Analysis and testing Flashcards

Question

How would you perform a method transfer of Assay method to another lab? What stats would you use?

Answer 1

ELISA stands for Enzyme-Linked Immunosorbent Assay. It is a quantitative or semi-quantitative analytical method used to detect and measure biological substances such as hormones, proteins, peptides, and antibodies, based on antigen–antibody interactions. ⸻ How it works: 1. A microplate (usually 96-well) is coated with a capture antibody or antigen specific to the target analyte. 2. The sample is added — if the target analyte (e.g., hormone, protein) is present, it binds to the coated antibody/antigen. 3. A secondary antibody, linked to an enzyme, is added and binds to the analyte. 4. A chromogenic substrate is introduced — the enzyme catalyses a color change. 5. The intensity of the signal (measured as absorbance) correlates with the amount of analyte present in the sample. ⸻ Types of ELISA: • Direct ELISA • Indirect ELISA • Sandwich ELISA (commonly used for large molecules like proteins or cytokines) • Competitive ELISA ⸻ Viva-style summary sentence: “ELISA is an immunoassay technique used to quantitatively or semi-quantitatively measure biological molecules like hormones or proteins. It uses antigen-antibody binding followed by an enzyme reaction to produce a measurable signal.”

Answer 2

Step 0: Gather Context * What is the product, its indication, and patient group (e.g., oncology, paediatrics)? * Are there market alternatives? * What other stability parameters (e.g., assay, known impurities, dissolution) look like? * Review prior stability trend data for the impurity (e.g., was it increasing gradually?). * Confirm whether the impurity was present at time zero (manufacturing issue) or only appears during storage. ⸻ Step 1: OOS Investigation (per MHRA OOS Guidance) * Phase 1a: Analyst-level checks — method, system suitability, sample prep, calculations, equipment calibration, reagent integrity. * Phase 1b: Supervisor-led review using an approved checklist — reprocessing using the same sample (not re-sampling), cross-checking method parameters, etc. * If no assignable cause is found, proceed to: * Hypothesis testing (e.g., using a second column or mobile phase) — protocol must be QA-approved in advance. * If still unresolved: raise a formal deviation — possible product quality issue. ⸻ Step 2: Immediate Containment and Risk Review * Quarantine affected batch(es) in stability chambers and market stock (if still in distribution). * Investigate the identity and structure of the unknown impurity: * Use MS/NMR techniques to identify. * Check if a Permitted Daily Exposure (PDE) is available or derivable. * Review toxicological data (consult QPPV and medical team). * Check: * Reference/retention samples for comparison. * Previous stability batches for similar impurity trends. * Customer complaints / signal detection systems for any related findings. * Environmental monitoring trends (if contamination suspected). ⸻ Step 3: Impact Assessment * Marketing Authorisation (MA): * This is an OOS for an unknown impurity, which may breach ICH Q3A(R2) limits — reportable to the MHRA and possibly to other global regulators. * GMP Impact: * Could indicate process control failure, cleaning validation failure, or contamination. * Patient Risk: * Unknown impurity = unknown toxicity. * Precautionary principle applies — involve QPPV and medical to assess benefit-risk. * Finished Product Risk: * Identify potentially affected batches (e.g., same lot, line, equipment, raw material). ⸻ Step 4: Recall Decision * Convene a recall risk assessment meeting with multidisciplinary team: QA, QP, QC, Medical, Regulatory, Supply Chain, and QPPV. * Based on outcome: * Recall class: likely Class III (no immediate safety risk, but quality defect). * Recall level: depends on distribution (pharmacy, hospital, wholesaler). * Notify MHRA DMRC before executing recall. * Consider DASH portal notification to DHSC if shortage expected. ⸻ Step 5: Root Cause Analysis (RCA) Use a structured method such as an Ishikawa diagram to evaluate: * QMS: prior deviations, change controls, CAPAs, PQR trends * Facility/Equipment: cleaning validation, qualification, maintenance logs, line clearance, temperature excursions * Documentation: batch records, cleaning records, analytical raw data * Personnel: training and supervision * Process: contamination control, hold times, process validation * Suppliers: COA review, supplier change, transportation deviations * Storage/Distribution: temperature/microbiological excursions * Audit Findings: any previous GMP findings relevant to the impurity? ⸻ Step 6: Corrective and Preventive Actions (CAPA) * Based on root cause, implement: * Supplier audits * Strengthened incoming goods testing * Enhanced cleaning validation or EM monitoring * Method revalidation (if analytical issue) * CAPAs must be tracked, effectiveness checked, and documented. ⸻ Step 7: Regulatory Reporting * Submit a closing investigation report to the MHRA DMRC, including: * OOS details * Root cause * Patient risk assessment * Recall outcome * CAPAs taken ⸻ Final Viva-Style Summary: “For an unknown impurity OOS at 18-month stability, I would investigate per MHRA OOS guidance, involve the QPPV and medical team, assess patient and MA impact, and convene a recall risk meeting. I would identify the impurity, evaluate risk, and report to MHRA. Root cause analysis would drive CAPAs, with effectiveness checks and regulatory follow-up.”

Answer 3

Potency in a Cell-Based Product: Potency is defined as the biological activity of the product and is a critical quality attribute (CQA), especially for cell-based therapies such as CAR-T products. It should reflect the intended mechanism of action (MoA). In the case of a CAR-T product, a common potency assay would be a cytotoxicity assay, where the ability of CAR-T cells to lyse target cancer cells (e.g., CD19+ lymphoma cells) is measured. One approach is to calculate potency by comparing the activity of the test sample against a qualified reference standard, expressed as a relative potency ratio. For example, you may assess the percentage of cancer cell lysis (using a dye release or flow cytometry-based assay) and report the ratio of the test product’s activity relative to that of a reference CAR-T batch. This can be further expressed using dose-response curves and EC50 values. ⸻ Robustness Testing in ELISA Method: Robustness is part of method validation under ICH Q2 and reflects the method’s reliability under a variety of deliberate variations. For ELISA: • Identify critical method parameters (e.g., incubation temperature, time, reagent concentration, washing steps). • Deliberately vary them within a realistic range (e.g., ±5°C, ±10% incubation time, alternate dyes or detection systems). • Assess the impact on the assay result (e.g., optical density or calculated concentration). • A common acceptance criterion is that the %RSD of replicate measurements should be ≤2% (or a defined acceptance range based on method variability and clinical relevance). ⸻ Statistical Tools for Robustness Evaluation: To evaluate the effect of each variable, the following statistical tools can be used: • Different Temperatures: • Use %RSD to assess repeatability within each temperature group. • Apply t-tests (if comparing two temperatures) or ANOVA (if more than two) to determine if there is a statistically significant difference in mean results. • Different Incubation Times: • Use %RSD for internal precision. • Apply ANOVA to assess whether varying incubation time has a statistically significant impact on assay performance. • Different Dyes (e.g., detection labels in ELISA): • Use t-tests to compare performance metrics (e.g., OD values or calculated concentrations) between dye conditions, assuming data is normally distributed. • Alternatively, use non-parametric tests (e.g., Mann-Whitney U) if normality cannot be assumed.

Answer 4

Differences Between Chemical and Biological Methods: 1. Molecular Size and Structure: * Chemical products are typically small molecules with well-defined, simple structures. * Biological products (biologics) are large, complex molecules such as proteins, enzymes, monoclonal antibodies, or hormones. 2. Method of Production: * Chemical products are manufactured via controlled chemical synthesis. * Biologics are produced using living cells or organisms through biological processes, e.g., antigen–antibody interactions, cell cultures, or recombinant DNA technology. 3. Stability and Variability: * Chemical molecules tend to be relatively stable and consistent between batches. * Biologics are inherently more variable and sensitive to changes in manufacturing conditions. Therefore, tighter in-process controls are essential (e.g., precise control of bioreactor parameters such as temperature, pH, oxygen, and CO₂ levels). 4. Sterilisation and Process Design: * Chemical products can often undergo terminal sterilisation (e.g., heat or radiation). * Biologics are usually produced in closed systems to prevent contamination, as many cannot tolerate terminal sterilisation—aseptic processing is typically required.

Answer 5

Immediate Actions and Data Review First, recognise that an 18‑month stability OOS (out‑of‑specification) result on a marketed batch immediately triggers an investigation per MHRA policy . Immediately ask: What was the actual assay value versus spec? Review previous stability results (e.g. at 6, 12 months) for trends or Out‑of‑Trend (OOT) signals. Examine the analytical data and method: confirm the correct, latest method version was used; check chromatograms, standard/reference identities, calculations and equipment logbooks. Verify sample chain of custody and storage conditions. Evaluate possible patient impact: e.g. if Atenolol content is low, patients may be undertreated. In summary, collect all relevant data quickly (prior time‑points, method validation, recent equipment checks, distribution history) to scope the problem . Quarantine and Containment All remaining stock of the affected batch – both stability samples and manufactured product – must be held under quarantine immediately to prevent use. Label the batch as “under investigation/OOS” and stop any further distribution or dispensing. Retain all unused stability samples (and any reference standards or reagents used) for re-testing. If any product has been released or shipped, inventory it and notify distributors or warehouse to hold supply pending investigation. Essentially, treat the batch as nonconforming until the investigation is complete. This aligns with GMP requirements to “not delay” handling of a defective product . (No references needed for standard holding procedure.) OOS/OOT Investigation (MHRA Phases I–III) MHRA guidance prescribes a stepwise investigation of OOS/OOT results . The investigation is conducted in phases: * Phase 1A – Analyst check (Immediate): The analyst who ran the assay first reviews raw data for obvious errors. Check calculations, weighing and dilution logs, instrument settings, power failures, column issues, leaks, sample mix‑ups or spills . Verify the correct sample was tested and recorded. If a simple lab error is found (e.g. calculation mistake, instrument fault, expired standard), correct it and re-calculate; if this resolves the OOS, document and close Phase I (no formal report needed beyond lab records). The MHRA notes that clear causes like technician error or equipment failures can invalidate a result . * Phase 1B – Supervisor check (Checklist review): If no obvious error was found, the QA/manager or supervisor conducts a joint checklist review with the analyst . Using the MHRA’s investigational checklist, verify key points: correct method (version, solvent), instrument calibration and system suitability, sample identity and integrity (chain of custody, storage), reagent/standard quality, analyst training and any deviations. Check that the retention/sample storage conditions (stability chamber temperature) were maintained. Restrict this initial review to data/equipment records only. Once the checklist is complete and documented, proceed to re‑measurement: perform a repeat assay on the original sample solution or additional aliquot as per SOP . Any retest should follow the documented hypothesis plan. If the repeat is in spec, evaluate whether the original was an outlier or whether to invalidate it based on evidence. If re-test confirms OOS, or if doubts remain, move to Phase II. * Phase 2 – Full Investigation: If Phase I finds no clear cause, launch a formal, hypothesis‑driven investigation under QA oversight . First, execute a manufacturing investigation: review batch records for process deviations, raw material certificates (API assay, impurity limits), equipment logs (granulator, tablet press), environmental controls, etc. Simultaneously, plan a laboratory investigation: write and QA‑approve a protocol detailing the hypothesis (e.g. degradation, sampling error, method issue) and testing plan . This plan must document exactly which samples/standards to retest, how (e.g. new extractions, alternate method), and data evaluation criteria . Perform additional analyses to confirm or eliminate each hypothesis: for example, retest another portion of the retained stability sample, prepare fresh solution from the sample, check alternate assay methods or stability‑indicating procedures, and test working standards. Also consider testing retention samples from other time points or other batches. MHRA emphasizes multiple hypotheses (e.g. equipment, method, sample prep) must be tested systematically . Any corrective retesting must not compromise the integrity of the remaining reference samples. * Phase 3 – Conclusion and CAPA: If, after investigation, the result remains outside spec with no assignable lab error, then the batch is formally rejected (batch disposition = “fail”), and broader actions follow . The Phase III investigation reviews all findings: it evaluates whether other batches or ongoing stability studies might be affected and determines product disposition . MHRA guidance states “once a batch has been rejected there is no limit to further testing… and the decision to reject cannot be reversed” . QA/Quality Unit should document all testing done and evaluate process implications. The Qualified Person (QP) must conclude whether the batch can ever be released (usually not, if one assay failed) . Crucially, CAPAs (Corrective/Preventive Actions) are identified and implemented: for example, process corrections, analytical method fixes, equipment maintenance or training. All conclusions, including impact on other products and CAPAs, are documented in the final OOS report . Regulatory Reporting and Recall Because this is a stability fail on a marketed product, it is a “suspected defect” that likely requires immediate notification to the MHRA (Defective Medicines Report Centre) and possibly a recall. Under GMP Chapter 8.15 and MHRA DMRC guidance, any defect that could lead to patient harm must be reported without delay . A stability OOS should not be held until the next timepoint – report it promptly . The licence holder should submit a defect report via the MHRA’s Yellow Card system or direct DMRC contact, including a description of the defect, batch/product details (PL number, batch number, expiry, distribution dates, quantities on hand), and an initial risk assessment . The report should state whether a recall is recommended. If product has been distributed, initiate a recall plan in consultation with QA and DMRC. Classify the recall level per MHRA guidelines: here, an Atenolol assay failure (likely lower potency) could reduce efficacy (risk of mistreatment but not immediate life‑threat). This scenario typically falls under a Class 2 Medicines Recall (“defect may cause harm but is not life-threatening”). MHRA’s table clarifies Class 2 is for “defect may cause harm; not life-threatening” . A Class 1 (NatPSA) patient‑alert would be for severe risk. The licence holder decides recall scope (patient, pharmacy, wholesale) based on risk and stock distribution. Notify the DMRC promptly with details of the recall actions and affected stock. If a MHRA-wide Medicines Recall Notification is warranted (widespread supply or serious risk), the agency may issue an alert . Throughout, coordinate with pharmacovigilance (for any reported lack of efficacy) and follow MHRA’s recall guidance. Root Cause Analysis (Fishbone Diagram) Use a systematic root-cause approach (e.g. Ishikawa “fishbone” diagram) covering all potential factors. On the diagram, consider categories such as Equipment (HPLC calibration, stability chamber sensors, blending/compression machines), Materials (API potency, impurity content, excipient quality, packaging), Methods (analytical method validation, stability‑indicating power, sample preparation, filter use), Manpower (analyst training, oversight, deviations in procedure), Environment/Facilities (laboratory conditions, stability chamber temperature/humidity control, transport/storage conditions), Process Controls (manufacturing process parameters, in-process sampling, batch uniformity), and Documentation (errors in recording, SOPs, label mix-ups). Brainstorm all plausible causes: for example, a decline in Atenolol content could be due to degradation (e.g. moisture or light effects), a formulation inconsistency (blending or coating issue), analytical underestimation (expired reference standard or incomplete extraction), or simple operator error . For each hypothesis, gather evidence: review equipment maintenance logs, re-test calibration standards, analyze retained API and excipients, and inspect stability chamber logs. MHRA guidance encourages testing hypotheses one by one (e.g. retesting with fresh standards, verifying sample prep, re-analyzing in a different lab) to confirm or eliminate each potential cause . Common root‑causes to explicitly consider (and have been noted by MHRA) include technician/calculation errors, sample or standard prep mistakes, analytical method issues, equipment failures, or procedural deviations . CAPA Development and Effectiveness Checks Based on the root‐cause analysis, implement targeted CAPAs. For example, if an unstable assay procedure or calibration issue was found, revise and revalidate the method or replace faulty equipment. If manufacturing issues (e.g. tablet homogeneity or moisture) are implicated, adjust process parameters or supplier specs. If human error (training or oversight) was a factor, retrain staff or strengthen SOPs. Document each CAPA with a clear action plan, responsible person, and timeline. Include preventive measures (e.g. more frequent stability chambers calibration, updated sampling SOPs) to avoid recurrence. Plan effectiveness checks: for instance, run extra stability points, audit the process, or perform trending on subsequent batches to verify that the issue is resolved. Update the Quality Management System records with the CAPAs and monitor their implementation. MHRA guidance stresses that corrective and preventive actions be recorded alongside the investigation conclusion . Investigation Closure and Reporting to DMRC Once all testing, root-cause analysis and CAPAs are complete, finalize the investigation. Prepare a formal investigation report summarizing findings, justification for the OOS, root cause(s) identified, actions taken, and the final disposition of the batch (usually rejection and destruction if the OOS holds). The QP (or QA head) reviews and signs off. If a recall was executed, include the recall/reconciliation report. Submit the final report to the DMRC, as MHRA requires the full conclusion of investigations into defects . Maintain all records in the batch file and GMP quality systems. Finally, confirm closure by verifying that CAPAs are completed and effective (e.g. no recurrence of similar OOS/OOT on future batches). References: MHRA’s current OOS/OOT guidance provides a detailed phased flowchart ; MHRA inspectorate and DMRC communications emphasize timely reporting and root‑cause rigor . Adhering to these steps (including full documentation and notifying DMRC) ensures compliance and patient safety.

Answer 6

Regression analysis is used to determine whether a dataset shows a linear relationship between variables. This is often assessed by calculating the coefficient of determination (R² value). An R² value greater than 0.99 indicates a very strong linear correlation. This method is commonly used in pharmaceutical stability studies to demonstrate that assay results, degradation products, or potency trends remain consistent over time.

Answer 7

1. Initiate Recall and Convene Recall Meeting A recall is initiated upon detection of a serious product defect. A multidisciplinary recall meeting is promptly organised, involving representatives from Pharmacovigilance (PV), Medical, Quality Assurance (QA), Manufacturing, and Regulatory Affairs. 2. Conduct Risk and Impact Assessment The team reviews the nature of the defect, assesses patient safety risks, supply chain impact, and potential regulatory obligations. Based on this, the recall class (I, II, or III) and level (wholesale, retail, patient) are determined in line with MHRA and the Guidelines for the Notification and Recall of Defective Medicines. 3. Notify the MHRA/DMRC The Defective Medicines Report Centre (DMRC) at the MHRA is contacted promptly. Formal notification is submitted using the Yellow Card system and the Defective Product Reporting Form. The recall must not proceed until the DMRC agrees on the recall level and communication content. 4. Execute the Recall Once approved, the recall is executed. Communications are sent to affected stakeholders with clear instructions for return, segregation, and quarantine of defective stock. Distribution records are reviewed to ensure traceability and completeness. 5. Reconciliation and Disposal All returned products are reconciled against distribution data. Any affected stock held on-site is segregated and clearly labelled. Defective stock is securely quarantined and disposed of in accordance with SOPs and waste regulations. 6. Root Cause, CAPA and Effectiveness Check A formal root cause investigation is conducted. Corrective and Preventive Actions (CAPAs) are raised for any process gaps identified. Effectiveness checks are performed to ensure CAPAs prevent recurrence. 7. Closure and Final Reporting A final recall report is prepared and submitted to the DMRC, including reconciliation data, investigation outcomes, CAPAs, and effectiveness verification. The recall is only closed when DMRC formally accepts the outcome.

Answer 8

1. Gather Initial Information: * Ask: What is the related substance? Confirm if it’s a known degradant, process impurity, or new/unknown peak. * Determine: Is the result OOS (Out of Specification) or OOT (Out of Trend)? * Confirm: What is the product, indication, dosage form, and patient population? This informs the risk level (e.g., oncology, paediatric). * Check if the value is still within the ICH specification or justified limits. 2. If OOS or OOT, Initiate Formal Investigation: * Follow MHRA Guidance for OOS Investigations (aligned with the FDA 2006 OOS Guide). * Conduct Phase 1a/1b review: analytical error, instrument calibration, sample prep, standard, etc. * If no assignable cause is found, escalate to Phase 2: cross-functional team investigation (QA, QC, production).

Answer 9

GOV.UK - OOS guidance

Answer 10

1. Initiate OOT Investigation: * Open a formal OOT investigation. * Conduct Phase 1a (by analyst): Check for analytical errors (e.g., sample prep, instrument calibration, calculation). * If inconclusive, escalate to Phase 1b (with supervisor/QA): Review method validity, equipment status, reagent quality, analyst training, etc. * If found to be a true OOT, proceed to Phase 2 – full investigation. 2. Evaluate Trend and Predict Shelf-Life Impact: * Review historical stability data. * Apply statistical trend analysis (e.g., regression) and calculate 95% confidence intervals to estimate when the impurity will breach specification. * Consider increasing stability test frequency (e.g., monthly) to monitor further progression. 3. Product Risk Assessment and Regulatory Impact: * Assess clinical risk based on impurity identity, ICH Q3B thresholds, and indication (e.g., chronic use, paediatrics). * If the product may breach its specification before expiry, initiate recall discussions (Class and Level) and contact MHRA/DMRC as needed. * Ensure QA oversight and consult with regulatory team on required notifications. 4. Deviation and Cross-Batch Impact Assessment: * Open a deviation and perform impact assessment: * Review all batches using the same API batch, packaging component, or manufacturing conditions. * Check reference samples and other stability batches for similar trends. * Review recent manufacturing or storage changes. 5. Root Cause Analysis and CAPA: * Conduct RCA: Focus on API impurity profile, vendor change, environmental storage, or formulation robustness. * Perform supplier audit or data review if linked to raw materials. * Implement CAPA, including long-term preventive measures. * Document and perform effectiveness check (e.g. future stability batches, audit closure).

Answer 11

1. Phase 1a – Performed by Analyst (Within 1 Working Day): The aim is to identify any obvious, assignable errors. Actions typically include: * Re-checking calculations and data transcription. * Confirming correct sample, method, and dilution were used. * Verifying equipment was calibrated, powered, and functioning. * Checking for sample preparation errors or analyst deviations from SOP. * Ensuring correct reference standards and reagents were used and in-date. This phase should be documented contemporaneously. If an assignable cause is found, and it is scientifically justified, the test may be repeated with QA approval. 2. Phase 1b – Performed with Supervisor/QA and Checklist: If Phase 1a is inconclusive, a supervised review using a structured checklist is carried out. This includes: * Reviewing equipment qualification and maintenance/calibration status. * Checking method validation/transfer suitability for the matrix and product. * Reviewing training and competency of the analyst. * Confirming consumables’ quality and expiry date. * Reviewing environmental conditions and logbooks. * Ensuring no mix-up of samples, standards, or reagents. If no assignable cause is found after 1b, the issue may be escalated to Phase 2 (full investigation), which involves QA, production, and possibly a cross-functional team.

Answer 12

1. Documentation Expected for Reworked API Batch: The QP must ensure the following documentation is available and reviewed prior to certification of the finished product: * Rework justification (with scientific rationale and risk assessment). * Deviation record (approved and QA-reviewed). * Approved rework protocol (including batch-specific procedures and additional testing). * Analytical data showing that the reworked API meets all registered specifications. * Impurity profile comparison (pre-/post-rework) to assess against ICH Q3A thresholds. * Stability data or bracketing/justification if stability is affected. * QP confirmation that the rework is in line with the Marketing Authorisation (MA) or supported by a post-approval change. * Reference to the QTA if a third-party API manufacturer is involved, clarifying who holds responsibility for approving and overseeing rework.Reprocessing Repeating an established process step to bring material back into specification. Recrystallising, drying again. Acceptable if within validated process. Must be documented. Rework Introducing a process not part of the approved manufacturing SOP to bring material into compliance. Using a different solvent or pH adjustment not in MA. Higher risk. Must be justified, documented, and may need regulatory notification or variation. 3. Considerations for Market Action: If there is concern over the reworked API and product quality cannot be assured: * Contact DMRC (MHRA) to agree recall class and level. * Recall should not be executed without MHRA/DMRC agreement. * Patient safety risk assessment must consider: * New impurity profile. * Indication (e.g., critical medicines, paediatrics). * Batch size and distribution status. * If alternative product not available: * Coordinate with the Marketing Authorisation Holder (MAH) and regulatory team. * Notify the Department of Health and Social Care (DHSC) through the DASH Portal to support shortage management.

Answer 13

a. Further information on questioning – 2 sets of assay results one OOT and one OOS, but mean within specification and the mean is what has been registered b. Discussed other time points, all ok c. Discussed other tests – expelled dose was also low 73% - medical have advised no lack of efficacy would be noted d. No increase in complaints from PV e. Asked about market – informed that all batch is used within 3-4 months of being certified and placed on market f. Went through oos/t investigation

Answer 14

j. Went into recall for any remaining stock on the market

Answer 15

a. Went through OOS/T – were interested in phase 2 investigation b. Went through investigation and potential probable root causes c. Stopped me at API as had another scenario later on this .

Answer 16

Analytical Method Transfer is the documented process of transferring a validated analytical procedure from a transferring (sending) laboratory to a receiving (testing) laboratory, to ensure the method can be performed reliably at the new site without the need for full revalidation. ⸻ Purpose: * Ensure consistent, accurate, and reproducible results at the receiving lab. * Required when: * Adding a new QC site. * Outsourcing to a contract lab. * Transferring testing within a company network. ⸻ Governing Guidance: * ICH Q2 (R2) – Validation of analytical procedures (ensure method is suitable and robust). * EU GMP Chapter 6 – Quality control. * EU GMP Annex 15 – Qualification and Validation (section on method transfer). * MHRA Inspectorate Blog – Practical expectations and common deficiencies. ⸻ Controlled by Change Control (CC): * A formal change control should be raised and approved. * The change control should include: * Gap analysis: Between transferring and receiving labs (equipment, reagents, training). * Impact assessment: On licence compliance, GMP status, and patient risk. * Method transfer protocol: Outlines method, number of replicates, sample types, acceptance criteria. ⸻ Typical Method Transfer Protocol Includes: * Description of test(s) (e.g., assay, related substances, dissolution). * Reference standards, samples to be tested, and testing schedule. * Acceptance criteria (e.g. %RSD, mean recovery, system suitability). * Transport and storage conditions of test items and standards. * Staff training and qualification at receiving site. * Handling of deviations or OOS results. * CAPA in case of failures or non-conformance.

Answer 17

1. Initiate Change Control (CC): * Open a formal change control to manage the transfer. * Justify the transfer (e.g., new testing site, outsourcing, business continuity). * Appoint responsible parties from both the transferring lab and receiving lab. 2. Conduct Gap Analysis and Impact Assessment: * Senior analytical staff perform a gap analysis covering: * Equipment differences * Reagents and standards * Environmental conditions * Analytical experience * Conduct an impact assessment on: * Product licence (MA/variation requirements) * GMP compliance * Finished product release timelines and patient impact 3. Prepare Analytical Method Transfer Protocol: Include: * Defined test: e.g., Assay of API by HPLC * Samples and reference standards to be tested * Test method (as per validated procedure) * Acceptance criteria (e.g., % recovery, %RSD, system suitability) * Number of replicates or runs to be performed * Transport and storage conditions for samples and standards * Any special handling instructions * Deviation management process * Requirement to raise CAPA if the transfer is unsuccessful * Training requirements for receiving lab analysts 4. Ensure Prerequisites Are Met: * The receiving lab must complete equipment qualification (IQ/OQ/PQ). * Analysts must be trained and qualified. * Perform partial revalidation as needed to demonstrate suitability (aligned with ICH Q2(R2) and Annex 15), typically covering: * Accuracy * Precision (repeatability, intermediate) * Linearity * Specificity * Robustness (if environmental or procedural conditions differ) 5. Execute the Comparative Study: * Test a common sample batch in both labs under identical conditions. * Compare results using statistical methods (e.g., t-test) to confirm equivalence. 6. Review and Conclude Transfer: * QA reviews all results and confirms success. * Document in a transfer report. * Update SOPs, method files, and quality agreements as needed.

Answer 18

A t-test is a statistical method used to compare the means of two sets of data to determine if they are significantly different from each other. It is commonly used in analytical method transfer to assess if the receiving lab generates results equivalent to the transferring lab. ⸻ Step-by-Step Explanation of a t-test: 1. Establish Hypotheses: * Null Hypothesis (H₀): There is no significant difference between the two sets of results. * Alternative Hypothesis (H₁): There is a significant difference. 2. Collect Data: * Use the same sample measured in both labs. * For each lab, calculate the mean (x̄) and standard deviation (SD). 3. Calculate the t-value: * For unpaired data (e.g., different analysts or conditions), use: t = \frac{|\bar{x}_1 - \bar{x}_2|}{\sqrt{\frac{s_1^2}{n_1} + \frac{s_2^2}{n_2}}} * Where: * \bar{x}_1, \bar{x}_2 = means of lab 1 and 2 * s_1, s_2 = standard deviations * n_1, n_2 = number of replicates 4. Determine Degrees of Freedom (df): * For unpaired tests: df \approx \frac{\left( \frac{s_1^2}{n_1} + \frac{s_2^2}{n_2} \right)^2}{\frac{(s_1^2/n_1)^2}{n_1-1} + \frac{(s_2^2/n_2)^2}{n_2-1}} 5. Find the Critical t-value from the t-distribution table: * Based on degrees of freedom and desired confidence level (e.g., 95% = p < 0.05). 6. Interpret the Result: * If the calculated t-value < critical t-value, fail to reject H₀ → no significant difference. * If calculated t-value ≥ critical t-value, reject H₀ → there is a significant difference. Alternatively, compare the p-value to your threshold (usually 0.05). If p < 0.05, it’s statistically significant. ⸻ Application in Method Transfer: * If the t-test shows no significant difference between labs, the method transfer can be considered successful. * This adds confidence in inter-lab equivalency, especially for critical tests like assay or content uniformity.

Answer 19

1. Specificity • Ability to measure the analyte response in the presence of potential impurities, excipients, or degradation products. • Demonstrated using blank, placebo, and forced degradation samples. 2. Linearity • Assesses the correlation between analyte concentration and detector response. • Typically performed at 5 concentration levels (e.g. 80%–120%), with r² ≥ 0.999. 3. Accuracy (Recovery) • Measures closeness of test results to the true value. • Performed by spiking known quantities of API into placebo at 3 concentration levels (e.g. 80%, 100%, 120% of target concentration), with 3 replicates each. • Total: 9 independent sample preparations, evaluated for % recovery (typically acceptable range: 98–102%). 4. Precision • Includes: • Repeatability: Same analyst, instrument, short time frame (usually 6 injections). • Intermediate precision: Different analysts, instruments, days within the same lab. • Reproducibility: Optional, between different labs. • Evaluated using %RSD. 5. Range • Interval where the method demonstrates suitable accuracy, precision, and linearity. • For assay methods: typically 80% to 120% of label claim. 6. Robustness • Assesses the method’s resilience to small, deliberate variations (e.g. ±10% flow rate, pH variation, mobile phase composition). • Identifies critical method parameters. 7. Solution Stability • Confirms that standard and sample solutions remain stable throughout analysis. • Evaluated at different time points (e.g. 0, 6, 24 hours).

Answer 20

• LOD and LOQ are not required for assay methods. Assay methods quantify the API at high concentrations (e.g. ~100% of label claim), where sensitivity is not the focus. • LOD and LOQ are required for methods that detect or quantify low levels of impurities or degradation products, especially when the impurity may have safety implications. Use cases: • Required for: • Impurity profiling (e.g. genotoxic impurities, degradation products) • Residual solvent analysis • Limit tests (e.g. elemental impurities) • Not required for: • API or drug product assay • Content uniformity (typically) LOD determines the lowest amount of analyte that can be detected (not necessarily quantified). LOQ is the lowest amount that can be quantified with suitable accuracy and precision.