Analytical Validation SOP-0014

Procedure Document
Number:
SOP-0014
Revision: 1
Effective Date:
Title: Assay Validation Page: 1 of 10

Property of Novartis Molecular Diagnostics (MDx)
May not be used, divulged, published, or otherwise disclosed without the consent of MDx

1.0 PURPOSE
The purpose of this procedure is to define the Novartis Molecular Diagnostics, (herein after referred to as
MDx) Assay Analytical Validation strategy during the product development cycle and to provide an
overview of the Assay Validation program.
2.0 SCOPE
All MDx locations are within the scope of this procedure.
The scope of this procedure is applicable to laboratories dealing with:
O Development of procedures to carry out assay performance characteristics studies during the
validation for aII MDx deveIoped assays onIy.
Excluded from the scope of this procedure are activities surrounding stability testing and analytical
method validation procedures. Also excluded from this procedure is "inter-lab variance evaluation. This is
done as part of clinical trials where multiple sites are running the study. Biostatistics group will analyze
the data and evaluate inter-lab variance.
3.0 RESPONSIBILITIES
Function Responsibility
Method Owner
O Method owner is responsible for writing, reviewing the method
(assay) validation plan, protocols and reports.
O Method owner is responsible for creating and reviewing appropriate
predetermined acceptance criteria and providing the appropriate
statistical rationale.
O Method owner is responsible for planning and organizing validation
execution resources. This includes scheduling, materials, equipment,
and operators.
O Method owner is responsible to ensure that the employees carrying
out validation have completed the training on relevant documents to
conduct validation.
O Method owner is responsible to ensure that all instruments used
during validation have been properly calibrated and serviced with
completed documentation.
":aIity Ass:rance
O Quality Assurance is responsible for reviewing/approving method
validation plans, protocols, reports, and periodic assessments.
O Quality Assurance is responsible for approval of protocol with
predetermined acceptance criteria.
Diagnostics
DeveIopment
O Diagnostics Development is responsible for environmental
monitoring of equipment and laboratories.
O The Diagnostic Development project team is responsible to execute
the validation plan.
Biostatistics
O Biostatistics is responsible for reviewing/approving method validation
plans, protocols, and reports when applicable.
O Biostatistics is responsible for reviewing the predetermined
acceptance criteria and for executing the protocol, if the validation
requires biostatistical analysis.

Procedure Document
Number:
SOP-0014
Revision: 1
Effective Date:


4.0 REFERENCES
4.1 SOP-0005 Risk Management, current revision
4.2 SOP-0013 Statistical Techniques and Methods, current revision
4.3 W-0037 Process Validation, current revision
4.4 21 CFR 11 820.75 Process Validation
4.5 Evaluation of Precision Performance of Quantitative Measurement Methods: Approved CLS
Guideline-Second Edition, EP05-A2 (Vol. 24, No. 25, 2004).
4.6 Evaluation of the Linearity of Quantitiative Measurement Procedures: A Statistical Approach:
Approved CLS Guideline, EP06-A (Vol. 23, No.16, 2003).
4.7 nterference Testing in Clinical Chemistry: Approved CLS Guideline-2
nd
Edition, EP07-A2 (Vol.
25, No. 27, 2005).
4.8 Method Comparison and Bias Estimation Using Patient Samples: Approved CLS Guidelines-
Second Edition (nterim Revision) EP-09A2-R (Vol. 30, No.17, 2010).
4.9 User Protocol for Evaluation of Qualitative Test Performance: Approved CLS Guideline-Second
Edition, EP12-A2 (Vol. 28, No. 3, 2008).
4.10 Evaluation of Matrix Effects: Approved CLS Guideline-Second Edition, EP 14A2 (Vol. 25, No.4,
2005).
4.11 NCCLS Protocols for Determination of Limits of Detection and Limits of Quantitation: Approved
CLS Guideline, EP17-A (Vol. 24, No. 34, 2004).
4.12 Defining, Establishing and Verifying Reference ntervals in the Clinical Laboratory: Approved
CLS Guideline-Third Edition, C28-A3c (Vol. 28, No. 30, 2008).

5.0 DEFINITIONS, ACRONYMS, ABBREVIATIONS
5.1 Predetermined Acceptance Criteria: Specified indicators or measures employed in assessing the
ability of a method (assay) to perform its intended function.
5.2 Method Validation: The process by which it is established, by laboratory studies, that the
performance characteristics of the method (assay) meet the requirements for its intended clinical
applications.
5.3 CLS: Clinical and Laboratory Standard nstitute.
5.4 NCCLS: National Committee for Clinical Laboratory Standards.
5.5 SST: Serum Separator Tube

6.0 SAMPLES AND CONTROLS AC"UISITION:
6.1 Specimen types: The Method Owner, in conjunction with the Medical Officer and Regulatory
Affairs, will acquire the samples (including patient samples) for the study. These samples should
cover the entire assay range; inclusive of all the sample types intended in the product insert

Procedure Document
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SOP-0014
Revision: 1
Effective Date:


claims (e.g., body fluids, urine, blood, serum, SST, Li-plasma, EDTA-plasma, cell or tissues,
swab and biopsy, etc).
6.2 Specimen collections and acquisitions: The patient samples could be collected from retrospective
or prospective studies based on the guidance from the Medical Officer and Regulatory Affairs.
According to the FDA guidelines, these samples would be collected either under RB approval or
at a minimum with "informed consent from the patients. f samples are acquired from external
vendors, the method owner will seek documentation from the vendor that the samples were
collected with informed consent.
6.3 Specimen Storage and Stability: Storage and stability of the patient samples is critical during the
assay validation. Stability of the analyte(s) is assay dependent and the method owner will
establish the timeline for the acquisition of the samples based on the shelf life and stability of the
samples. The stability of the samples and analyte is also affected by storage conditions (e.g.,
multiple freeze-thaw cycles) and should be taken into account during the validation studies.
6.4 Diagnostics Development will maintain the environmental monitoring data that corresponds to the
storage location of the samples. This provides evidence that the integrity of the samples has not
been affected during the storage and until the validation studies have been completed.
6.5 Contrived or Simulated samples: n the instance that true patient samples cannot be obtained
(extreme upper end or lower end of the assay range); contrived (artificiaIIy spiked anaIyte)
sampIes can be used for the study. However in general, FDA consensus is that no greater than
5% of the samples in the study shall be contrived. t should be taken into account that the
contrived samples do not mimic a true patient, because for example, the endogenous metabolites
in the sample matrix have been voided.
6.6 Controls and Calibrators: Selection of the controls and their matrix should be similar to the
sample types intended for the assay (such as serum or plasma). The selection of control levels
(concentration) should cover entire assay range (low, medium, high) as well as clinically
significant concentrations for the assay.
6.7 Sample processing: DNA, RNA, protein and other analytes isolated and prepared.

7.0 ANALYTICAL PERFORMANCE CHARACTERISTICS
The analytical performance characteristics to be validated are assay specific and based on the output of
the specific assay. Most, but not all of the following analytical performance characteristics are to be used
to analytically validate diagnostic assays. Additional assay characteristics, not listed herein may be
included in the validation plan and protocols as needed for specific assays.
7.1 Specificity: For a quantitative assay, the ability to assess unequivocally the analyte in the
presence of components that may be expected to be present, such as host cell protein or nucleic
acids, medium component impurities, degradation products, and matrix components. For a
qualitative assay, the method's ability to obtain negative results in concordance with negative
results obtained by the reference method. Assay specificity can be determined according to the
CLS guidance documents EP12A2 or EP14-A.
7.2 Sensitivity: The ability to assess unequivocally the analyte in presence of components that may
be expected to be present, such as host cell protein or nucleic acids, medium component
impurities, degradation products and matrix components. t is expressed as the percentage
(number fraction multiplied by 100) of subjects with the target condition (as determined by the

Procedure Document
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diagnostic accuracy criteria) whose test value is positive. Assay sensitivity can be determined
according to the CLS guidance documents EP12A2 or EP14-A2 and EP 17-A2.
7.2.1 Limit of BIank (LoB): Highest apparent analyte concentration expected to be found when
replicates of a blank sample containing no analyte are tested. The LoB study is carried out
according to the CLS guidance document EP17-A.
7.2.2 Limit of Detection (LoD): The lowest amount of analyte that can be detected, but not
necessarily quantitated, under the stated experimental conditions. The following is an
example of an approach that can be used to determine LoD. Other approaches may be
acceptable. The LoD study is carried out according to the CLS guidance document EP17-A.
7.2.2.1 Signal-to-Noise: The LoD is determined by comparing measured signals from samples
with known concentrations of analyte with those of blank samples and establishing the
minimum concentration at which the analyte can be reliably detected. Signal-to-noise
ratios (dependent of the assay type: immuno, PCR, calorimetric, etc) are used for
estimating the LoD. The acceptance criteria for each specific assay type is captured in
the validation plan. Limit of detection can also be measured by testing an independent
panel of precision pool samples as part of CLS recommended 20 day precision
studies. This panel contains the sample at the low end of the assay range, with lowest
concentration sample spanning below the assay range. This approach can only be
applied to instrumental analytical procedures that exhibit background noise.
7.2.3 Limit of ":antitation (Lo"): The lowest amount of analyte that can be quantitatively
determined in a product sample by a procedure with suitable precision and accuracy. The
following are examples of approaches that can be used to determine LoQ. Other approaches
may be acceptable. The LoQ study is carried out according to the CLS guidance document
EP17-A.
7.2.3.1 Signal-to-Noise: The LoQ is determined by comparing measured signals from samples
with known low concentrations of analyte with those of blank samples and establishing
the minimum concentration at which the analyte can be reliably quantified. Signal-to-
noise ratios (dependent of the assay type: immuno, PCR, calorimetric, etc) are used for
estimating the LoQ. The acceptance criteria for each specific assay type are captured
in the validation plan. Limit of Quantitation can also be measured by testing an
independent panel of precision pool samples as part of the CLS recommended 20 day
precision studies. This panel contains the sample at the low end of the assay range,
with lowest concentration sample spanning below the assay range. This approach can
only be applied to analytical procedures that exhibit background noise.

7.3 Linearity: The ability of an assay to obtain test results (within a given range), which are directly
proportional to the concentration of analyte in the clinical sample. The correlation coefficient,
coefficient of determination, y-intercept, slope of the regression line, and residual sum of squares
may be considered when performing linearity analysis. n order to establish linearity, testing of a
minimum of five (5) concentrations is recommended. The samples should range from the upper
limit of the assay range and the linearity dilution should extend to the reportable Limit of Detection
(LoD). f linearity is not attainable, a nonlinear model may be used. The goal is to have a model,
whether linear or nonlinear, that describes closely the concentration-response relationship. The
linearity study is carried out according to the CLS guidance document EP6-A.

Procedure Document
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Revision: 1
Effective Date:


7.4 Assay Range: The interval between the upper and lower concentrations of an analyte in the
clinical sample (including these concentrations) for which it has been demonstrated that the
procedure has a predetermined level of precision, accuracy and linearity. The assay range is
established based on the clinical utility of the assay as well as taking into account predicate
device and market analysis on the customer need.
7.5 NormaI range: The normal range of the assay (99
th
percentile) is established using normal donor
samples (retrospective). The donor population should have equal distribution of the gender and
age that is consistent with the assay's intended use population. n some instances based on the
FDA 510K guidance, some of the special population samples might be necessary to be tested as
part of normal range study (e.g., pediatric, adolescent or neonatal samples). The sample size and
the distribution are analyzed in conjunction with Biostatistics group and include the data analysis
to determine the normal range and 99
th
percentile. The normal range study is carried out
according to the CLS guidance document C28-A3c.
7.6 Acc:racy (Method Comparison): The closeness of agreement between the value which is
accepted either as a conventional true value or an accepted reference (predicate device) and the
value found. The selection of patient samples should include all the sample types claimed for the
assay; with even distribution through out the assay range. Some of the high samples should be >
95% of the assay range. These samples could be contrived as stated in section 6.0. Accuracy
shall be established across the assay range. The accuracy also referred as correlation or method
comparison is carried out according to the CLS guidance document EP9-A2.
7.7 Interfering S:-stances: The study is conducted by screening for potential interfering
substances, to quantify interference effect and confirm interference in patient samples. Potential
interfering substances may originate from endogenous and exogenous sources. Some of the
most common endogenous interfering substances in human blood are Bilirubin (conjugated and
unconjugated), Lipids, triglycerides, protein and RF factor; however potential clinical disease
conditions, drugs and metabolites of drugs to which the intended use population may be routinely
exposed should also be considered. Examples of exogenous sources include contaminants
introduced during sample collection and handling or sample preparation. An assay developed
with murine antibody may be tested for HAMA interference. Concentrations at which these
interfering substances are tested are assay specific and captured in the validation plans. The
study is carried out according to the CLS guidance document EP7-A2.
7.8 Cross-Reactivity: A cross-reactivity study examines interference which is structurally similar to the
analyte being measured and hence may cross react or block the assay from correctly measuring
the targeted analyte. Concentrations at which these interferences or cross reactants are tested is
assay specific and is captured in the validation plans. The study is carried out according to the
CLS guidance document EP7-A2.
7.9 Reprod:ci-iIity or TotaI Error Estimate (TEE): Describes the variability within laboratories. This
includes different lots, different days, different analysts, and different equipment and multiple
calibrations. The study evaluates the total variance coming from various contributing factors. The
total error estimate gives the measure of allowable variance at clinically significant concentrations
without affecting outcome or interpretation. The study is carried out according to the CLS
guidance document EP5-A2.
7.10 Precision (Repeata-iIity): The closeness of agreement (i.e., the standard deviation, coefficient
of variation, and confidence interval) between a series of measurements obtained for multiple
sampling of the same sample under prescribed conditions. Precision may be a measure of either
the degree of reproducibility of the assay under normal operating conditions. The controls and

Procedure Document
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samples selected for precision studies should cover the assay range as well as target key
clinically significant target concentrations. The precision studies are carried out according to the
CLS guidance document EP-5A2.

7.11 Tracea-iIity: The assay traceability is analyte specific. Concordance of the assay calibrators is
established with analyte specific reference standard (WHO or USP or Gravimetric or GC-MS).
The traceability of the assay is documented. There will be no acceptance criteria for the % bias
with reference standard. t is a tool used to verify the calibration and anchor the assay calibrators
over the period of time (over several years or lots of calibrators).
7.12 High Dose Hook Effect (Prozone effect): Applicable for the immuno-based sandwich assay
format. The high dose hook effect refers to measured levels of antigen displaying a significantly
lower signal than the actual level present in a sample. This appears when an assay is saturated
by a very high concentration of sample antigen binding to all available sites on both the capture
antibody as well as the detection antibody and thereby preventing the sandwich-formation. The
antigen-saturated detection antibodies in solution are washed off giving a falsely low signal. A
"hook is observed in the curve when data is plotted as a signal versus antigen concentration. A
Hook sample with analyte concentration 10X or 100X greater than the highest level calibrator is
tested as unknown. The signal from this sample should be greater than the signal from the
highest calibrator.
7.13 SampIe Type Eq:ivaIence: n the case of multiple sample types are recommended (for
example, serum, Serum Separator Tube, Li-Heparin or EDTA plasma), concordance must be
shown between various sample types (serum vs. Li-Heparin plasma or serum vs. EDTA plasma).
A minimum number of samples should be based on statistical rationale as described in assay
validation plan and in conjunction with Biostatistics group, with the samples spanning over the
entire assay range. These paired samples are coming from the same donor and are contrived to
span the entire assay range. n the case, where such concordance between the sample types
can not be established, a disclaimer must be claimed in the customer insert that the patient
sample types are not interchangeable.

8.0 DOCUMENT TYPES
8.1 Validation Plan: A written approved plan that summarizes the scope and validation approach for a
specific validation project. Major validation projects may require a validation plan which may
include but are not limited to new systems, replacement of current systems, or multiple validation
projects of a similar type. The validation plan guides management on a project and defines
systems, timelines, resources, priorities, and responsibilities with regards to the validation effort.
8.2 Validation Protocol: An approved, written plan stating how the validation will be conducted,
including a test matrix and reference to the method specific SOP, which specifies the validation
characteristics to be tested, statistical design, pre-defined statistical method(s) and criteria to
identify and remove outliers with valid justification, an acceptance criteria for the validation
results.
8.3 Validation Report: An approved, written document that summarizes the validation activities,
including specific test methods, results for each test performed, protocol deviations and protocol
generation errors and a conclusion which states whether or not acceptance criteria were met.

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.0 VALIDATION STRATEGY AND RE"UIREMENTS
9.1 Project Specific Validation Plan
9.1.1 For complex validation projects, a project specific validation plan may be prepared to specify
the required validation activities. f a project specific validation plan is written a summary
report must be written to close out the validation activities.
9.2 Validation nstruments or Equipment Qualification
9.2.1 nstruments or equipment used to perform a given assay validation must be qualified prior to
performing the studies. The qualification of the validation instrument or equipment must be
performed according to document W-0037 Process Validation, current revision.
9.3 Assay Validation Protocol
9.3.1 The protocol is a written, pre-approved document stating how validation will be executed. The
pre-approved assay validation protocol according to CLS guidelines and an approved test
procedure, , must be available prior to conducting the validation study. Validation protocols
may cover several test procedures. Ensure that the protocol is updated with corrections made
for any protocol generation errors or deviations identified in the previous validation protocol.
9.4 During the development of a new assay validation protocol, the Method Owner and QA will use
Table 1 as a guide to determine the performance characteristics to be validated for a given
method which will include number of lots to be used to perform each study. This will be 383.
with what is needed for regulatory submissions (FDA 510K or PMA). The associated validation
protocol will define the assay type and the appropriate characteristics to be tested. t is important
that FDA feedback and regulatory consensus for number of lots used for each study are finalized,
especially for regulatory submission in China. A minimum of requires three consecutive validation
lots of reagents is suggested, however there must be statistical rationale used to determine the
number of lots of reagents required..
9.5 StatisticaI AnaIysis: The Assay Development group will seek guidance from the Biostatistics
group with regards to the size of the sample population, rationale for number of samples to be
tested during the study. This is particularly applicable for accuracy (method comparison) and the
assay range study. The sample population selection is assay specific and the details are captured
in assay validation plan. n certain instances, it may be necessary to test special population
samples (i.e: Assay specific or dictated by FDA response). The assay validation plan incorporates
these studies.
9.5.1 Table1: Assay Validation Chart
Type of Study
Validation Lot
1
Validation Lot
2
Validation Lot
3

Acc:racy v v v (opt|ona|)
Precision v v v
Reprod:ci-iIity
(TotaI Error)
v
v v

Procedure Document
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Type of Study
Validation Lot
1
Validation Lot
2
Validation Lot
3

Interference &
Cross reactivity
v

Specificity v v
Sensitivity v v
LoB v v v
LoD v v v
Lo" v v v
Linearity v
Tracea-iIity v v
NormaI range v v
High Dose
Hook
v
v
Assay Range v
SampIe
Eq:ivaIence
v

9.5.2 Predetermined Acceptance Criteria may be derived from historical validation data, and /or
criteria specified in the method SOP. Validation, and Subject Matter Experts (SME) identify
the parameters and the acceptance criteria with concurrence from QA, and when necessary,
Biostatistics. Specific acceptance criteria for each assay are identified in the validation
protocols.
9.5.3 A risk-based approach and valid statistical rational must be used when designing validation
sampling plans. The level of testing must be commensurate with the risk posed by the assay
as determined from the project specific risk management activities as defined by SOP-0005
Risk Management.
9.5.3.1 Analytical performance characteristics associated with high levels of risk must have
sampling plans designed to demonstrate reliability at a 95% level of confidence as
defined in the assay validation plan.
9.5.3.2 Analytical performance characteristics associated with moderate levels of risk must
have sampling plans designed to demonstrate reliability at a 95% level of
confidence as defined in the assay validation plan.
9.5.3.3 Analytical performance characteristics associated with low levels of risk must have
sampling plans designed to demonstrate reliability at a 95% level of confidence as
defined in assay validation plan.

Procedure Document
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Revision: 1
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9.6 Protocol Deviations and Protocol Generation Errors
9.6.1 All deviations that occur during analytical assay validation must be reported. During re-
qualification activities, deviations other than protocol generation errors, that occur, must be
captured in an exception report.
9.7 Testing
9.7.1 Personnel performing validation studies must be qualified and appropriately trained in the
relevant procedures. The following general requirements apply:
O The equipments used must be qualified/calibrated before any testing is performed.
O During validation work, if required by the assay, a system suitability test must be
performed. The extent this test depends on the objective of the method and the
technique being used.
O Testing must be carried out according to the tests defined in the respective validation
protocol and according to the appropriate SOP.
O All raw data and derived results obtained must be documented.
O Any acceptance criteria failure or procedural deviation during validation study must be
reviewed and assessed in an exception report and assessed in the validation report
to identify the root cause.
O Action to be taken following a test failure/procedural deviation must be documented,
reviewed and approved..
9.8 Assay Validation Final Report
9.8.1 When Validation activities for a given method are complete, a final report must be prepared.
At a minimum the method validation report must contain the following.
O Reference to the document used for the study
O Reference to the validation protocol and or plan
O Results fro the characteristics defined in the protocol
O Review of data and the evaluation of results against acceptance criteria
O Exception reports for invalid Assays or Deviations (if any)
O Summary and conclusion
O Formal statement of validation status
9.8.2 All the Assay Validation Final Reports must be submitted and routed for approval.
9.9 Retest
9.9.1 n the case, one of the validation lot or a particular study does not meet acceptance criteria;
retest of the study can be performed. Prior to the retest, a technical assessment must be
performed by the Method Owner for the methods capability to ensure it is still fit for its
intended purpose. The assessment must be documented in the validation report.
9.10 Change Control
9.10.1 Any proposed changes are reviewed and approved by QA and the original approvers of the
validation protocol or plan.

Procedure Document
Number:
SOP-0014
Revision: 1
Effective Date:


10.0 REVISION HISTORY
REV Author Revision Description
1 Girish Ranadive New procedure required for Assay validation activities.

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