TGS-4 Guidance on Test Method Validation for IVDs · Technical Guidance Series for WHO...

Technical Guidance Series (TGS)

for WHO Prequalification – Diagnostic

Assessment

Guidance on Test Method

Validation of in vitro diagnostic

medical devices

TGS–4

Draft for comment 20 December 2016

© World Health Organization 2016

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Contact: Irena Prat, EMP Prequalification Team Diagnostics

WHO - 20 Avenue Appia - 1211 Geneva 27 Switzerland

mailto:[email protected]

Technical Guidance Series for WHO prequalification – Diagnostic assessment: Guidance on Test method validation of in vitro diagnostic medical devices TGS–4

of 21 Draft for comment 20 December 2016

Contents

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



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



Acknowledgements

The draft document “Technical Guidance Series for WHO Prequalification – Diagnostic Assessment:

Guidance on Test method validation of 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.



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



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



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



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



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



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



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



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



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



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



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


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


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


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



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




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


The characteristics required are sensitivity (range and uncertainty at specific concentrations), trueness 440

(accuracy, bias) and precision. 441


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



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



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

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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. 529

International Organization for Standardization; Geneva, Switzerland: 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

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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

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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

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(http://www.eurolab.org) 542

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(http://www.ich.org/products/guidelines/) 544

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Supplementary guidelines on good manufacturing practices : validation - WHO, 2006 546

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2001 & September 2013 (draft update) 548

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Switzerland: International Organization for Standardization; 2012 550

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Principles for Performance studies TGS–3. Geneva: World Health Organization; 2016. Available 552

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http://www.imdrf.org/docs/ghtf/final/steering-committee/procedural-docs/ghtf-sc-n4-2012-definitions-of-terms-121109.pdf

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studies: making decisions with confidence intervals in random and mixed ANOVA models. ASA & 565

SIA Math: ISBN 0898715881 566

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reagents. Geneva, Switzerland: International Organization for Standardization; 2011. 568

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Establishing stability of an in vitro diagnostic for WHO Prequalification TGS–2. Geneva: 570

World Health Organization. 2016. Available at: 571

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