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Drug Metab Pharmacokinet. 1991 Draft Guidance on Bioanalytical: USFDA. 1999 Bioanalytical Workshop. 2000 Guidance for Industry: Bioanalytical: USFDA. 2001 DeSilva B et al.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Draft Guidance on Safety Testing of Drug Metabolites: USFDA. 2005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