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Technical Guidance Series (TGS)
for WHO Prequalification – Diagnostic
Assessment
Guidance on Test Method
Validation for in vitro diagnostic
medical devices
TGS–4
Draft for comment 20 December 2016
© World Health Organization 2016
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be liable for damages arising from its use.
Contact: Irena Prat, EMP Prequalification Team Diagnostics
WHO - 20 Avenue Appia - 1211 Geneva 27 Switzerland
Technical Guidance Series for WHO Prequalification – Diagnostic Assessment: Guidance on Test method validation for in vitro diagnostic medical devices TGS–4
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Table of contents
Table of contents 3
Acknowledgements 5
1 Definitions 6
2 Introduction 8
3 Scope 8
4 Terminology for test method validation 8
4.1 Explanation of the terms characterisation, verification and validation .................... 8
4.2 Explanation of the terms accuracy, trueness and precision ................................... 10
5 Uses of test method validation in the lifecycle of the IVD 11
6 Test methods 11
6.1 Categories of test methods ..................................................................................... 11
6.2 Statistics and test methods ..................................................................................... 11
6.3 Quantitative and qualitative assays ........................................................................ 11
6.4 Specimen panels and test methods ........................................................................ 12
7 Variability in the test method 12
8 Planning for test method validation 13
9 Examples of test methods and their validation 14
9.1 Validation of test methods related to cleaning processes ...................................... 14
9.2 Validation of test methods for raw materials ......................................................... 16
10 References 20
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WHO Prequalification – Diagnostic Assessment: Technical Guidance Series
The World Health Organization (WHO) Prequalification Programme is coordinated
through the Department of Essential Medicines and Health Products. The aim of WHO
prequalification of in vitro diagnostic medical devices (IVDs) is to promote and facilitate
access to safe, appropriate and affordable IVDs of good quality in an equitable manner.
Focus is placed on IVDs for priority diseases and their suitability for use in resource-
limited settings. The WHO Prequalification Programme undertakes a comprehensive
assessment of individual IVDs through a standardized procedure aligned with
international best regulatory practice. In addition, the WHO Prequalification Programme
undertakes post-qualification activities for IVDs to ensure the ongoing compliance with
prequalification requirements.
Products that are prequalified by WHO are eligible for procurement by United Nations
(UN) agencies. The products are then commonly purchased for use in low- and middle-
income countries.
IVDs prequalified by WHO are expected to be accurate, reliable and be able to perform
as intended for the lifetime of the IVD under conditions likely to be experienced by a
typical user in resource-limited settings. The countries where WHO-prequalified IVDs
are procured often have minimal regulatory requirements. In addition, the use of IVDs
in these countries presents specific challenges. For instance, IVDs are often used by
health care workers without extensive training in laboratory techniques, in harsh
environmental conditions, without extensive pre- and post-test quality assurance
capacity, and for patients with a disease profile different to those encountered in high
income countries. Therefore, the requirements of the WHO Prequalification Programme
may be different to the requirements of high-income countries, and/or of the regulatory
authority in the country of manufacture.
The Technical Guidance Series was developed following a consultation, held on 10-13
March 2015 in Geneva, Switzerland which was attended by experts from national
regulatory authorities, national reference laboratories and WHO prequalification dossier
reviewers and inspectors. The guidance series is a result of the efforts of this and other
international working groups.
This guidance is intended for manufacturers interested in WHO prequalification of their
IVD. It applies in principle to all IVDs that are eligible for WHO prequalification for use in
WHO Member States. It should be read in conjunction with relevant international and
national standards and guidance.
The TGS guidance documents are freely available on the WHO web site.
WHO
Prequalification
– Diagnostic
Assessment
Procurement of
prequalified
IVDs
Prequalification
requirements
About the
Technical
Guidance
Series
Audience and
scope
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Acknowledgements
The draft document “Technical Guidance Series for WHO Prequalification – Diagnostic Assessment:
Guidance on Test method validation for in vitro diagnostic medical devices” was developed with support
from the Bill & Melinda Gates Foundation and UNITAID. This draft was prepared in collaboration with Dr
J Duncan, London, United Kingdom; D Healy; R Meurant, WHO; and with input and expertise from Dr V
Alcón; D Lepine; IVDD Section, Medical Devices Bureau Health Canada, Ottawa, Canada; Dr S Hojvat,
MD, USA; and Dr S Norman, CA, USA. This document was produced under the coordination and
supervision of Robyn Meurant and Irena Prat, Prequalification team – Diagnostic Assessment, WHO,
Geneva, Switzerland.
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1 Definitions
The section below provides definitions which apply to the terms used in this document. 1
Accuracy: The closeness of agreement between a test result and the accepted reference value. 2
(1) 3
Lot: Defined amount of material that is uniform in its properties and has been produced 4
in one process or series of processes. 5
NOTE: The material can be either starting material, intermediate material or finished 6
product. (2) 7
Characteristic: Distinguishing feature 8
Note 1 to entry: A characteristic can be inherent or assigned. 9
Note 2 to entry: A characteristic can be qualitative or quantitative. ( 3) 10
Note 3 Characterisation: a description of the distinctive nature or features of 11
something. ( 3) 12
Control material: Substance, material or article used to verify the performance characteristics of an in 13
vitro diagnostic medical device. ( 4) 14
Control procedure: Activities at the point of use to monitor the performance of an IVD medical device. 15
Note 1 In the IVD medical device industry and in many laboratories that use IVD 16
medical devices, these activities are commonly referred to as quality control. 17
Note 2 Quality control may monitor all or part of the measurement procedure, from 18
the collection of samples to reporting the result of the measurement. ( 4) 19
In vitro diagnostic medical device (IVD): A medical device, whether used alone or in combination, 20
intended by the manufacturer for the in vitro examination of specimens derived 21
from the human body solely or principally to provide information for diagnostic, 22
monitoring or compatibility purposes. 23
Note 1 IVDs include reagents, calibrators, control materials, specimen receptacles, 24
software, and related instruments or apparatus or other articles and are used, for 25
example, for the following test purposes: diagnosis, aid to diagnosis, screening, 26
monitoring, predisposition, prognosis, prediction, determination of physiological 27
status. 28
Note 2 In some jurisdictions, certain IVDs may be covered by other regulations. (5) 29
In vitro diagnostic reagent/IVD reagent: Chemical, biological or immunological components, solutions, 30
or preparations intended by the manufacturer to be used as an IVD. (2) 31
Life-cycle: All phases in the life of a medical device, from the initial conception to final 32
decommissioning and disposal. (6) 33
Limit of detection, detection limit: Measured quantity value, obtained by a given measurement 34
procedure, for which the probability of falsely claiming the absence of a component 35
in a material is β, given a probability α of falsely claiming its presence. 36
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Note 1 IUPAC recommends default values for α and β equal to 0.05. 37
Note 2 The term analytical sensitivity is sometimes used to mean detection limit, but 38
such usage is now discouraged. Modified from ( 7) 39
Limit of quantitation, quantitation limit: Lowest value of measurand in a sample which can be 40
measured with specified measurement uncertainty, under stated measurement 41
conditions. ( 7) 42
Measurand: Quantity intended to be measured. (7) 43
Objective evidence: data supporting the existence or verity of something 44
Note 1 Objective evidence can be obtained through observation, measurement, test, 45
or by other means. (2) 46
Performance claim: Specification of a performance characteristic of an IVD medical device as 47
documented in the information supplied by the manufacturer. 48
Note 1 This can be based upon prospective performance studies, available 49
performance data or studies published in the scientific literature. ( 2) 50
WHO Note “Information supplied by the manufacturer” includes but is not limited 51
to: statements in the instructions for use, in the dossier supplied to WHO and / or 52
other regulatory authorities, in advertising, on the internet referred to simply as 53
“claim” or “claimed” in this document. 54
Precision: The closeness of agreement between independent test results obtained under 55
stipulated conditions. (1) 56
Quality: Degree to which a set of inherent characteristics of an object fulfils requirements. ( 3) 57
WHO Note: for the purpose of this document these requirements include fitness-for-58
use, safety and performance. 59
Quality assurance: Part of quality management focused on providing confidence that quality 60
requirements will be fulfilled. (3) 61
Ruggedness (robustness): A measure of an analytical procedure’s capacity to remain unaffected by 62
small but deliberate variations in method parameters and provides an indication of 63
its reliability during normal usage. (8) 64
Standard method: A method that is (metrologically) traceable to a recognized, validated method. 65
Non-standard method: A method that is not taken from authoritative and validated 66
sources. This includes methods from scientific journals and unpublished laboratory-67
developed methods. (8) 68
Trueness: The closeness of agreement between the average value obtained from a large series 69
of test results and an accepted reference value. (1) 70
Validation: Confirmation by examination and provision of objective evidence that the 71
requirements for a specific intended use have been fulfilled. (3) 72
Verification: Confirmation through the provision of objective evidence that specified 73
requirements have been fulfilled. (3) 74
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2 Introduction
The purpose of test method validation is to ensure that a method consistently provides results fit or 75
adequate for a specific purpose. Testing must have a useful purpose and the result from the test must 76
be shown to be meaningful and to give the expected (and appropriate) information. In order to ensure 77
meaningful results, the test method must be validated; otherwise the measurement has little purpose 78
and no economic value. By using validated test methods, a manufacturer can have confidence that 79
claims made in respect to the quality and performance of an IVD are supported by objective evidence. 80
3 Scope
This document is intended to provide guidance on the validation of the test methods used in 81
manufacturing of an IVD. Sometimes test methods are referred to as analytical methods but in the 82
context of establishing the design, the development and manufacture of an IVD, “test method” is the 83
more commonly used and a more appropriate description since not all testing is analytical. Minimal 84
specific guidance relating to test method validation is available for IVD manufacturers despite the 85
abundance of guidance for the analytical chemistry or pharmaceutical industries (e.g. those from 86
Eurachem ( 10), Eurolab ( 11), ICH ( 12), WHO ( 13) and FDA ( 14)) or for clinical laboratories compliant with 87
ISO 15189 ( 15). This document provides information on validating the test methods used by 88
manufacturers of IVDs in their research and development (R&D), quality control and quality assurance 89
laboratories; it must be read as an adjunct to those formal guides mentioned previously. 90
This document is not intended to give guidance on validation of the IVD itself. For this, it is 91
recommended to refer to “TGS3 Principles of performance studies” ( 16) in this series. Qualification of 92
instrumentation is outside the scope of this document although the test methods used in qualification 93
must be validated ( 17). This document does not outline statistical methods for analysis of the required 94
data. 95
4 Terminology for test method validation
4.1 Explanation of the terms characterisation, verification and validation 96
Although internationally accepted definitions exist for the terms characterisation, verification and 97
validation, the following explanation is provided to give greater clarity with relation to test method 98
validation. 99
Characterisation, verification and validation are essential terms. For the purposes of this guidance 100
document “characterisation of a test method” refers to an experimental procedure and the 101
documentation of its characteristics. It is undertaken in order to provide objective evidence of what a 102
method is capable of consistently achieving under defined conditions. The characteristics of the assay 103
are the numerical values proven and documented for each of the method attributes such as sensitivity, 104
specificity, limit of detection etc. Each attribute should be evaluated using an appropriate, validated test 105
method. 106
Verification is the documentary proof that particular specifications have been met. When designing and 107
developing an IVD, relevant attributes such as cost, and those for performance such as precision, 108
sensitivity and stability are identified and given numerical specifications in design input documentation. 109
It is subsequently the role of the R&D department to design an IVD that will meet those specifications. 110
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The R&D department consequently identifies valid test methods to demonstrate that the specifications 111
have been met (verification) in the new design. Once design has been established, further numerical 112
specifications are produced by the R&D department to ensure that the specifications of each attribute 113
will be met consistently in routine production to ensure quality manufacturing. These new specifications 114
are assigned to control critical production points and may include those for acceptance of raw materials, 115
in-process materials, cleanliness of equipment, qualification of instrumentation and for the finalised IVD 116
to verify its manufacture. Again, it is also the role of the R&D department to identify appropriate test 117
methods to monitor these specifications. An example of verification is related to incoming goods 118
inspections; each time a raw material is purchased its properties will be verified against the specification 119
using a validated test method. 120
Validation is the documentary proof that the particular requirements for a specific intended use can be 121
consistently fulfilled ( 9). VIM ( 7) defines validation as “verification against needs for a specific use” (i.e. 122
the specification for that use). Within this guide, consistency is essential: it is an expectation that every 123
lot of an IVD will behave as all other lots and will continue to meet design inputs. To ensure this, it is 124
necessary to have validated test methods for measuring and/or monitoring specifications that will 125
consistently produce results fit for purpose. The test methods must be validated to ensure that the 126
results of measuring and/or monitoring are meaningful. For example, the need for accurate 127
measurement of a raw material weighed in micrograms will not be achieved by using a weighing device 128
with tolerance measured in grams. A test method using such an instrument would not be valid for the 129
intended use. Thus, for the example provided, a test method should be specified that has the necessary 130
accuracy and precision for measuring such weights, and an instrument and procedure identified that will 131
consistently achieve this requirement during its use. The test method is then validated to produce 132
results fit for purpose. 133
Validation of a test method is distinct from its characterisation. Characterisation is documentation of 134
some or all of the features of the method; validation is ensuring that the relevant characteristics are 135
appropriate for the specific intended use. Validation of a method to be used widely, and for standard 136
methods, often begins with complete characterisation. However, for each specific intended use it is 137
likely that only a subset of the characteristics will be relevant and must be evaluated. 138
Published guides to test method validation provide the broad characteristics of assays as set out in Table 139
1. 140
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Table 1: Examples of characteristics of assays 141
Characteristic Attributes
Trueness bias, recovery, accuracy, matrix effects
Precision repeatability, intermediate precision, reproducibility
Selectivity specificity, interferences
Sensitivity traceability, working range, linear range, limit of detection, limit of
quantitation, uncertainty at clinically significant threshold values
Reproducibility precision under defined conditions: repeatability, ruggedness, robustness
Stability reagents, analyte (specimen types)
Productivity† speed, hazards, cost
† Productivity is not usually mentioned in test method validation texts but is important in
manufacturing environments. Although cost must not be a factor considered in risk minimization ( 6), it
should be a consideration in choice and validation of test methods.
Usually only a small selection of the possible characteristics and attributes will ever be studied for a test 142
method specifically developed for a single purpose. 143
4.2 Explanation of the terms accuracy, trueness and precision 144
The terms accuracy, trueness and precision have specific meaning for technical documentation. 145
Trueness and accuracy are the values obtained for the method under investigation and relative to a 146
value accepted as truth, being established through the use of an accepted traceable calibrator or derived 147
by testing using an accepted reference measurement method on the same item as measured by the 148
method under test. Without an accepted value neither trueness nor accuracy can be given for a test 149
method, only a percent (positive and/or negative) agreement. 150
Trueness is a measure of the closeness of agreement between the accepted value and the average of a 151
large [infinite] number of results from a test or assay method under review. It is expressed as a bias: “the 152
result from this test method has a bias of ±units”. Trueness is a characteristic of the method. 153
Precision is a comparison of the results obtained on the same test method. It does not encompass 154
comparison to another value obtained using a method associated with trueness. It is a measure of the 155
closeness of agreement between independent test results obtained under stipulated conditions using 156
the same test method and encompasses concepts such as repeatability and reproducibility, depending 157
on the specified conditions. It is expressed in terms of a standard deviation or related measures “the 158
precision (or repeatability or reproducibility etc.) of this test method under these conditions is ±y-units”. 159
Precision is a characteristic of the method. 160
Accuracy is a measure of the closeness of agreement between the accepted value as documented and 161
the result of a measurement using the item (i.e. test equipment etc.). It is a characteristic of that single 162
measurement and has components from both trueness and precision of the test method. Each time the 163
test method is performed the accuracy of the measurement is likely to be different because of 164
experimental error and the imprecision of the test method. If a test method requires the documented 165
result to be the average of several individual measurements, the accuracy is related to that average; the 166
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fact of limited replication does not convert accuracy to trueness although it may improve the accuracy. 167
Accuracy is expressed in terms of bias: “the accuracy of that measurement was -z-units” (1, 7). 168
Both precision and trueness for a particular method, and subsequently the accuracy of an assay by that 169
method, can be influenced by the concentration of the measurand. As such, when characterising a 170
method, knowledge of the performance of the assay should be obtained over a range of foreseeable 171
measurand concentrations, to ensure the validity of any assumptions regarding the performance of the 172
method. 173
5 Uses of test method validation in the lifecycle of the IVD
Testing in the R&D phase of the life-cycle of a commercial IVD is often to ensure that the work in 174
progress will meet the input requirements or to verify that the test development meets those 175
requirements. Design requirements such as a claim of lack of interference from similar analytes will need 176
to be supported by validated test methods. 177
During production, testing is usually employed to ensure that the material being tested meet its 178
specifications. Test methods will use classical analytical chemistry or biochemistry to evaluate the quality 179
of materials coming into, or synthesised by the factory e.g. commercial chemicals, enzymes, 180
recombinant proteins, peptides or nucleic acids. Validation of the test methods will ensure that the 181
correct attributes are measured appropriately. 182
6 Test methods
6.1 Categories of test methods 183
Test methods can be categorized as standard or non-standard. Standard methods are metrologically 184
traceable to a recognized, validated method and do not require additional characterisation by IVD 185
manufacturers. Pharmacopoeia and various national regulations document approved standard methods. 186
In contrast, non-standard methods must be individually characterised and validated for the intended 187
use. However, all methods must be assessed as appropriate for the specific intended use, and must be 188
verified as being used correctly ( 6, 13, 19). 189
6.2 Statistics and test methods 190
It is recommended to seek expert statistical advice during the planning stage of all experiments to 191
ensure that sufficient numbers of specimens are tested to provide statistically powered results. These 192
are required to justify any claim, and to provide reasonable estimates of uncertainty. 193
Frequently statistical differences will be found that have no practical consequence. For that reason 194
practical differences, or limits of confidence, should always be defined before experiments are 195
performed. 196
6.3 Quantitative and qualitative assays 197
Most test methods will produce numeric, quantitative results, but some assays can only produce 198
qualitative output: the binary result of analyte present or analyte absent relative to a particular cut-off 199
value. For qualitative assays some of the characteristics listed in section 4 Table 1 cannot be enumerated 200
without applying advanced statistical methods. For guidance on this issue see Valcárcel et al. 2002 ( 20). 201
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Some IVDs are intended only to produce qualitative results in users’ environments but it is usual (and is 202
probably essential) that the test methods used in manufacture (quality assurance, quality control) will 203
provide a quantitative result. In most cases qualitative assays can be adjusted to provide a quantitative 204
result, either from an instrumental reading, e.g. for an enzyme immunoassay, or against a graduated 205
reference scale (semi-quantitative reporting of a present/absent result as is the case with many rapid 206
diagnostic tests) . If a quantitative result cannot be obtained then experiments and results must be 207
designed to be analysed by appropriate qualitative statistical methods. Documenting an outcome as 208
merely positive or negative without giving an uncertainty estimate is rarely sufficient, particularly for 209
test methods intended to characterise an IVD (e.g. for stability, precision, sensitivity) or for release-to-210
sale testing. 211
6.4 Specimen panels and test methods 212
Test methods used to verify design and consistent production will frequently involve the choice and use 213
of panels of specimens in order to determine and/or monitor quality characteristics of an IVD. Panels 214
must be designed and specimens selected to ensure that data generated usefully demonstrates that the 215
specifications have been met. Designing a valid method to assess sensitivity of antibody detection for 216
example, will need to take into account the fact that testing of dilutions of a strong positive specimen 217
will not produce results that reflect the performance of the assay with respect to seroconversion 218
sensitivity. Similarly, the panel composition for release-to-sale and stability testing must employ panels 219
utilising specimens demonstrated to reflect the state of an IVD relative to real, critical specimens to be 220
valid. It is useful to note that test methods used for an IVD in both the design and development phases 221
as well as during production can have various applications, for example the experiments undertaken at 222
release-to-sale can be also be used with adjusted criteria in proving the stability of the IVD. 223
7 Variability in the test method
A critical attribute of all test methods is that they must be less variable than the parameter being 224
evaluated (i.e. have a higher precision). The variability of the test method must not conceal variability in 225
that which is tested. This requirement is usually studied as “gauge R&R” (Gauge Repeatability and 226
Reproducibility, refer to Burdick et al ( 21)) but the process and methodology applies to any measuring 227
system, not just to gauges. As a rule of thumb, the gauge should have a variance of less than 20% of the 228
variance of the “test-piece”. In this context, it is unreasonable to make claims based on one lot of an IVD 229
evaluated in one or two similar laboratories, regardless of how many individual specimens are tested. It 230
is important to understand the variability between lots of IVD and the test method must be capable of 231
revealing it. For instance, it is an accepted published practice to use three lots of an IVD to demonstrate 232
stability ( 22), however, no guidance is provided on what actions are required when significant lot-to-lot 233
variability is identified during stability testing. Shelf-life should be assigned statistically taking into 234
consideration the variance between lots (see “TGS-2 Guidance Document for establishing stability of in 235
vitro diagnostics assays and components” in this series ( 23)). Due to the requirement of high precision of 236
the test method, using a previous lot of the IVD as a simple comparator is unlikely to meet the 237
requirements of being a validated test method unless there is sufficient knowledge and control of the 238
variability associated with each lot. The concerns regarding variability apply equally to all claims, 239
including specificity and sensitivity. 240
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8 Planning for test method validation
Figure 1: Test method validation process 241
The flow of the test method validation process is 242
shown in Figure 1. The steps will be described in 243
detail in the examples of test method validation to 244
follow. 245
Understanding the real, intended purpose of the test 246
method is critical. The US FDA ( 24) states that: 247
“Design input is the starting point for product design. 248
The requirements which form the design input 249
establish a basis for performing subsequent design 250
tasks and validating the design. Therefore, 251
development of a solid foundation of requirements is 252
the single most important design control activity”. 253
ISO 13485 ( 25) requires that “design and 254
development outputs shall meet the input 255
requirements”. Compiling requirements and 256
comparing outputs to inputs is essential for activities 257
that require detailed planning and execution. 258
Once the input requirements which define the exact 259
purposes for the test method are documented and 260
agreed (e.g. to ensure a particular claim will be met 261
for the whole shelf-life of the IVD), the required 262
characteristics of the test method can be specified 263
and given measurable attributes, usually following 264
risk assessments and development work. 265
The following are examples of input requirements: 266
ability to detect an increase of 0.5% in the invalid 267
result rate of an IVD; ability to detect 10% loss of 268
sensitivity for a particular epitope; evidence that 269
infectivity of a positive control material is reduced by >100-fold. 270
The method can then be developed and validated against these predetermined needs. Without the 271
predetermined needs the method cannot be validated, merely characterised. 272
The analysis of incoming raw materials is usually supported by well-established standard methods 273
and/or published routine test method validation. There are usually a greater array of test methods (e.g. 274
chemical analyses, protein, peptide or nucleic acid sequencing or terminal analyses, spectroscopy, 275
chromatography, electrophoresis, electro-blotting) than for routine quality control or quality assurance 276
of an IVD itself but the link between specifications, predetermined test method characteristics, the 277
capability of the method and the utility of results must still be documented. 278
For quality assurance and quality control of an IVD, the test method frequently requires decisions on the 279
attributes to be measured and numbers of specimens for testing panels. The finalised test methods and 280
specimens to be used are usually developed together during the R&D for an IVD. Given the important 281
Identify the required
characteristics of the test method
Compare performance with
requirements
Is themethod fit for
purpose?
YESYES
Use method
Definethe purpose of the
testing
Select or develop the test method
NOselect a different
method or redevelop
Assign numerical values to the
attributes
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relationship between the chosen test method and the testing panel, it is almost always too late to try to 282
find appropriate specimens for the panels after R&D is completed. 283
9 Examples of test methods and their validation
This section gives examples of test methods and their validation for some aspects of IVD manufacture. 284
The examples were generated following WHO analysis of the deficiencies in evidence provided dossiers 285
submitted to support prequalification. The analysis concluded poor understanding of the importance of 286
test method validation, and its necessity in support of claims (for example on lot to lot reproducibility, 287
stability, specimen types). A manufacturer will need to evaluate each phase of work, processes and 288
materials and adapt the procedure outlined in section 0 8 to the particular IVD. The examples in this 289
document are neither authoritative nor complete. However, if the test method is not valid and 290
documented, the claim is not supported. 291
9.1 Validation of test methods related to cleaning processes 292
Introductory discussion 293
This example of test method validation is of validation of the methods used in verifying cleanliness, not 294
validation of the cleaning process itself. The specifications of what constitutes “clean” must be 295
ascertained on a case by case basis. This is usually from risk analysis based on chemical knowledge of the 296
reasons the cleaning process is necessary and some experimental evidence: why cleaning is essential, 297
the cleaning agents used and the probable subsequent uses of the cleaned item. Once the specifications 298
for cleanliness are known, proven and documented, the requirements of the test methods can be 299
defined. 300
As a simple example consider cleaning a vessel used for preparing conjugates, last used for a conjugation 301
of a monoclonal antibody with an enzyme and now to be used for preparing other conjugates. 302
a) Define the purpose of the testing 303
The residual conjugation chemicals, antibody and enzyme and subsequently any cleaning agents must all 304
be removed in order not to contaminate the next solutions in the vessel. 305
The vessel will be cleaned with pressurised hot water containing an organic anionic detergent followed 306
by alkali and acid rinses and finally rinsing with distilled water and drying. 307
b) Identify the required characteristics 308
The characteristics required are trueness, sensitivity, selectivity and precision for each of the possible 309
analytes. 310
The criteria for successful cleaning could be based in a standard operating procedure requiring vigorous 311
extraction of the cleaned vessel with a defined volume of distilled water prior to any drying stage in 312
order to avoid artefacts from an unclean vessel appearing clean because of difficulty in detecting dried-313
on contaminants. The methods must be able to detect any contamination of the water that could in 314
principle affect subsequent use of the vessel. 315
c) Assign numerical values to the attributes 316
Typically the specification for the water after rinsing could be: less than 10 ppm of total organic carbon, 317
less than 5 ppm of residual protein, less than 1 ppm of residual detergent, less than 1 µM in conjugation 318
related reagents and a conductivity of less than 0.5µS. 319
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These are typical specifications for equipment used for fermentation or protein handling and experience 320
shows that if they are achieved the vessel is likely to be acceptably clean for these purposes. However 321
the utility of the cleaning process with these specifications would need to be validated ( 26) for the 322
specific use before finalising and validating the test methods. 323
d) Select or develop the method(s) 324
The extraction of the water from the vessel is part of the test method: it is required to demonstrate 325
during validation by R&D that a second, similar extraction would contain unmeasurable amounts of the 326
potential contaminants and, independently, that nothing of practical importance would leach into the 327
next solution to be used in the vessel. This aspect of the work requires different methods to those used 328
for the routine verification. These more sensitive methods are required to be validated in the R&D phase 329
of the work (validated for use in the conjugation solution). 330
The validation of the (non-standard) test methods used in this type of work is thoroughly exemplified in 331
the formal pharmaceutical test method validation guides. Validation of the total organic carbon 332
measuring system originates from the manufacturer’s specification and the subsequent performance 333
qualification. 334
As the measurements are made in almost pure water it would not be necessary to verify lack of 335
interference from other constituents of the matrix. Similarly if the conductivity meter was specified as 336
being capable of accurate readings superior than the requirement, further validation would not be 337
necessary. 338
Residual detergent would be measured using an instrument, for example high performance liquid 339
chromatography (HPLC). The method chosen would require characterisation of the sensitivity, accuracy 340
and precision for this purpose and the specific detergent involved. Functionality and conformation of 341
proteins would not survive the acid and alkali washes so any contaminating protein would be measured 342
by a chemical technique, defined in the standard operating procedure for the cleaning process. The 343
definition of the test method is essential as each method of protein quantitation (Lowry, biuret, binding 344
of various dyes and HPLC) provides marginally different results for specific proteins. The sensitivity of the 345
method would be demonstrated to be capable of meeting the requirement and the precision to show 346
that the stated level of protein could be determined with sufficient accuracy. Chemical methods may be 347
used to analysis cleanliness relating to the conjugation reagents. The test method must be defined and 348
the precision and sensitivity in the matrix of distilled water demonstrated to be appropriate. 349
e) Compare performance with requirements and use the methods if adequate 350
Once the methods have been characterised and proven to meet the required numerical attributes they 351
can be used in routine verification of the cleaning process. 352
Over time as the process is shown to consistently produce cleanliness within the required 353
specification, testing would be minimised: i.e. the process itself would be validated 354
(consistently providing cleanliness fit for purpose). However this can only be done by prior 355
use of validated test methods. 356
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9.2 Validation of test methods for raw materials 357
9.2.1 Routine commercial materials 358
Most commercial chemicals (salts, acids, alkalis, sugars) have standard analytical methods from 359
pharmacopoeia, (needing no further evaluations except for verification of proper use and documentary 360
evidence of the required level of quality in the materials. The scope of testing for routine commercial 361
chemicals requires individual assessment. However this should be easy with reputable suppliers. 362
9.2.1.1 Components 363
Components to accompany the IVD such as sachets of drying agents, specimen collection devices and 364
tubes, transfer pipettes or dropper bottles will require testing (and documentation) against the specific 365
requirements of the IVD. 366
Example: validation of an incoming test for transfer pipettes used to drop specimen into an IVD. 367
a) Define the purpose of the testing 368
The purpose of the testing is to demonstrate that across the lot of transfer pipettes, the volume 369
delivered meets the specification provided by the R&D department during the development of the IVD. 370
The specifications provided by the R&D department would have been validated to demonstrate that all 371
the claims of the assay (sensitivity, specificity, precision, etc.) are met through the assigned life of the 372
IVD using the pipettes which provide a volume within the specification. 373
b) Identify the required characteristics 374
The required characteristics are the trueness and the precision of the lot of transfer pipettes, for each 375
specimen type claimed. 376
Further specifications that need to be evaluated for such pipettes may include: orifice diameter and 377
overall length (measures of trueness and sensitivity required), ability to deliver discrete drops easily by 378
untrained individuals (i.e. an in-use precision measure). Each of these requires a validated specification, 379
numerical limits and consequently a test method validated as giving the required information. The 380
following example below is only for the volume measurements. 381
c) Assign numerical values to the attributes 382
The specification for volume delivered into the IVD, validated by the R&D department, might be “not 383
less than 30 µL and not more than 45 µL of specimen to be delivered in two drops from the pipette” 384
which in the instructions for use would be translated to “add two drops of specimen using the dropper 385
pipette provided”. The specification of the pipettes would be “to deliver 35-40 µL ± 2 µL in two drops 386
and evaluated across the lot”. This would have been validated by R&D for each specimen type claimed. 387
From that specification, the requirement of the test method would be a bias of < 1 µL in the range 30 – 388
45 µL and a precision of < ± 0.8 µL (variance ≈20% of that allowed in the volume specified for the 389
pipettes). 390
d) Select or develop the method 391
The test method (weighing drops of water) is unlikely to introduce bias or imprecision beyond that in the 392
specification (based on the assumption that properly maintained and calibrated weighing 393
instrumentation is accurate) so the most important validation aspect is the relationship between drops 394
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of water and drops of each specimen type from the pipettes. The number of randomly selected pipettes 395
and the proportion of lots to be tested are calculated on the basis of acceptable risks ( 27) as confidence 396
in the supplier increased. 397
It is unlikely that the quality assurance incoming goods inspection team have access to the specimens 398
claimed for the IVD (e.g. fresh whole blood, fresh serum, cerebrospinal fluid). Hence any volume 399
measurements on a substitute liquid (e.g. water) with volume estimated by weight must be validated. 400
Drop volumes and variances of different liquids differ due to density and surface tension effects. 401
The exact method and required specifications would be documented in a standard operating procedure 402
in addition to data recording, monitoring requirements and a reference to the validation of the test 403
method. 404
e) Compare performance with requirements 405
The required characteristics of the test method can be measured and compared against the specification 406
(the precision and the number of pipettes to be tested). Consequently the method may be used if found 407
to be fit for purpose. 408
As can be seen, method validation at this level require planning, experimental work and documentation 409
beyond merely defining the method (“weigh some drops”) and the specification for the component. 410
9.2.1.2 Package labels, instructions for use, vials, stoppers etc. 411
Ancillary materials (e.g. printed matter, packing materials, containers, stoppers) will at a minimum, need 412
inspection prior to acceptance. Inspection is a test method. As usual, the requirements of the test 413
method (the inspection) must be defined once the purpose of the testing is understood. The attributes 414
of the test method can be evaluated (e.g. the pre-defined consumer risk and hence the proportion of the 415
incoming delivery to examine, the capability of the inspectors to distinguish and record the attribute) 416
and the method validated to consistently assure appropriate quality. Regardless of inspection method 417
chosen, its capability to detect flaws at the required level of risk must be documented as must be the 418
standard operating procedure for performing the inspection. 419
9.2.2 Constituents critical to IVD performance 420
Critical constituents must be decided on a risk assessment basis. Nitrocellulose membranes, some 421
detergents and all complex biological reagents (peptides, proteins, and oligonucleotides) are assumed to 422
be critical (unless there is evidence to the contrary). 423
Testing critical constituents typically involves techniques such as HPLC, spectroscopy, mass-424
spectrometry, sequencing and various forms of electrophoresis. All instrumentation is assumed to be 425
correctly documented, qualified and operated and the instrumentation itself will not contribute to bias 426
or uncertainty in the example below. The latter assumptions are usually true for the biological systems 427
described here; it is the nature of the measurements made that requires validation. Assessment of 428
critical constituents should be more detailed than for non-critical constituents (even if the critical 429
materials are obtained commercially). A commercial supplier cannot know the exact use of the material 430
and can only give a general certificate of analysis (COA). 431
9.2.2.1 Example of acceptance testing for a low molecular weight constituent 432
Some detergents (those containing a polyether bond e.g. Tween, Triton) easily and quickly generate 433
peroxides ( 28). 434
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a) Define the purpose of the testing 435
Peroxides can disrupt enzyme activity and the conformation of recombinant proteins and some 436
peptides. For this reason it may be considered necessary to monitor the peroxide content of detergents 437
used in an IVD, either at the incoming goods check or often just prior to use. 438
b) Identify the required characteristics 439
The characteristics required are sensitivity (range and uncertainty at specific concentrations), trueness 440
(accuracy, bias) and precision. 441
c) Assign numerical values to the attributes 442
R&D should have proven the stability of the IVD in studies using various lots, some of these lots at the 443
end of their shelf-lives ( 18, 23). Preliminary stability experiments should lead to knowledge of the 444
maximum permissible concentration of peroxide, (specified as, for example, < 6.0 µM) in the detergent 445
used routinely in the manufacture of the IVD. 446
An example of specifications for the test method derived from the R&D requirements could be “no bias, 447
sensitivity of 5 µM ± 1 µM at a concentration near 55 µM “(i.e. ability to distinguish between 50 and 55 448
µM and to allow for 10-fold dilution of a stock solution), with a peroxide specification of <55 µM in the 449
stock solution: to give an acceptable margin of safety relative to the permissible concentration and the 450
test method variance. 451
d) Select or develop the method 452
Several suitable methods for measurement of peroxide in aqueous detergent solutions are available. 453
However, as they are not standard methods, they necessitate characterisation and validation of the 454
required sensitivity, bias and precision near the permissible concentration of peroxide in solutions of the 455
particular detergent. 456
e) Compare performance with requirements 457
Clear specifications and justification of both method and expected result are required. 458
9.2.2.2 Acceptance testing of molecules with defined structures 459
For short peptides and oligonucleotides, the COA from an established and reputable supplier usually 460
gives sufficient structural detail (e.g. proof of sequence and terminal residues (usually by mass 461
spectrometry) and freedom from synthetic artefacts and residues (usually by HPLC)). As result, further 462
acceptance measurements are usually not required. However, for peptides containing cysteine (or 463
cystine) residues it might be necessary to monitor the state of the received material to ensure lack of 464
oxidation (or reduction), requiring a validated method for measurement of sulphydryl content. 465
Monoclonal immunoglobulin G class antibodies (but not polyclonal antibodies) are normally robust in 466
production and a COA of identity and purity is generally sufficient. Polyclonal antibodies, which vary in 467
avidity and precise epitopic dependence from animal to animal, may require similar functionality testing 468
to that suggested in the following for recombinant proteins. This is also true for immunoglobulin M class 469
antibodies, whether monoclonal or polyclonal. 470
9.2.2.3 Acceptance testing of recombinant proteins and polynucleotides 471
Recombinant proteins and polynucleotides require more complex testing than for molecules with a 472
simple sequence. The functionality of recombinant proteins and polynucleotides is frequently 473
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dependent on the conformation of the macromolecules and specificity depends on the precise 474
impurities present (among other things). The material’s specification must include requirements for 475
functionality, for purity based on similarity of contaminants between lots, measures of sequence 476
integrity and molecular conformation. 477
The test methods involved in preparing satisfactory COAs, or of providing evidence of satisfactory in-478
house preparations, are much more complex than those in the elementary examples given above. 479
However, validation of the methods follows exactly the same principles. Usually the methods are well 480
known analytical procedures. It may be the case that they are not “standard” methods but adequately 481
known and characterised so that if used appropriately they do not require further validation. 482
A problem observed in many submissions to WHO prequalification is that either the methods are not 483
used at all, or the output is not appropriate for the task. The following section discusses these major 484
issues. It does not provide detail of the process of validation, but an expectation of how the well-known 485
test methods will be used. 486
Both purity and conformation are critically lot dependent and are not usually documented in sufficient 487
detail in a commercial COA to provide objective evidence of inter-lot reproducibility. A standard 488
commercial COA for a recombinant protein usually provides a result for purity from a gel after 489
electrophoresis (e.g. “>95 %”) without specifying the exact concentrations and molecular weights of the 490
impurities (so allowing lot-to-lot variation within the specification and hence potential for specificity and 491
stability issues). A COA will also usually provide a molecular weight which is determined from a gel or a 492
Western blot. However, neither technique are adequately sensitive to demonstrate minor post-493
translational modifications, nor capable of providing any information about conformation (allowing 494
potential sensitivity and selectivity issues). Quantitation of results from both stained and blotted gels is 495
not reproducible without special techniques and gives only approximate values ( 29). A COA should 496
always give some measure of uncertainty in the stated values of both molecular weight and quantity. A 497
competent COA should also include amino- and carboxy- terminal amino acid analyses to ensure 498
absence of minor proteolysis during purification. 499
Choice of correct methods, and knowledge of their limitations, is a major deficiency in most WHO 500
prequalification submissions. There is insufficient proof that there is no lot-to-lot variability, neither in 501
those critical materials nor in the final IVD made from them. 502
Before committing to purchase or process a substantial amount of a new lot of recombinant protein, a 503
careful manufacturer will need to check that the new lot will detect the difficult specimens 504
(seroconversion, latent stage, unusual serotype specimens) with the sensitivity and specificity claimed 505
for the IVD and with approximately the same utilization (devices per milligram) as the lots used to 506
validate the IVD itself. Proficient manufacturers develop testing of identity, integrity and functionality of 507
polynucleotides to be used in IVD. 508
These tests for complex critical reagents can only be specified for commercial or for in-house reagents 509
by the manufacturer of the IVD, since the requirements are unique to the IVD and its validated claims. 510
Nevertheless, the methods used must be validated as suitable for use. 511
The sensitivity, specificity and utilization measurements on new lots of recombinant proteins are usually 512
made by preparing the IVD on a small scale and testing against defined panels of specimens proven to 513
monitor the stated parameters with satisfactory efficiency. Test method validation is to ensure that the 514
panels do indeed monitor the expected parameters. 515
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10 References
1. ISO 5725-1:1994: Accuracy (trueness and precision) of measurement methods and results — 516
Part 1: General principles and definitions. Geneva, Switzerland: International Organization for 517
Standardization; 1994 518
2. ISO 18113-1:2009. In vitro diagnostic medical IVDs – Information supplied by the manufacturer 519
(labelling) – Part 1: Terms, definitions and general requirements. Geneva, Switzerland: 520
International Organization for Standardization; 2009. 521
3. ISO 9000:2015. Quality management systems – Fundamentals and vocabulary. Geneva, 522
Switzerland: International Organization for Standardization; 2015. 523
4. ISO 15198:2004. Clinical laboratory medicine – In vitro diagnostic medical IVDs – Validation of 524
user quality control procedures by the manufacturer. Geneva, Switzerland: International 525
Organization for Standardization; 2004. 526
5. GHTF/SC/N4:2012 (Edition 2). Glossary and Definitions of Terms Used in GHTF Documents. 527
Global Harmonization Task Force (GHTF) Steering Committee; 2012. 528
6. ISO 14971:2007. Medical devices – Application of risk management to medical IVDs. Geneva, 529
Switzerland: International Organization for Standardization; 2007. 530
7. ISO/IEC Guide 99:2007: International vocabulary of metrology -- Basic and general concepts and 531
associated terms (VIM). Geneva, Switzerland: International Organization for Standardization; 532
2007. 533
8. US FDA Volume II: Methods, Method Verification and Validation ORA-LAB.5.4.5 October 2003, 534
Revised August 2014. http://www.fda.gov/ScienceResearch/FieldScience/ucm171877.htm 535
9. United States CFR - Code of Federal Regulations Title 21. Sec. 820.3 Definitions. Washington DC, 536
United States of America; 2010. 537
10. Magnusson, B., and Örnemark, U. (eds.) Eurachem Guide: The Fitness for Purpose of Analytical 538
Methods – A Laboratory Guide to Method Validation and Related Topics, (2nd ed. 2014). ISBN 539
978-91-87461-59-0. (http://www.eurachem.org) 540
11. Eurolab: Validation of Test methods. General principles and concepts: EL1545/96 541
(http://www.eurolab.org) 542
12. ICH: Validation of analytical procedures: text and methodology {Q2(R1)} November 1996 543
(http://www.ich.org/products/guidelines/) 544
13. WHO Technical Report Series, N°937, Appendix 4, Analytical method validation, of Annex 4 545
Supplementary guidelines on good manufacturing practices : validation - WHO, 2006 546
14. US FDA Center for Drug Evaluation and Research (CDER): Bioanalytical Method Validation, May 547
2001 & September 2013 (draft update) 548
15. ISO 15189:2012. Medical laboratories -- Requirements for quality and competence. Geneva, 549
Switzerland: International Organization for Standardization; 2012 550
16. WHO Prequalification – Diagnostic Assessment. Technical Guidance Series (TGS). Principles for 551
Performance studies TGS–3. Geneva: World Health Organization; 2016. Available at: 552
Technical Guidance Series for WHO Prequalification – Diagnostic Assessment: Guidance on Test method validation for in vitro diagnostic medical devices TGS–4
Page 21 of 21 Draft for comment 20 December 2016
http://www.who.int/diagnostics_laboratory/guidance/technical_guidance_series/en/, accessed 553
15 July 2016. 554
17. CLSI. Quality Management System: Equipment; Approved Guideline. CLSI document QMS13-A 555
Wayne, PA: Clinical and Laboratory Standards Institute; 2011 556
18. CLSI. Evaluation of Stability of In Vitro Diagnostic Reagents: Approved Guideline. CLSI document 557
EP25-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2009 558
19. CLSI. User Estimation of Precision and Estimation of Bias CLSI document EP15-A3 Wayne, 559 PA: Clinical and Laboratory Standards Institute; 2014 560
20. Valcárcel, M., Cárdenas, S., Barceló, D. et al. Metrology of qualitative chemical analysis, KI-NA-561
20-605-EN-C, ISBN 92-894-5194-7; 2002 Available free of charge from: 562
http://bookshop.europa.eu/en/metrology-of-qualitative-chemical-analysis-pbKINA20605/ 563
21. Burdick, R.K., Borror, C. M., & Montgomery, D. C. (2005). Design and analysis of gauge R and R 564
studies: making decisions with confidence intervals in random and mixed ANOVA models. ASA & 565
SIA Math: ISBN 0898715881 566
22. ISO 23640:2011. In vitro diagnostic medical IVDs - Evaluation of stability of in vitro diagnostic 567
reagents. Geneva, Switzerland: International Organization for Standardization; 2011. 568
23. WHO Prequalification – Diagnostic Assessment. Technical Guidance Series (TGS). Establishing 569
stability of an in vitro diagnostic for WHO Prequalification TGS–2. Geneva: World Health 570
Organization. 2016. Available at: 571
http://www.who.int/diagnostics_laboratory/guidance/technical_guidance_series/en/, 572
Accessed 15 July 2016. 573
24. US FDA Center for Devices and Radiological Health (CDRH): Design control guidance for 574
medical device manufacturers March 1997. Available at: 575
http://www.fda.gov/medicaldevices/deviceregulationandguidance/guidancedocuments/uc576
m070627.htm Accessed 20 December 2016 577
25. ISO 13485:2003. Medical devices – Quality management systems – Requirements for regulatory 578
purposes. Geneva, Switzerland: International Organization for Standardization; 2003. 579
26. WHO Technical Report Series, N°937, Appendix 3, Cleaning validation, of Annex 4 580
Supplementary guidelines on good manufacturing practices : validation - WHO, 2006 581
27. ANSI/ASQ Z1.4–2003 (R2013): Sampling Procedures and Tables for Inspection by Attributes, 582
2013. Available at: https://www.ansi.org/ Accessed 20 December 2016. 583
28. Jaeger J, Sorensen K, Wolff SP. Peroxide accumulation in detergents. J Biochem Biophys 584
Methods. 1994;29:77–81. doi: 10.1016/0165-022X(94)90058-2 585
29. Taylor, SC, Posch, A. (2014) The Design of a Quantitative Western Blot Experiment BioMed 586
Research International, volume 2014, Article ID 361590 8 pages, 2014 587
http://dx.doi.org/10.1155/2014/361590 588