boston regulated bioanalysis
TRANSCRIPT
Regulated Bioanalysis:Science and Art of Meeting the Requirements
Dr. Bhaswat S. Chakraborty30.10.2007
India Technology SeminarBoston, 29 October – November 1, 2007
Bioanalytical Guiding Principles Used in quantitative determination of drugs and
their metabolites in biological fluids BE, PK, TK and Meatbolomics studies
Often the Study is as good as the bioanalytical Refinement of all aspects in last 20 years Implemented as though regulations More and more demanding
From 1980s to date US FDA setting the standards
Key Guiding Documents & Publications Shah VP et al. Analytical methods validation…. Eur J
Drug Metab Pharmacokinet. 1991 Draft Guidance on Bioanalytical: USFDA. 1999 Bioanalytical Workshop. 2000 Guidance for Industry: Bioanalytical: USFDA. 2001 DeSilva B et al. Validation of ligand-binding assays…
macromolecules. Pharm Res. 2003
Draft Guidance on Safety Testing of Drug Metabolites: USFDA. 2005
FDA-AAPS Bioanalytical Workshop. 2006
Scientific Questions behind Guidance Does the method measure precisely and accurately the
analyte(s)?
If so, how was it validated? How complete and accurate is the validation?
Were adequate CC and QC used during incurred sample runs? Was each and every batch acceptable?
Were the incurred samples (& repeats) accurately and reproducibly measured and were they stable at all times?
Is the documentation comprehensive and reconstructive?
Non-Chromatographic Assays: Issues E.g., RIA & other ligand binding assays Selectivity
Specific Non-specific or Matrix Often analyte is also present endogenously
Quantification Non-linear CC More points than Chromatographic CC Accuracy is questionable, so replicates required
Cross validation Stability is a complex issue
Stabilty of receptor binding activity as well?
Non-Chromatographic Assays: Validation
Deplete the matrix of the analyte or employ a “surrogate” matrix
5 or more validation concentrations Intra- and interbatch variation LLOQ, ~3 times LLOQ, mid [geometric mean], 75% of
ULOQ,and ULOQ At least 2 independent determinations
Interbatch imprecision (%CV) and inaccuracy (%RE): ±20% (25% at LLOQ and ULOQ).
Total target error (imprecision and inaccuracy) ≤30% (40% at LLOQ &ULOQ)
Non-Chromatographic Assays: Batch Runs CCs
At least 75% of the standard points should be within 20% of nominal concentrations (25% at LLOQ)
Does not apply to “ anchor calibrators, ” those outside the anticipated validation range and improve “ sigmoidal ” fitting
QCs LQC, MQC, HQC in duplicate 4 – 6-20 rule
4 of the 6 QCs must be within 20% of nominal At least one QC per conc. meets this criterion If additional QCs are used, then 50% of them be within 20%
of nominal
Non-Chromatographic Assays: Cross Validation
E.g., RIA vs LC-MS The comparisons can be done both ways. Cross-validation with spiked matrix and subject
samples be conducted at each site or laboratory Interlaboratory reliability
if analyses within a single study are conducted at more than one site or lab
If data generated using different analytical techniques
Chromatographic and Hyphenated Assays
HPLC, UPLC, GC, LC-MS More selective and accurate than LBAs Used for small molecules
Drugs, metabolites, toxicants Linear response LC-MS: highly sensitive High capacity Highly reproducible response stability
LC-MS or LC-MS-MS LC coupled with one or mass analyzers
Most popular, very accurate, high throughput Quadrupole mass analyzers: most widely used
Aligns different m/z according to their retention times Time-of-flight mass analyzers
Analyzes different m/z according to the different times taken to traverse a fixed distance
FTICR mass analyzers Applies a radiofrequency voltage at the same cyclotron
frequency of a m/z, the later latter is moved at a larger radius than the RF
Validation Batches
Analyze at least 3 batches for accuracy and precision
At least 1 validation batch should be made as large as the largest anticipated sample analysis batch
Calibration Samples & Acceptance Criteria
CC concentrations A minimum of 6 non-zero standards Matrix blank: Matrix sample without internal standard Zero standard: Matrix sample with internal standard
Acceptance criteria Intra- and inter-batch imprecision (%CV) and inaccuracy
(%RE) ≤15% except at LLOQ where up to 20% can be allowed
CC: Intra-Batch
N 4 4 4 4 4 3 4 4
MEAN 68.858 56.445 51.053 36.433 15.498 7.957 0.983 0.490
SD() 4.5328 4.0874 4.2432 2.8389 1.1250 0.4143 0.0785 0.0183
%CV 6.58 7.24 8.31 7.79 7.26 5.21 7.99 3.73
%NOMINAL 93.08 95.38 103.51 98.49 104.71 107.52 99.24 100.00
QC Samples & Acceptance Criteria QC concentrations
LLQC: About 3 times the LOQ; MQC: ~geometric mean of LQC & HQC; HQC: ~70% to 85% of ULOQ; Dilution QC: sufficient to cover highest anticipated dilution
Acceptance criteria Intra- and inter-batch imprecision (%CV) and
inaccuracy (%RE) ≤15% except at LLOQ where up to 20% can be allowed
QC: Inter-Batch
Selectivity (Non-interference from Matrix)
Review noninterference in at least 6 sources of matrix for non-MS assays
For MS assays determine MFs in 6 sources if the nonisotopically labeled IS is used
If isotopically labeled IS is used, demonstrate that IS normalized MF is close to unity
Interference in blank matrix ≤20% of LOQ
Example
Reproducibility of the Method
Precision and accuracy: Inter-run precision and accuracy of the QCs
Second column or instrument verification: Reproducibility of the method on an alternate column or
instrument A batch of precision and accuracy samples is analyzed on a
different column or instrument on one of the days of validation. Good practice but not mandatory
Reproducibility using incurred samples: Sample availability can be an issue
Example: Column to Column Ruggedness
Analyte Recovery
Detector response of extracted vs direct input Extent of recovery of analyte and IS
Demonstrate that it is Consistent Precise Reproducible
Determine recovery at 3 concentration levels Does not have to be close to100%
Example: Drug Recovery
Low QC Extracted response Unextracted response
325712 605788 330450 611672 332840 621078 309710 607364 345282 606242
375431 602309 N 6 6
Mean 336570.83 609075.50 SD (±) 22257 6608 CV (%) 6.61 1.08
Mean recovery (%) 55.26
Stability of Samples Stock solution:
Minimum of 6 hours at room temp. & Fridge temp. for 24 hr Postpreparative (extracted samples/autosampler tray):
Longest time from preparation through analysis. Vs. fresh standards
Benchtop: At ambient temp. (or processing temp.) – for extraction duration
(typically ~4-24 hr) Freeze-thaw:
QC samples at least 2 conc., 3 cycles, completely thawed, refrozen for 24/12 hr, at anticipated storage temp.
Long-term: Can be postvalidation For longest time – collection to for any sample (3 aliquots; low
and high conc. with fresh standard curves); assess vs. nominal
Example
Dilution Integrity for Concentrations >ULQ
One or more additional QC >>ULQ prepared and diluted with blank matrix to bring the concentration to within the calibration range and then analyzed
The acceptance criteria for the diluted QC are the same as for other QCs Intra- and inter-batch imprecision (%CV) and
inaccuracy (%RE) ≤15%
Example
Dilution integrity accuracy (as QC %nominal value)
For 1/5th dilution factor is 96.13 For 1/10th dilution factor is 111.49
Dilution integrity precision (as QC %CV) For 1/5th dilution factor is 6.82 and For 1/10th dilution factor is 4.76
MS Techniques: Matrix Factor
Syringe Pump
Autosampler
MSDrug ISTD
Ion Suppression
Ion Enhancement
MS Techniques: Matrix Factor (MF) A quantitative measure of the matrix effects due to
suppression or enhancement of ionization in an MS detector
MFs can be determined for the analyte and the IS Ratio is called IS-normalized MF for the analyte
IS-normalized MFs using stable isotope labeled IS Usually close to unity for bioanalytical samples
MF or IS-normalized MF be determined in 6 independent lots of matrices with desirable CV <15% (not for stable isotope labeled IS using methods)
PK Repeats SOP
All PK repeats are chosen before interim analysis of data
Based on sound PK and Bioanalytical principles without any bias
Original value is retained if the repeat value is within 15%
Repeats with one or more “correct” estimates
Ct
t
CC Algorithm & Estimation of the Unknown
Data
Conc. (ng/ml) Peak Area Ratio
1501 1.541002.5 0.91
703 0.77249.5 0.2599.5 0.1330 0.0410 0.015 0.01
Comparison: LS vs. MLE
…very similar in this case but can be very different in other cases
Least Squares MLE
Intercept 0.009557334 0.009426942
Slope 0.00099529 0.001014858
Std. Error 0.023385281 0.029742873
df 6 6
Least Squares vs. MLE
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 200 400 600 800 1000 1200 1400 1600Conc. (ng/ml)
Pea
k A
rea
Rat
io
MLE
Least Squares
Comparison: LS vs. MLEAn Example where differences can be substantial
X Y Least Squares MLE5.66 59
4.63 43 Intercept 3.192698019 10.362372545.21 41 Slope 7.424965403 6.6212443576.01 47 Std. Error 0.157779577 3.3001033426.17 53 df 10 104.82 435.08 465.51 454.95 415.13 424.66 394.74 45
Linear Weighting (1/X2 and 1/Y2) & MLE
Least Squares MLE Weighted (1/X^2) Weighted 1/Y^2
Intercept 0.009557334 0.009426942 0.00393 0.00262
Slope 0.00099529 0.001014858 0.00104 0.00102
Std. Error 0.023385281 0.029742873 -- --
df 6 6 6 6
Conclusions
Current Guidelines are based on
Expertise Correct understanding of the isolation of the analyte from
matrix, metabolites & noise Relevant approach to method development & validation, e.g.,
target bias, establishment of method characteristics
Ethics Non-deviation from protocol, QA, QC, audit trail
Focus Specificity, accuracy, precision, reproducibility & stability
You are Successful when Your Data
Enable the regulators know exactly what you know & come to the same conclusions
Provide evidence of ‘correct’ determination of unknown concentrations
Present validation and analytical report Are traceable, accounted for and of proven
integrity Are complete
In addition ..
Be aware of CC algorithms, their strengths & pitfalls Peak smoothing techniques & expertise Peak instability, non-reproducibility of a specific assay Having correct & enough SOPs Long term sample storage Assays where replication of clinical samples assay may be
necessary Regulatory queries & how to respond to them
Acknowledgments:
Dhriti ChakrabortyChinmoy GhoshAdinarayana Andy
THANK YOU