FIT-FOR-PURPOSE BIOANALYTICAL VALIDATION AND SAMPLE PROCESSING - - PowerPoint PPT Presentation

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FIT-FOR-PURPOSE BIOANALYTICAL VALIDATION AND SAMPLE PROCESSING

Raymond H. Farmen, Ph.D. Vice President, Global Bioanalytical Services Kirk Newland Bioanalytical Principal Investigator for Tobacco Products

SLIDE 2

Goals

• When a study is GLP
• Bioanalytical guidances
• Bioanalytical batch
• Value of a system suitability process
• Requirement of standards
• Relationship between standards and quality control samples
• Various types of regression parameters used to define a batch
• The importance and timing of proper chromatographic integration
• Batch acceptance criteria
• Dilution integrity
• Incurred sample reproducibility
• How to report sample concentrations

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SLIDE 3

GLP Cornerstones

• Say what you’re going to do
• Do what you said you would do
• Document it

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SLIDE 4

GLP Cornerstones

• Say what you’re going to do
• Write a study plan that is based upon your SOPs
• SOPs must be based upon a detailed understanding of

international bioanalytical guidances

• Do what you said you would do
• Document it
• Who, when and with what equipment
• Data change control – who made the change? when did it
• ccur? what was the original value? why was the change made?
• QC reviewed
• QA audited

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SLIDE 5

Bioanalytical Guidance’s – Assay must be:

• Selective
• Accurate
• Precise
• Stable
• Sample collection and handling
• Freezer (-20
• C or -80
• C)
• Freeze/Thaw
• UV light sensitivity
• Benchtop
• Pre-extraction
• Post-extraction

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Nicotine selectivity vs. contamination: Selectivity also means that sources

• f contamination are minimized!

Every surface (transfer tubes, pipette tips, injection vials, etc.) must be rinsed with methanol.

SLIDE 6

Bioanalytical Batch History

• 1st Crystal City Meeting in 1990 – Bioanalytical

foundation was created

• GMP guidelines existed – verifying a known concentration
• Clinical chemistry guidelines existed – normal or abnormal
• Bioanalytical chemistry needed accuracy and precision over a

broad concentration range for PK analysis

• Bioanalytical Batch!
• Standards & Quality Control (QC) Samples in every batch
• If the standards and the QCs passed acceptance criteria then all
• f the systems, equipment and personnel used to generate that

batch were working successfully that day

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SLIDE 7

Bioanalytical Batch

• Stand alone entity
• No data from a different experiment can be used as supportive

data

• Standards, QCs and study samples are all processed at the

same time in the same experiment by the same analyst using the same equipment

• What about hydrolyzing samples for glucuronides & aglycones?
• Standards and QCs must be prepared in matrix that has been

shown to be free of interferences

• In contrast - some clinical chemistry assays use one standard

curve/week

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SLIDE 8

Bioanalytical Batch on LC-MS/MS

• LC-MS/MS System Suitability
• Mass spectrometers have a tendency to drift over time
• Process must be developed to assure the instrument

response is stable during a batch

• The following MS parameters must be consistent

between method development and sample processing:

• Smoothing factor
• Dwell time

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SLIDE 9

Bioanalytical Batch – Standards & QCs

• Each batch must contain its own standard calibration curve
• Minimum of six different calibrator concentrations
• Range of the standard curve should reflect the expected range of the

study sample concentrations

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• 5

15 35 55 75 95 115 135

• 1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Standard QC

Clinical Sample Results

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Bioanalytical Batch – Standards & QCs

• Each batch must contain its own standard calibration curve
• Minimum of six different calibrator concentrations
• Range of the standard curve should reflect the expected range of the

study sample concentrations

• Each batch must contain a minimum of 3 QC concentrations in

duplicate (low, middle and high)

• What’s the difference between standards and QCs?
• Philosophically – standards create the calibration curve and QCs

demonstrate that non-standards can be accurately measured – so they must be different

• Different weighings – getting standards and QCs to match each other is

sometimes very difficult

• QCs are stored with study samples to verify sample integrity

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SLIDE 11

Bioanalytical Batch – Regression Parameters

• Regression curve model can be linear or quadratic for

chromatographic assays and a 4-parameter logistic (4PL) non-linear model for ELISA type assays.

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Linear Quadratic 4PL

• The regression model can be weighted by no-weight, 1/x or 1/x2 where

x = concentration.

• The simplest regression model and weighting must be used and it is

selected during method validation.

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Bioanalytical Batch Chromatographic Integration

• Big Deal – the vast majority of serious audit findings involve

scientists modifying integration parameters for QCs to get them to pass acceptance criteria.

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Consistent with other samples Original computer generated Inconsistent with other samples Manually drawn QC = +15.7% QC = +14.9%

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Bioanalytical Batch – Chromatographic Integration

• Big Deal – The vast majority of serious audit findings

involve scientists modifying integration parameters for QCs to get them to pass acceptance criteria.

• Best Practice – Review all chromatographic integration

and manually redraw baselines then “lock-it-down” prior to performing regression analysis. It should be very difficult to modify integration following regression analysis.

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SLIDE 14

Bioanalytical Batch Batch Acceptance Criteria

• Review
• All standards, QCs and study samples were analyzed on

the same day by the same analyst(s) using the same equipment

• For LC-MS/MS methods, the system suitability process

proved that the instrument response was stable during injection of the entire batch

• All chromatographic baseline integration has been

reviewed, was consistent and “locked-down”

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Now you are ready to review and accept the batch

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Bioanalytical Batch Batch Acceptance Criteria

• Standard Curve Acceptance
• Must use the same regression model and weighting option that

was chosen during method validation

• Must have an SOP in place that describes how to reject

standard(s)

• Only sample concentrations that are within the range of your

accepted standards can be reported

• QC Acceptance
• The predicted concentration of 2/3 of the QC samples must be

within 15% of their nominal concentration (20% for ligand binding assays)

• At least 50% of the QCs must be accepted at each QC level

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SLIDE 16

Bioanalytical Batch Batch Acceptance Criteria

• Blank (no internal standard) and standard zero samples
• Prepared with matrix that was free of interferences
• At least 50% of these samples must have a response that is <20% of

the response of the lowest standard – reject low standard

• Sample Dilution
• Can only report sample concentrations that fall within the range of the

standard curve

• Sample concentrations that exceed the concentration of the highest

standard must be diluted

• Must prepare dilution QCs and dilute them using the same respective

dilution (for example, 1/10 dilution)

• Dilution QCs are used for acceptance of the diluted samples
• 50% of dilution QCs must be within 15% of their nominal concentration

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SLIDE 17

Incurred Sample Reproducibility (ISR)

• Purpose is to demonstrate that re-assaying study

samples is reproducible.

• Recommended by FDA and EMA
• Generally accepted practice:
• minimum 20 samples
• If n < 1000 samples then reanalyze 10%
• If n >1000 samples then reanalyze 100 samples + 5% of

samples beyond 1000

• Select more subjects with fewer samples per subject
• Acceptance Criteria: The difference between the repeat value

and the original value < 20% (< 30% for ligand binding assays) for 2/3 of the samples

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How to Report Sample Concentrations

• Demonstrate the sample analysis plan was followed
• Must have an SOP to select samples for re-analysis
• Must show that the acceptance criteria for each batch was

met

• Demonstrate inter-batch reproducibility: List all of the QCs by

batch and perform statistical analysis (% mean deviation and %CV) on each QC level for the study

• Report sample concentrations that were < the concentration
• f the lowest standard as < LLOQ
• Report sample concentrations that were > the concentration
• f the highest standard as >ULOQ
• Prepare a table by batch as either passed or failed

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SLIDE 19

Conclusion

• It’s easy to say that you follow GLP. It’s challenging to

have GLP processes in place that remain current.

• Selective, accurate, precise, stable and documented!
• Bioanalysis is all about compliance!
• Sample processing revolves around the “BATCH”
• Stand alone experiment – uses the same analysts(s) and stable

equipment on the same day

• Must have SOPs that minimize data “interpretation”
• Strict acceptance criteria for standards, QCs and blanks
• ISR analysis is a requirement

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• Reminder – Bioanalysis is all about compliance!