GENERAL I.

1. PREPARING FOR A METHOD VALIDATIONSteven S. Kuwahara, Ph.D.GXP BioTechnology, LLCPMB 506, 1669-2 Hollenbeck AvenueSunnyvale, CA 94087-5042Tel. & FAX: (408) 530-9338e-Mail: stevekuwahara@yahoo.comWebsite: www.gxpbiotech.org IVTMethVal1205

2. 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. IVTMethVal1205

3. 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. IVTMethVal1205

4. SYSTEM 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. IVTMethVal1205

5. SYSTEM 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. IVTMethVal1205

6. 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. IVTMethVal1205

7. Schering-Plough Products, LLC. 2001 San Juan District Office, FDA • According to the firm, theAE method for thenew 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. IVTMethVal1205

8. 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. IVTMethVal1205

9. 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. IVTMethVal1205

10. 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. IVTMethVal1205

11. 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. IVTMethVal1205

12. 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. IVTMethVal1205

13. 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. IVTMethVal1205

14. Case Study. Part 1. Initial ConditionsTaken 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, IVTMethVal1205

15. Case 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. IVTMethVal1205

16. 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.) IVTMethVal1205

17. Case 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. IVTMethVal1205

18. Case Study. Initial Investigation. Part 2 • 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. IVTMethVal1205

19. Case Study. Initial Investigation. Part 3 • 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. IVTMethVal1205

20. Case Study. Initial Investigation. Part 4 • 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. IVTMethVal1205

21. Case 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. IVTMethVal1205

22. Case Study. Further Investigation. Part 2 • 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.” IVTMethVal1205

23. Case Study. Further Investigation. Part 3 • 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. IVTMethVal1205

24. Case 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. IVTMethVal1205

25. 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) IVTMethVal1205

26. 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.) IVTMethVal1205

27. 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. IVTMethVal1205

28. 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. IVTMethVal1205

29. 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. IVTMethVal1205

30. WIL Research Laboratories, Inc. 1999 GLP and Bioequivalence Investigations BranchDivision of Scientific Investigations, Office of Medical Policy, CDER • 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, IVTMethVal1205

31. 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. IVTMethVal1205

32. Analytical Procedures and Methods Validation. CMC Guideline: 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. IVTMethVal1205

33. 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.” IVTMethVal1205

34. 483s 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 or evaluated.” “Systematic identification of degradation products has not been performed.” IVTMethVal1205

35. 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? IVTMethVal1205

36. 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. IVTMethVal1205

37. 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. IVTMethVal1205

38. 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? IVTMethVal1205

39. 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. IVTMethVal1205

40. 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. IVTMethVal1205

41. 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.? IVTMethVal1205

42. IT IS A CAPITAL MISTAKE TO THEORIZE BEFORE ONE HAS DATA • Sir Arthur Conan Doyle, aka Sherlock Holmes IVTMethVal1205

43. 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. IVTMethVal1205

44. 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. IVTMethVal1205

45. 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. IVTMethVal1205

46. Tri-State Analytical Laboratory LLC 2003FDA New Orleans District, Nashville 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. IVTMethVal1205

47. 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. IVTMethVal1205

48. 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. IVTMethVal1205

49. 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. IVTMethVal1205

50. 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. IVTMethVal1205