Course: BioT 006 Quality Control & Validation Assay Validation Course: BioT 006 Quality Control & Validation

Definitions: (Dictionary: Relative to quality endeavors) Validation: An act, process, or instance to support or corroborate something on a sound authoritative basis. Verification: An act or process of establishing the truth or reality of something. Qualification: An act or process to assure something complies with some condition, standard, or specific requirements.

Why Validate? Quality by design, built in Can’t inspect quality in Demonstrate control of process Good science Good business It’s the law

Considerations Prior to Validation Suitability of Instrument Status of Qualification and Calibration Suitability of Materials Status of Reference Standards, Reagents, Placebo Lots Suitability of Analyst Status of Training and Qualification Records Suitability of Documentation Written analytical procedure and proper approved protocol with pre-established acceptance criteria

Analytical Methods Validation Considered a critical step in the manufacturing process Requirements for validated analytical methods explicitly written into the CFR’s 211.165 – Testing and release for distribution FDA: Requires that the: Accuracy Sensitivity Specificity Reproducibility of test methods etc. Employed by the firm shall be established and documented. Such validation and documentation: Accomplished in accordance with 21 CFR 211.194 (a)(2)

What is not an Analytical Method Validation? Calibration The Process of Performing Tests on Individual System/ Components to Ensure Proper function For example: HPLC Detector calibration, pH meter Analytical Balance, Centrifuge Particle counter etc.

Compendium Compendium: Compendial: A collection of concise but detailed information about a particular subject, especially in a book or other publication. Compendial: Pertaining to compendium

Verification vs. Validation Compendial vs. Non-compendial Method Compendial methods: Verification Non-compendial methods: Validation requirement

Validation protocol for analytical method Statement of purpose and scope Responsibilities Documented test method List of materials and equipment Procedure for the experiments for each parameter Statistical analysis Acceptance criteria for each performance parameter

Validation Component Specificity (Selectivity) Accuracy Precision Linearity Detection limit Quantitation Limit Range Robustness Ruggedness

Specificity The ability of the analytical method to quantify (differentiate) the analyte in the presence of other components in the sample Does the analytical method detect the component it is supposed to detect? e.g. Cross reactivity in antibody based methods Demonstrate specificity by conducting analytical method on materials that may mimic analyte of interest Looking for “false positives”

Specificity, contd. Specificity may be affected by: Interference: Excipients or impurities Spike effect: Adding appropriate levels Assay results should be unaffected Chromatography or Electrophoresis Impurities get separated

Accuracy/ Trueness Ability of analytical method to accurately determine the presence and amount of the analyte of interest The closeness of mean test results to the true value of the analyte. It is determined by replicate analysis of samples containing known amounts of the analyte. Accuracy should be measured using a minimum of five determinations per concentration. A minimum of three concentrations in the range of expected concentrations is recommended.

Accuracy Accuracy is hitting the part of the bulls eye for which you’re aiming at.

Precision The closeness of agreement (degree of scatter): Between a series of measurements obtained from multiple sampling of the same homogeneous sample How much variability does the assay exhibit when analyzing the same sample Precision should be measured using a minimum of five determinations per concentration. A minimum of three concentrations in the range of expected concentrations Measured by parameters of variation, mostly CV (Coefficient of variation)

Coefficient of variation Also called Relative Standard Deviation CV = Coefficient of variance It is a measure of dispersion of a probable distribution RSD = SD (or Sigma) X 100 Mean = CV

Precision… Considered at 2 Levels Repeatability (Intra assay) Intermediate Precision (Inter assay)

Precision: Intra-assay (Repeatability) Express the precision under the same operating conditions over a short interval of time. Should be assessed using minimum of 15 determinations 5 concentrations/ 3 replicates Minimum of 5 determinations within the range.

Precision: Inter-assay Express variations within-laboratory. Expressed in terms of relative standard deviation coefficient of variation Studies should include varying days, analysts, equipment, etc. Also called Intermediate precision

Linearity & Range Linearity: The ability of the assay (within a given range) to obtain test results which are: Directly proportional to the concentration/amount of the analyte Range: The interval between the upper & lower concentrations of an analyte for which: the assay has suitable levels of Precision Accuracy Linearity

Linearity & Range (cont.) Linearity and Range can be evaluated simultaneously Range is established by confirming acceptable degrees of: Linearity Accuracy Precision within or at the extremes of a specified range

Linearity Should be Evaluated By Visual Inspection of plot of signals vs. analyte concentration By Appropriate statistical methods Linear Regression (y = mx + b) Requires a minimum of 5 concentration levels

Method Validation LOD & LOQ III Lowest level at which method can detect analyte (LOD or DL) Lowest level at which method can accurately quantify analyte (LOQ or QL) Precision and LOQ related Lower LOQ will typically result in lower precision

Method Validation LOD & LOQ IV A method is not acceptable for accurate detection or quantitation if: the analyte level is likely be fall beneath the limit(s) calculated based upon the blank signal and its standard deviation Mean sample signal must be sufficiently larger than the blank so that positive detection or accurate quantitation is possible

Ruggedness The effectiveness of an analytical process in face of small environmental/operating conditions, such as: Different analysts Different equipment Different labs Different time

Method Validation Ruggedness Methods need to be shown to be rugged A replicate set of data produced at a particular time point by an operator working with a particular set of equipment in a given laboratory will verify repeatability. To show reproducibility, the method must produce similar results when any of these parameters are changed. The most likely changes are to time and operator.

Validation: Robustness Robustness of an analytical method refers to: It’s ability to remain unaffected when subjected to small changes in method parameters. e.g. in PCR: Defining the critical range of: Primer concentration Mg2 Concentration Thermo cycler temperature range e.g. in HPLC: A perfect mobile phase is one which allows small changes in the composition without affecting the selectivity or the quantitation of the method. Usually a part of the assay optimization Prior to the validation process

Characteristics of Analytical Procedures Ruggedness: It is due to factors external to the method Robustness It is due to factors internal to the method. Variability caused by: Day-to-day variations Analyst-to-analyst Laboratory-to-laboratory Instrument-to-instrument Chromatographic column-to-column Instability of analytical reagents Reagent kit-to-kit

Validation: Method Performance Characteristics Purity Impurity Detection Impurity Quantitation Identity Content Accuracy + Specificity Precision Repeability Precision Intermediate Linearity Range LOQ LOD

Extent of Validation New methods require complete validation Pharmacopoeia methods require partial validation (or verification) Significant changes mean partial revalidation equipment changes formula changed changed suppliers of critical reagents

Bradford Assay for Total Protein Well known colorimetric assay that relies on: the binding of Commassie Blue G-250 dye to the proteins in an acidic solution Dye binding is proportional to number of positive charges in protein Color (absorbance) is read at 595 nm Proteins up to 3000 Da not detected Assay linear over a short range, typically from 0 µg/mL to 2000 µg/mL, often making dilutions of a sample necessary before analysis. Simple, quick, wide range, few interfering agents

Bradford Assay Accuracy Specificity Precision Linearity & Range Limit of detection (LOD) Limit of quantification (LOQ) Robustness

Validation: Analytical method transfer Method transfer protocol and procedure Example: R&D lab to QC Lab Precision Accuracy Ruggedness Written and approved specific test method Proficiency check Three assays compared to original lab assay Formal acceptance by new laboratory

Chemical Laboratory Validation Requirements Balances Chromatography HPLC, GC, TLC Dissolution or disintegration apparatus Karl Fischer moisture determination Melting, softening or freezing point apparatus Ovens, refrigerators, incubators pH meter Spectrophotometer UV/Vis, IR, Amino Acid Analyzer Timers Viscometer Volumetric equipment/glassware

Typical validation of HPLC assay Method validation Specificity: that the method is free of interference from excipients, impurities, etc. Accuracy: that the method gives closeness to true results. Precision: by checking that the method is precise. Linearity: that the method will produce results that are directly proportional to the concentration of analyte in the samples. Robustness: by checking that the method will withstand deliberate changes.

Microbiological Testing: Validation Microbial limit testing: indicator organisms such as S. aureus, P. aeruginosa etc. Microbial count: Total viable count in starting and finished products Sterility testing Preservative effectiveness testing: After inoculation, bacteria should not grow in up to 28 days Environmental monitoring program: Water air and surface monitoring

Validation: Microbial test procedures Incubation temperature and time Media may not grow all microorganisms Variations in media may affect recovery: shelf life Inhibitory disinfectants or preservatives: water with chlorine must be neutralized Sample procedures handling, storage, transport In case the sample is contaminated when the sample is drawn, or very long storage and very hot or cold storage temperatures are used, then the sample will be affected.

Sterility Testing Validation Media growth promotion, sterility, pH Product validation Stasis testing: Also called inhibition test,  It is performed to ensure that there are no inhibitory substances remaining in the product and  that the media is still capable of supporting the growth of microorganisms at the end of the sterility test incubation period.  Environmental monitoring Negative controls Challenge organisms: Staphylococcus aureus, Bacillus subtilis, Pseudomonas aerugihosa, Aspergillus niger, Clostridium sporogenes, and Candida albicans. For each challenge organism, the inoculum level is 10-100 CFU. Validation tests using live microorganisms must not be undertaken in the sterility testing area.

Re-Validation When What Method parameters have been changed The scope of the method has been changed Synthetic methods have been changed Impurity profile has been changed What Preferably everything Exceptions should be scientifically justified