IVTMethVal12051 PREPARING FOR A METHOD VALIDATION Steven S. Kuwahara, Ph.D. GXP BioTechnology, LLC...

IVTMethVal1205IVTMethVal1205 11

PREPARING FOR A METHOD PREPARING FOR A METHOD VALIDATIONVALIDATION

Steven S. Kuwahara, Ph.D.Steven S. Kuwahara, Ph.D.

GXP BioTechnology, LLCGXP BioTechnology, LLCPMB 506, 1669-2 Hollenbeck AvenuePMB 506, 1669-2 Hollenbeck Avenue

Sunnyvale, CA 94087-5042Sunnyvale, CA 94087-5042Tel. & FAX: (408) 530-9338Tel. & FAX: (408) 530-9338

e-Mail: e-Mail: [email protected]@yahoo.comWebsite: www.gxpbiotech.orgWebsite: www.gxpbiotech.org


GENERAL I.GENERAL I.

• In general, when a validation is planned, there should be enough information available that the workers will have reason to believe that the validation will be successful.

• The method validation study should not be used to “discover” the assay parameters such as accuracy, precision, and linearity.

• The validation study should confirm proper performance under normal conditions of use or suitability for the intended use under normal conditions, including extremes.


GENERAL II.GENERAL II.

• Quite often the method development or adaptation takes place in a R & D laboratory or under other conditions where dedicated equipment and personnel are used.

• This may result in the “discovery” of new factors when the method is transferred to the user laboratory, but this should be unexpected.

• No matter how well the assay performed in the development laboratory, if it does not perform well in the user laboratory, it is useless.


SYSTEM SUITABILITY vs VALIDATIONSYSTEM SUITABILITY vs VALIDATION

• System suitability testing is not the same as validation. Therefore while the development and setting of system suitability criteria may be a part of an assay development and validation program, it cannot be considered to be the equivalent of an assay validation.

• A full validation study will consider many factors in addition to the criteria included in system suitability testing, and must involve testing of the actual sample, not system suit. samples.

• Some organizations regard an assay for which system suitability criteria have been developed and emplaced as being “qualified but not validated.”

• The same is true for assays that have not been tested with the specific sample.


SYSTEM SUITABILITY vs VALIDATIONSYSTEM SUITABILITY vs VALIDATION

• 15. METHODS VALIDATION• “System suitability data alone is insufficient for

and does not constitute method validation.”

• GUIDE 1. GUIDE TO INSPECTIONS OF PHARMACEUTICAL QUALITY CONTROL LABORATORIES

• Note: This document is reference material for investigators and other FDA personnel.


WHAT ARE YOU VALIDATING?WHAT ARE YOU VALIDATING?

• If the test method will be used for an analyte in different products (e.g. you are testing for an excipient used in different products) or for an analyte in different matrices ( e.g. an active substance in samples from intermediate stages of manufacture) then you must use all of these products in your validation study.

• This will expand your study, but it’s better to do it all now than to go at the validation in a piecemeal fashion.


Schering-Plough Products, LLC. 2001 Schering-Plough Products, LLC. 2001 San Juan District Office, FDASan Juan District Office, FDA

• According to the firm, the AE method for the new product, Nasonex Unscented Nasal Spray (MAA) is considered validated based on the AE method validation for KTL. This validation was completed on 3/96 according to protocol dated 12/85. On 12/98 the firm approved a new SOP for the AE method validation. Even when this new SOP includes the use of negative and positive controls as well as the gram stain of test organisms during the AE method validation, the firm has not revalidated the existing AE method for KTL.


WHEN TO VALIDATE? I.WHEN TO VALIDATE? I.

• The best time is at the end of the method development process or at the point of transfer to the user laboratory.– At this point the reagents and equipment are still

available, the procedure is fresh in people’s memory, and the method developer should be available.

• Validate at this point, do not put things off until “late phase III.”– Work piles up and then a whole bunch of validations

must be done under time pressure. – Poor planning or a change in schedules can create

major problems, then work is put off and forgotten.– Putting off work to save money only works if the

product fails.


WHEN TO VALIDATE? II.WHEN TO VALIDATE? II.

• If you wait, people forget, equipment may be modified.– This will result in a need for re-training, re-

calibrations, re-conditioning.– Time will be wasted, and additional resources

consumed.

• If the validation study reveals problems, and you are in late Phase III, you may not have the time to go back and fix the assay.– There may be intense pressure to complete the

validation and ignore the problems, but FDA may see it in their labs.


WHO VALIDATES? I.WHO VALIDATES? I.

• The end users should do the validation, since the validation should reflect the actual working conditions.– This means that real samples, with real sample

collection and preparation should be used.

– Real analysts working under real conditions.

– If a senior analyst is taken aside and only works on the method validation to the exclusion of other work, this is an artificial situation.

– The real situation may be a junior analyst running the test while doing other tests.


WHO VALIDATES? II.WHO VALIDATES? II.

• Personnel are very important. In system suitability testing, the analyst should be considered to be a part of the system.

• If the laboratory has a “validation specialist” who does only validation work to the exclusion of other work, this is an artificial situation.

• If there are five analysts, any one of whom could be called upon to run the test, all five of them should participate in the analyst-to-analyst part of the repeatability and intermediate precision studies.


WHERE IS THE VALIDATION DONE? I.WHERE IS THE VALIDATION DONE? I.

• The validation should be done in the working laboratory using the equipment and facilities that will be normally used.

• If special equipment will need to be transferred to the QC lab, then do that before the validation.

• If equipment will be shared among different assays, that should be factored into the intermediate precision study.


WHERE IS THE VALIDATION DONE? II.WHERE IS THE VALIDATION DONE? II.

• If an instrument will be parked under the air conditioner vent and next to a drying oven, then it should be there during the validation study.

• If a shared HPLC will mean that a column will need to be changed and re-conditioned before the assay can be run, that event should be included in the validation study.

• If the lab operates 24/7 the shifts and days should be included.


Case Study. Part 1. Initial ConditionsCase Study. Part 1. Initial ConditionsTaken from: Dolan, J.W., LCGC (2000) 18: 1136.Taken from: Dolan, J.W., LCGC (2000) 18: 1136.

• Assay development and validation done by Methods Development Laboratory.

• Procedure transferred to QC Laboratory for routine use.

• Isocratic LC with UV detection at 214 nm.• Impurity peaks at 5, 6, and 15 min.• Peaks at 5 & 6 min have USP tailing factor <2.

Peak at 15 min is marginal with tailing factor of 2.1,


Case Study. Part 2. Initial ConditionsCase Study. Part 2. Initial Conditions

• Method transfer protocol consisted mainly of having QC repeat the separations obtained by Methods Development.

• Results of Method Transfer Study showed that QC obtained the same results and transfer was considered to be successful.

• A few days after the transfer study, manufacturing of the product increased and QC work load increased.


Case Study. Problem Case Study. Problem

• The chromatograms now show irregular peaks. • In some cases, the peak shapes fail requirements

for the tailing factors.• In addition to the irregular shapes, there is some

indication of a rising baseline during some of the runs.

• The results are clearly OOS, and the irregular peak shapes suggest that the impurities are not pure. (Remember this was an impurity assay.)


Case Study. Initial Investigation. Part 1Case Study. Initial Investigation. Part 1

• When peak distortions such as tailing occur for all peaks, one possibility is a blocked frit or bubble at the column head.

• A quick solution was to change the column.– When this was done, the problem appeared to go away,

but quickly came back. Flushing the column head, manually, had no effect.

– Therefore the column was probably not the source of the problem.



• To check the irregularity of the peak shape, an autosampler precision check was performed by repeatedly injecting the same sample under standard run conditions.

• The result was a relative standard deviation of less than 1% when checking the peak areas, despite their irregular shape.

• This was well within specifications.



• The autosampler was cleaned and reassembled to see if the problem was being caused by carryover or cross contamination or a bad sampler.– There was no change.

• There was some suggestion that the baseline noise was occurring in a regular pattern, and this led to several suggestions and checks.



• A bubble or leak in the pump should reduce the flow rate, but there was no evidence of a significant change in the retention times of the peaks.

• The possibility of bubbles in the detector was checked by extra degassing of the solvent.

• The detector lamp was replaced. • Nothing changed, suggesting that there were no

bubbles in the detector and the lamp was not failing.


Case Study. Further Investigation. Part 1Case Study. Further Investigation. Part 1

• The baseline should be constant in an isocratic run, so the rising baseline suggests that the samples contain a contaminating material that “bleeds” off the column.

• In an isocratic separation, a contaminated mobile phase will result in a shift of the baseline, but it should not rise throughout the separation.



• Interviews with the Methods Development analyst revealed little except that it was customary to make two runs in the morning and two in the afternoon. During lunch and overnight, the pump was left running at a lower rate “to keep the column conditioned.”

• This procedure was eliminated after the method transfer study because the HPLC was needed for other work and “to save on the cost of solvents.”



• It was decided to check the baseline change by letting the column continue to run past the normal 20 min run time. In addition, the flow rate was increased.

• The run was allowed to go for 180 min. It was found that the baseline continued to rise in an irregular manner over the whole 180 min with several changes in its slope. In addition there were two, very broad peaks centered around 70 and 150 min. They were superimposed on the rising baseline.


Case Study. ConclusionCase Study. Conclusion

• In isocratic runs, it is known that peak widths increase as the retention time increases. Consequently, the presence of the broad peaks suggests that the samples contain two impurities that “bleed” through several cycles of separation.

• During Method Development, the extended “washing” of the columns eluted the contaminants.

• The introduction of a “flushing” step with a strong solvent at the end of the 20 min run eliminated the problem.


Case Study. Corrective Action and Follow-Up.Case Study. Corrective Action and Follow-Up.

• The flushing step required “pushing” a strong solvent through the column for 5 min after the 20 min separation run. – As this changed the column conditioning and

the programming, it was felt that a revalidation was needed, but this was not done because of a bigger problem. (See Below)


Case Study. Follow-Up. 1.Case Study. Follow-Up. 1.

• Part of the problem here arose from the fact that the validation and method transfer studies were not conducted “under actual conditions of use.” (21 CFR 211.194(a)(2) The suitability of all testing methods used shall be verified under actual conditions of use.)


Case Study. Follow-Up. 2.Case Study. Follow-Up. 2.

• The “bigger” problem here, of course, is the fact that two, new, impurities were found. It was now necessary to identify these substances, decide on their importance, and redesign the chromatographic procedure to include them in the routine analytical run.

• A new validation was needed.


21 CFR 312.23(a)(7)(iv)(a)21 CFR 312.23(a)(7)(iv)(a)

• (iv) Reflecting the distinctions described in this paragraph (a)(7), and based on the phase(s) to be studied, the submission is required to contain the following:

• (a) Drug substance. A description of the drug substance, including its physical, chemical, or biological characteristics; the name and address of its manufacturer; the general method of preparation of the drug substance; the acceptable limits and analytical methods used to assure the identity, strength, quality, and purity of the drug substance; and information sufficient to support stability of the drug substance during the toxicological studies and the planned clinical studies. Reference to the current edition of the United States Pharmacopeia--National Formulary may satisfy relevant requirements in this paragraph.


21 CFR 312.23(a)(7)(iv)(b)21 CFR 312.23(a)(7)(iv)(b)

(b) Drug product. A list of all components, which may include reasonable alternatives for inactive compounds, used in the manufacture of the investigational drug product, including both those components intended to appear in the drug product and those which may not appear but which are used in the manufacturing process, and, where applicable, the quantitative composition of the investigational drug product, including any reasonable variations that may be expected during the investigational stage; the name and address of the drug product manufacturer; a brief general description of the manufacturing and packaging procedure as appropriate for the product; the acceptable limits and analytical methods used to assure the identity, strength, quality, and purity of the drug product; and information sufficient to assure the product's stability during the planned clinical studies.


WIL Research Laboratories, Inc. 1999 WIL Research Laboratories, Inc. 1999 GLP and Bioequivalence Investigations BranchGLP and Bioequivalence Investigations Branch

Division of Scientific Investigations, Office of Medical Policy, Division of Scientific Investigations, Office of Medical Policy, CDERCDER

• Your response indicates that none of the FDA 483 observations suggest non-compliance with the GLP regulations. The response also indicates that, as a contract laboratory, WIL Research cannot be held responsible for sponsor conducted activities (analyses of dosing formulations) which are not under WIL Research’s direct control.

• This response does not satisfy the requirements as set forth in Section 58.31(d) which requires that testing facility management shall assure that test and control articles or mixtures have been appropriately tested. Specifically, when analytical tests for homogeneity, concentration, and stability of dosing formulations are performed by another laboratory, it is the testing facility’s responsibility to assure that such tests are performed,


Sec. 58.31(d) Testing facility management. Sec. 58.31(d) Testing facility management.

• For each nonclinical laboratory study, testing facility management shall:

• (d) Assure that test and control articles or mixtures have been appropriately tested for identity, strength, purity, stability, and uniformity, as applicable.


Analytical Procedures and Methods Validation. CMC Analytical Procedures and Methods Validation. CMC Guideline: August, 2000Guideline: August, 2000

• C. Stability-Indicating Assay: A stability-indicating assay is a validated quantitative analytical procedure that can detect the changes with time in the pertinent properties of the drug substance and drug product. A stability-indicating assay accurately measures the active ingredients, without interference from degradation products, process impurities, excipients, or other potential impurities. . . . Assay analytical procedures for stability studies should be stability-indicating, unless scientifically justified.


483 Observation:483 Observation:

“There is no data to show that the method used for XX stability testing has been validated as stability-indicating with respect to acid and base hydrolysis and photolysis; there was inadequate data for oxidation and thermal degradation.”


483s on Impurities/Degradants483s on Impurities/Degradants

“Analysts can use “suppress unknowns” to omit reporting of unknown peaks in HPLC run”

“Unidentified HPLC peaks found during stability testing of the validation lots were not identified orevaluated.”

“Systematic identification of degradation products hasnot been performed.”


DEVELOPMENT REPORT I.DEVELOPMENT REPORT I.

• You should not attempt to validate an undeveloped or poorly developed test.

• What tests were considered? Is this a compendial test? Is this the right test?

• What is known about the test?– Problems, interferences, cross reactions, chemistry.

• What are the literature references behind the test?• Are preliminary estimates available for validation

parameters?


DEVELOPMENT REPORT II.DEVELOPMENT REPORT II.

• The development report should have enough information that you feel sure that the validation will be successful.

• The stability of reagents and intermediate solutions and the “stopping points” should be identified for the test.

• The standards and controls should have been defined, SOPs written and material transferred to the validation workers.


FACILITY I.FACILITY I.

• Is your laboratory ready?• Do you have space for the work, the personnel, the

instrument? Is the space clear or will it need to be shared?

• This is sort of an IQ for the lab. Can you install your validation study without disrupting things?

• The work will need to be done within the context of existing work. You cannot shove it off into a corner and then expand it later.


FACILITY II.FACILITY II.

• What about data storage? • If you do the validation, where are you going to

store the records of the work?– Do you at least have a fireproof file cabinet?

– Is there space in the document storage facility?

• Are there any special records such as electrophoresis gels, animal or cell culture tissues, computerized data that are recorded on discs.– How will you store these?

– Do you have a back up location to store copies?


EQUIPMENT I.EQUIPMENT I.

• Do you know if the needed equipment and utensils are ready and suitable for use. (how about a DQ?)

• All of the important instruments should have been qualified (IQ and OQ at least).

• Do you have calibration and maintenance SOPs available for the instruments?

• At the point where you start the validation, the equipment should have been recently calibrated and required maintenance should have been done.


EQUIPMENT II.EQUIPMENT II.

• If the normal calibration and maintenance cycles will call for this work to be done again during the validation study, do it. – Do not hold off on calibration and maintenance just

because you are in the middle of a validation study.

• Do you have spare parts and other material that may be needed for calibration and maintenance work?

• Do not overlook small items. Pipettors may need calibration and maintenance too.


EQUIPMENT III.EQUIPMENT III.

• If computers and computerized equipment are used, they should be compliant with 21 CFR 11 at least to the extent required by the guidelines.

• How will you validate any special software, especially if it is used to work up final data?

• How will you archive the computer records?• Do you have computer techs or other assistance

available if needed?• What about consumables such as paper, discs,

tapes, flashdrives, etc.?


IT IS A CAPITAL MISTAKE IT IS A CAPITAL MISTAKE TO THEORIZE BEFORE ONE HAS TO THEORIZE BEFORE ONE HAS

DATADATA

• Sir Arthur Conan Doyle, aka Sherlock Holmes


Test Method Procedure (SOP) I.Test Method Procedure (SOP) I.

• If there is no SOP for the test, how do you know what you are validating?– Note that the validation study is only valid for the test

as described in the SOP.

• Is the SOP clearly written? Does it describe reagent preparations? Is the procedure for data work up clear?

• The SOP should clearly specify the supplies needed, the length of time the test will require.

• The SOP MUST describe safety precautions and potential hazards.


Test Method Procedure (SOP) II.Test Method Procedure (SOP) II.

• Reagent stability should be clearly stated.• Stopping points in the assay should be clearly

identified. – If the analyst needs to take a break, where can the

procedure be stopped without affecting the test results?– Where are the steps when a break in the activity

sequence cannot be tolerated or where stopping the test will affect the outcome?

• The SOP should contain references so that analysts can refer to the original literature, if necessary.


STANDARDS STANDARDS

• If a compendial or other well-recognized standard exists, your working standard for the validation should have been calibrated against it.

• There should be an SOP describing the preparation of the working standard even if it is nothing more than a solution of a purchased preparation. The solvent and purchase specifications should be stated.

• If it had to be “prepared” by synthesis or special purification this should be described.

• The stability of the standard in its “working form” should be known and documented.


Tri-State Analytical Laboratory LLC 2003Tri-State Analytical Laboratory LLC 2003FDA New Orleans District, FDA New Orleans District, Nashville Branch OfficeNashville Branch Office

• Analytical results were reported to [redacted] stating that a sample met specifications when either out-of-specifications (OOS) results were obtained on the sample analysis or on the quality control samples used to determine the validity of the analytical results. These OOS results were not investigated/documented properly to assure results reported to [redacted] were accurate and valid.

• Use of reference standards and reagent solutions for extended periods of time without data in the analytical records supporting time of use.


CONTROLS I.CONTROLS I.

• The control should resemble the actual test sample to the degree that it can act as a suitable surrogate for the actual samples.

• Several controls may be needed if samples can come in several forms, e.g. the analyte may be dissolved in different solvents or the sample matrix may vary.

• Negative controls should be the sample matrix without the analyte.

• A positive control should be a real sample.– Positive controls prepared by adding a standard to the

negative control are not as good.


CONTROLS II.CONTROLS II.• Positive and negative controls should be used. • Do not use the solvent blank for the “zero” standard

as the negative control, unless it is real.• Controls can be prepared by using “practice” runs

from your development work. Later, positive controls can be obtained from failed lots or validation lots.

• Ideally, you can prepare standard curves by diluting your positive control with the negative control. At least try to have “high/low” controls by diluting the positive control with the negative.

• System suitability samples can be prepared by spiking controls. You want a difficult sample.


TRAINING I.TRAINING I.

• Everyone who will participate in the validation must be properly trained to run the assay.– This includes the supervisor. If the supervisor does not

understand what is being supervised, how can supervision be done?

– Analyst to analyst variation should not be due to different levels of training. Analyst skills will change and mature over time.

– If a reproducibility (lab to lab variation) study will be done, it is important to show that all analysts were equally trained and qualified.


TRAINING II.TRAINING II.

• The training should be completed BEFORE the validation study is launched.

• Do not use the validation study as your training platform. Otherwise training activities will create artificial situations that are not related to the validation or normal testing.

• Specialist training (statistics, instrument maintenance and calibration) should be completed before the validation is attempted, so that the specialists can have proper input to the validation protocol.


TRAINING III.TRAINING III.

• The training must be documented and proof of training supplied.

• Simple signatures to the effect that analysts have read protocols or that they have been observed to perform the test correctly are not sufficient.

• Proof should be provided in the form of test results obtained with standards and controls. This should include calculations and use of spreadsheets. System suitability or precision and accuracy specifications can be employed.


TRAINING IV.TRAINING IV.

• Statistics is an important body of knowledge for performing method validations and good training in statistics should be provided for all QC analysts. Simple basic statistics is sufficient.

• Specialist training in DOE or fractional factorial designed experiments is needed for at least a few members of the method validation team.

• Intermediate precision and robustness are best studied using fractional factorial designs. The DOE studies will minimize the total number of tests runs that are needed.


Germiphene Corporation 2005 Germiphene Corporation 2005 Division ofDivision of Manufacturing and Product Quality Manufacturing and Product Quality

Center for Drug Evaluation and ResearchCenter for Drug Evaluation and Research

• Several observations cited a lack of following procedures even though there was documentation that your employees were trained in the procedure. For example, when asked about the procedure for determining the status of equipment prior to use, the employee could only say that the analysts were trained to check the calibration prior to use. However, the piece of equipment in question was in use and out of calibration for 1.1 months


RAW MATERIAL I.RAW MATERIAL I.

• If you have special raw material needs, you will need to control these items just as you would with manufacturing raw material.– For instance, if you have a nucleic acid primer or a

monoclonal antibody that is custom made for your assay, you will need to have purchasing specs. that define the properties needed. If you need ACS grade do you have it? Will USP grade substitute?

– You will need raw material testing SOPs to verify that the material delivered is what you ordered and what you need.


RAW MATERIAL II.RAW MATERIAL II.

• If you do not have test procedures for these, how did you qualify the vendor? Reputation is not enough!– Did you audit the vendor?– How about confirming the vendor’s certificate of

analysis? NEVER TRUST a CoA from a new source.

• The raw material qualification should be done before you use the test material in the validation study. – What if the validation goes bad because the reagent

does not perform properly?


REAGENTS I.REAGENTS I.

• In addition to qualifying your raw material, you should have SOPs describing how solutions and other test reagents are made and, if necessary, checked for proper concentration or potency.

• If the procedure is simple, like making 0.85% (w/v) NaCl or 0.1 N HCl, then you could write it into the test method SOP.

• If a reagent requires extensive preparation or where careful checking needs to be done, an SOP will be needed.


REAGENTS II.REAGENTS II.

• Consider a monoclonal antibody that needs to be isolated from culture supernatant, derivatized with a fluorescent tag or an enzyme, and have its binding constant determined. More than one SOP may be needed.

• If culture media are needed, how are you going to qualify the media in your laboratory? The manufacturer may obtain good results, but what about your laboratory?


REAGENTS III.REAGENTS III.

• What is the stability of these reagents? Even solutions of inorganic salts will evaporate over time and with use.– You need to specify an expiration date for all prepared

solutions. A real stability study may be required.

• Are there special storage requirements?– Is the solution photosensitive?

– Does it need refrigeration?

– If it needs refrigeration, can it work if added cold? Does it need to be brought to room temperature? If so, how long before it expires at room temperature?


PLANNING I.PLANNING I.

• Have the costs for the validation study been factored into the budget?

• What facilities and equipment will be needed and when? How can these needs be woven into the normal laboratory activities?

• What people are needed and when? How will these needs be woven into normal laboratory activities?

• Can the needs really be superimposed on normal laboratory operations? Are additional personnel and facilities needed? Is the budget sufficient for all the activities?


PLANNING II.PLANNING II.

• Do the supervisors understand their responsibilities and have plans for dealing with the needs in their individual areas of responsibility?

• Remember that simply imposing a study on a lab by decree and expecting them to deal with the added workload without a plan and supporting structure, is a sure recipe for disaster and is a clear symptom of bad management.

• BY FAILING TO PLAN, YOU ARE PLANNING TO FAIL!

– Deming?


Validation Protocol I.Validation Protocol I.

• A method validation should be a PROSPECTIVE study, so a protocol should be written BEFORE the work is started.

• The protocol needs to state what is being validated– The test method (SOP # and effective date), the types of

material to be studied (different matrices or concentrations).

• What elements will constitute the validation?– Based on ICH Q2A or other works, what are you

checking? Accuracy, linearity, LOD or LOQ?


Validation Protocol II.Validation Protocol II.

• The sequence of work to be done and by whom.– What do you study first? Who does this study?

– Who works up the data?

– How is the work to be done?

• How is the information to be recorded and how is it to be stored?

• Who is responsible for supervising the work, preparing the protocol, and preparing the final report? Responsibilities must be assigned otherwise things fall into cracks.


Validation Protocol III.Validation Protocol III.

• What are the criteria that must be met for a successful validation?

• A timetable for activities should be proposed with intermediate check points for verifying that the work is progressing well or for clearing away problems that may develop.

• The final protocol approval should be done by an individual or group who verify that the validation study is ready for execution.


SPECIAL NEEDS I.SPECIAL NEEDS I.

• All tests should be validated, but not all tests follow the same general pattern for execution. Microbiological tests, dissolution tests, content uniformity and endotoxin tests are often cited as examples. With content uniformity and dissolution tests, there are actually two stages of testing. The second stage, where the analyte is being measured can follow the usual procedures, but validating the sample acquisition or preparation may be a problem.


SPECIAL NEEDS II.SPECIAL NEEDS II.

• With any test, the accuracy and reproducibility must be established over various test conditions.

• For accuracy, it is important to establish the fact that the test really does measure the factor itself in the magnitude that is present. For instance, in dissolution testing, the assay may do a really good job of measuring the analyte, but that is not the question; it is the rate of appearance of the analyte in the solution that is critical. It is very important to know what is being measured.


SPECIAL NEEDS III.SPECIAL NEEDS III.

• Reproducibility is important because a highly variable test will cast doubt on the accuracy of the test result. If the test is highly variable, the results will be virtually irreproducible. In which case it will be useless for manufacturing or dosing control.

• There is always one reproducibility check that can be done. If the test is working at all, it should be possible to choose some concentration of analyte and some test condition that should give a result within the normal response range.


SPECIAL NEEDS IV.SPECIAL NEEDS IV.

• This condition can be created and reproduced about six times and the test could be run, even without a standard curve, as long as there is some measurable response. This can be done with any test, including whole animal or cell culture tests.

• This check, which gives repeatability (or within run precision) should show the minimum possible variation. If the variation here is large, then it shows the presence of uncontrolled factors in the test, and these must be found and controlled during the method development process.


SPECIAL NEEDS V.SPECIAL NEEDS V.

• There are some who run animal or tissue culture based tests who have been known to accept potency test results ranging from 5% to 160% of the expected values.

• They take the fatalistic attitude that “that’s the way it is with biological tests.”

• The data are so variable that it is not possible to assign sensible values for doses or therapeutic windows.

• Often an investigation will show that virtually no effort was expended in trying to reduce the variability in the assay.


SPECIAL NEEDS VI.SPECIAL NEEDS VI.

• High variability can only be accepted when a development laboratory shows that it made serious attempts to reduce the variability of the test. Often the problem arises because non-analysts are placed in the position of developing tests.

• If the previous repeatability test shows a low variability, then the high test variability is due to factors normally covered in the intermediate precision portion of the validation study.


The Dogbert ConjectureThe Dogbert Conjecture

• “If your controlled tests have never found psychic powers, how do you know the tests

work for that sort of thing?” • “Isn’t that like using a metal detector to find out if there are unicorns in your sock drawer?”

• - Dogbert (1/20/98).

IVTMethVal12051 PREPARING FOR A METHOD VALIDATION Steven S. Kuwahara, Ph.D. GXP BioTechnology, LLC...

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