**National Cancer Institute**

**Division of Cancer Prevention**

**Requirements for:**

**PHARMACOKINETIC AND BIOMARKER **

**METHODS DEVELOPMENT**

**1.0 PHARMACOKINETIC AND BIOMARKER METHODS DEVELOPMENT AND VALIDATION
REPORTS **

All pharmacokinetic and biomarker laboratory techniques, assays, and
procedures shall be developed and validated according to the parameters
described in this section, and a draft report prepared for NCI, DCP
approval. The approved Method Development and Validation Report will be
incorporated into the contract Final Report. The format of this section
should be followed when preparing Pharmacokinetic and Biomarker Methods
Development and Validation Reports.

Summaries of specific recommendations for methods development and
validation are presented in this section. In general, establish the
following for each method:

\_C Conditions to ensure stability of the analyte or antigen in the
matrix during collection, processing, storage and analysis.

\_C Linear range: The range over which the procedure has been
demonstrated to produce a reproducible, linear response. (A
reproducible, nonlinear response may also be acceptable.)

\_C Specificity: The ability of the method to measure only what it is
intended to measure.

\_C Sensitivity: Limit of detection, limit of quantitation, and 95%
confidence interval of standard curve.

\_C Inter-day and intra-day accuracy: The closeness of determined value
to the true value.

\_C Inter-day and intra-day precision: The variability of replicate
determinations.

\_C Ruggedness: Variability between operators, instrument, columns,
*etc*.

Draft Methods Development and Validation Reports shall include all
analytical and computational documentation resulting from these efforts.
In addition, sample chromatograms or endpoint measurements from method
development should be submitted; densitometric scans, photographs,
*etc.,* may be presented as appropriate. These samples should include
the following: blank plasma, urine, or tissue; analyte in buffer; and
analyte in plasma, urine, and/or tissue.

When sample analysis is conducted at more than one site, it is necessary
to validate the method at each site and provide appropriate validation
information for different sites to establish interlaboratory
reliability. Any modification of an analytical method would require
revalidation of the procedure.

**2.0 PHARMACOKINETIC ANALYTICAL METHODS DEVELOPMENT AND VALIDATION**

Recommendations for pharmacokinetic methods development and validation
are derived from the Conference Report *Analytical Methods Validation:
Bioavailability, Bioequivalence and Pharmacokinetic Studies* \[1\].

Analytical methods employed for the quantitative determination of drugs
and their metabolites in biological samples play a significant role in
the evaluation and interpretation of pharmacokinetic data. It is
essential to employ well-characterized and fully validated analytical
methods to yield reliable results which can be satisfactorily
interpreted. Specific validation criteria are needed for methods
intended for analysis of each analyte (drug and/or metabolite). Method
validation includes all of the procedures required to demonstrate that a
particular method for the quantitative determination of the
concentration of an analyte is reliable for the intended application.
The parameters essential to ensure the acceptability of the performance
of an analytical method are stability of the analyte in the biological
sample under study storage conditions, accuracy, precision, sensitivity,
specificity (selectivity), response function, and reproducibility.

** **2.1 Specific Recommendations for Pharmacokinetic Analytical Method
Development and Validation

2.1.1 A specific detailed description of the analytical method (standard
operating procedure; SOP) must be prepared, including a statement of
purpose, a listing of necessary reagents, test solutions and mixtures,
and directions for preparation, storage, and expiration dating of
required materials. It should also include a listing of the required
instruments and instrumental parameters. Representative chromatograms
and calculations, if used, should also be provided. The SOP should
contain sufficient information to allow repetition of the method by a
qualified investigator.

2.1.2 Each step of the method should be investigated to determine the
extent to which environmental, matrix, material, or procedural
variables, from the time of collection of the material up to and
including the time of the analysis, may affect the estimation of analyte
in the matrix. Whenever possible, the same biological material as that
in the intended samples should be used for validation purposes.
Variability of the matrix due to its physiological state, and the
influence of freeze-thaw cycles should be considered, if appropriate.
The results of these investigations will define acceptable conditions
and restrictions for collection, storage and analysis of samples; these
conditions are then stated in the SOP.

2.1.3 The concentration range over which the analyte can be determined
must be defined in the method, based on evaluation of actual standard
samples, including their statistical variation. This defines the
standard curve. The standard curve consists of at least five
concentrations, excluding blanks, and should cover the entire range of
expected concentrations. The simplest relationship for response as a
function of concentration should be determined and the fit statistically
tested; this relationship must be demonstrated to be continuous and
reproducible. The equation of the function should be given and
represented graphically.

2.1.4 The limit of detection (LOD) is the lowest concentration of an
analyte that the method can reliably differentiate from background
levels. The lower limit of quantitation (LOQ) is the lowest
concentration on the standard curve that can be measured with acceptable
accuracy, precision and variability, respectively. The LOQ is determined
by using at least five samples independent of standards and by
determining the coefficient of variation (CV) and/or appropriate
confidence interval. The lower LOQ serves as the lowest concentration of
the standard curve.

2.1.5 Accuracy and precision with which known concentrations of analyte
in the biological matrix can be determined must be demonstrated. Within-
and between-run accuracy and precision should be calculated with
commonly accepted statistical procedures. Determination of accuracy and
precision can be accomplished by analysis of replicate sets of analyte
samples of known concentrations from an equivalent biological matrix,
using a minimum of five determinations per concentration. Specific
criteria must be set for accuracy and precision over the range of the
standard curve. At least three concentrations representing the entire
range of the calibration curve should be studied---one near the lower
LOQ, one near the center, and one near the upper boundary of the
standard curve. The mean value should be within ±15% of the actual value
except at LOQ, where it should not deviate by more than ±20%. The
precision around the mean value should not exceed 15% CV, except for
LOQ, which should not exceed 20% CV.

2.1.6 If possible, the specificity of the assay methodology should be
established using six independent sources of the same type of biological
sample.

2.1.7 If there are intermediate steps between the biological matrix and
the final assay, such as extraction of biological samples, and if
parallel processed standards are not being used, recovery should be
established and used to determine results.

2.1.8 It is suggested that validation include investigation of samples
from dosed subjects. If possible, stability of the analyte in the matrix
from dosed subjects should be confirmed.

2.2 Recommendations for Routine Application of Pharmacokinetic Methods

2.2.1 A standard curve should be generated for each analytical run for
each analyte and should be used to calculate the concentration of
analyte in unknown samples within that run.

2.2.2 Acceptance criteria for application of the assay and a protocol
for repeat analysis should be established prior to sample analysis.
Accuracy should be not more than 15% deviation from the nominal value
and precision should be not more than 15% CV. At the LOQ, 20% is
acceptable for both precision and accuracy.

2.2.3 Quality-control (QC) samples are matrix spiked with analyte. QC
samples in duplicate at three concentrations (one near the LOQ, one in
midrange, and one approaching the high end of the range) should be
incorporated into each run. Results of QC samples provide the basis for
accepting or rejecting the run.

2.2.4 Estimating unknowns by extrapolations below the low standard or
above the high standard should be avoided. Instead, the standard curve
should be redetermined or samples reassayed after dilution.

**3.0 BIOMARKERS METHODS DEVELOPMENT AND VALIDATION**

For biomarker assays in general, the recommendations in 1.0 should be
followed. Though many types of assays exist, only immunohistochemical is
specifically discussed. Immunohistochemistry is used to characterize a
wide range of biomarkers. This technique is not linear, it saturates at
higher intensities of staining, and is accentuated at the lower
intensities of staining. Dr. Grizzle has extensive experience with
immunohistochemical techniques and has developed a semiquantitative
index which incorporates both the intensity of staining of individual
cells and the percent of cells staining at each intensity \[2\]. The
parameters essential to ensure the acceptability of this technique
include determining stability of the antigen in the biological sample
(*e.g*., tissue biopsy) under study storage conditions, sensitivity of
the assay, specificity of the antibody, and intra- and inter-assay
reproducibility.

3.1 Specific Recommendations for Immunohistochemical Methods Validation

3.1.1 A specific detailed description and protocol of analytical method
and scoring methodology (standard operating procedure) includes a
statement of the method, a listing of necessary reagents, test solutions
and mixtures with directions for their preparation, storage conditions
and usable shelf life specified. It should also include a listing of the
required instruments and instrumental parameters, which contains
sufficient information to allow repetition of the method by a qualified
investigator.

3.1.2 Stability of the antigen in the biological sample is determined
under identical study storage conditions (*e.g*., temperature, duration)
and sample conditions (*e.g*., frozen tissue biopsy, paraffin-embedded
tissue).

3.1.3 Sensitivity of the assay should be determined using at least three
sequential antibody dilutions.

3.1.4 Specificity of the assay should be determined by using at least
two different antibodies to the same antigen, if feasible. It is also
important to perform the assay in the absence of antibody in order to
determine nonspecific staining.

3.1.5 Intra-assay reproducibility should be determined by staining
different serial sections from the same biological sample and/or by
showing that different antibodies to the same antigen give similar
patterns and intensities of staining.

3.1.6 It is necessary to purchase a sufficient amount of reagents
(*e.g.*, antibodies) with the same lot number to complete the study.

3.1.7 Inter-assay reproducibility should be determined by staining the
same tissue sections for the same antibodies during multiple different
assay runs on different days. For example, Dr. Grizzle demonstrated
reproducibility by staining seven biological samples with three
dilutions of various antibodies of interest. The staining was repeated
four times each on different days and the results were analyzed by a
qualified investigator without knowledge of the stain, concentration, or
repeat status of the stain. Several parameters can influence
reproducibility. First, variability in antigen expression within the
tissue can be minimized by using adjacent sections. Second, day to day
variability due to differences in staining procedure and conditions can
be minimized by using an automated stainer.

Third, variability introduced by the evaluator can be minimized by
averaging the scores of two or more observers.

**4.0 REFERENCES**

1\. Shah, V.P., Midha, K.K., Dighe, S., McGilveray, I.J., Skelly, J.P.,
Yacobi, A., Layloff, T., Viswanathan, C.T., Cook, C.E., McDowall, R.D.,
Pittman, K.A., and Spector, S. Analytical methods validation:
Bioavailability, bioequivalence and pharmacokinetic studies. *Pharm.
Res.* [9]{.underline}: 588--592, 1992.

2\. Myers, R.B., Lampejo, O., Herrera, G.A., Srivastava, S.,
Oelschlager, D., Brown, D.A., Waterbor, J.W., Grizzle, W.E. TGF\"
expression is a relatively late event in the progression of prostatic
adenocarcinoma. *J. Urol. Pathol.* [3]{.underline}: 195--203, 1995.