effect.

Suggested Reading Carroll MC. A multifaceted look at the microbial limits test. In: R Prince, editor. Microbiology in Pharmaceutical Manufacturing. Baltimore, MD: PDA; 2001. pp. 519–535.

PDA. Evaluation, validation and implementation of new microbiological testing methods: PDA Technical Report No. 33. PDA Journal of Pharmaceutical Science and Technology 2000; 54(Suppl. TR33).

Petitti DM. Practical considerations for the development, validation, and transfer of analytical test methods. In: R Prince, editor. Microbiology in Pharmaceutical Manufacturing. Baltimore, MD: PDA; 2001. pp. 723–746.

Steven S. Kuwahara, Ph.D., is the principal consultant and founder of GXP BioTechnology LLC, PMB 506, 1669-2 Hollenbeck Avenue, Sunnyvale, CA 94087-5042, 408.530.9338