tgs2 Stability

Technical Guidance Series (TGS)
Establishing stability of
an in vitro diagnostic for TGS–2
WHO Prequalification
Draft for public comment 14 December 2015
Working document 14 December 2015

© World Health Organization 2015
All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, World Health
Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail:
bookorders@who.int). Requests for permission to reproduce or translate WHO publications – whether for sale or for non-
commercial distribution – should be addressed to WHO Press, at the above address (fax: +41 22 791 4806; e-mail:
permissions@who.int).
The designations employed and the presentation of the material in this publication do not imply the expression of any
opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city
or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent
approximate border lines for which there may not yet be full agreement.
The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or
recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors
and omissions excepted, the names of proprietary products are distinguished by initial capital letters.
All reasonable precautions have been taken by the World Health Organization to verify the information contained in this
publication. However, the published material is being distributed without warranty of any kind, either expressed or implied.
The responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health
Organization be liable for damages arising from its use.
Contact: Irena Prat, EMP Prequalification Team Diagnostics
WHO - 20 Avenue Appia - 1211 Geneva 27 Switzerland
Working document 14 December 2015

Establishing stability of an in vitro diagnostic for WHO Prequalification TGS–2
Contents
1 1 Introduction .......................................................................................5
2 2 Definitions and abbreviations ...........................................................6
3 3 WHO prequalification requirements ...............................................10
4 4 Basic principles for stability testing .................................................11
5 5 Shelf-life studies ..............................................................................15
6 6 Component stability studies ............................................................16
7 7 Stability during transport ................................................................19
8 8 In-use stability studies .....................................................................21
9 9 Production lots used in stability studies..........................................22
10 10 Stability plan ....................................................................................24
11 11 Stability report .................................................................................29
12 12 Changes to a Prequalified IVD .........................................................30
13 13 Authors and acknowledgements .....................................................32
14 14 References .......................................................................................33
15 Appendix 1: Example stability protocols ..............................................35
16 Appendix 2: Specimens for the stability testing panel ..........................43
17 Appendix 3: Summary table of standards relevant for stability
18 studies ...................................................................................................47
Working document 14 December 2015 3

WHO Prequalification of IVDs
WHO Prequalification Programme: IVD Technical Guidance Series

WHO The World Health Organization (WHO) Prequalification Programme is coordinated
Prequalification through the Department of Essential Medicines and Health Products. The aim of
of IVDs
WHO prequalification of in vitro diagnostics (IVDs) is to promote and facilitate
access to safe, appropriate and affordable in vitro diagnostics of good quality in an
equitable manner. Focus is placed on in vitro diagnostics, known commonly as
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.
Use of The findings of the WHO Prequalification of IVDs Programme are used to provide
prequalified independent technical information on safety, quality and performance of in vitro
IVDs
diagnostics, principally to other United Nations (UN) agencies but also to WHO
Member States and other interested organizations. The WHO prequalification
status, in conjunction with other procurement criteria, is used by UN agencies,
WHO Member States and other interested organizations to guide their
procurement of in vitro diagnostics.
Prequalification In vitro diagnostics (IVDs) prequalified by WHO are expected to be accurate,

requirements 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 a resource-limited Member
State. The countries using WHO-prequalified IVDs often have minimal regulatory
requirements. In addition, the users of IVDs in these countries have different
needs resulting in different requirements. For instance, the IVDs are often used in
patients with a different disease profile, by health care workers without extensive
training in laboratory techniques, in harsh environmental conditions and in
settings without extensive pre- and post-test services. Therefore, the
requirements of the WHO Prequalification Programme may be different to the
requirements of the users and regulatory authority in the country of manufacture.
About the The Technical Guidance Series was developed following a consultation, held on
Technical 10-13 March 2015 in Geneva, Switzerland attended by experts from national
Guidance
Series regulatory authorities, national reference laboratories and WHO prequalification
dossier assessors and inspectors. The guidance series is a result of the efforts of
this and other international working groups.
Audience and This guidance is intended for manufacturers interested in WHO prequalification of
scope 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.
4 Working document 14 December 2015

19 1 Introduction
20 1.1 Key concepts

21 Stability is the ability of an IVD reagent to maintain its performance characteristics
22 over a defined time interval [1,2]. The purpose of stability studies is to verify the
23 time interval and the storage conditions over which stable performance
24 characteristics of an IVD can be claimed.
25 1.2 Rationale of stability studies

26 The stability of an IVD is fundamental for its reliable performance over a defined
27 period of time. It is a regulatory requirement for the manufacturer to provide
28 objective, scientifically sound evidence to support all claims made regarding the
29 stability of an IVD. In addition a manufacturer can use stability studies to show
30 that all lots manufactured during the commercial life of the IVD will meet
31 predetermined user needs (inputs).
32 1.3 Purpose of this document

33 The purpose of this document is to provide IVD manufacturers with guidance on
34 possible approaches to determine stability. More specifically it describes the
35 requirements for WHO prequalification in terms of stability 1.
36 1.4 Limitations of this guidance

37 This guidance document should not be taken as a prescriptive checklist of what
38 should be performed, but as a guide on how to improve processes and generate
39 the evidence needed to ensure a comprehensive, systematic procedure with an
40 appropriate risk management plan.
41 The examples included throughout the document apply to the principles outlined
42 in this document only. Manufacturers must still perform their own product-
43 specific risk assessment for each of their IVDs.
44 It is possible that, depending on the particular categorization of the product and
45 on the particular jurisdiction that additional requirements may apply. These
46 regulatory and legal issues are specific for each regulatory authority and are
47 beyond the scope of this document.
1
See Instructions for Compilation of a Product Dossier, Prequalification of Diagnostics, PQDx_018 v3
27.08.2014, Section 7.2, Stability (excluding specimen stability).

48 2 Definitions and abbreviations

49
50 2.1 Definitions
51 The definitions given below apply to the terms used in this document. They may have different
52 meaning in other contexts.
53 Accelerated stability evaluation: Study designed to increase the rate of chemical and/or physical
54 degradation, or change, of an IVD reagent by using stress environmental
55 conditions to predict shelf-life.
56 NOTE: The design of an accelerated stability evaluation can include extreme
57 conditions of temperature, humidity, light or vibration.
58 Source: [1], definition 3.1
59 Acceptance criteria: A defined set of conditions that must be met to establish the performance of a
60 system.
61 Source: [2]
62 Numerical limits, ranges, or other suitable measures for acceptance of the results
63 of analytical procedures.
64 Source: [3]
65 Accuracy of measurement: Closeness of the agreement between the result of a measurement and a
66 true value of the measurand.
67 NOTE 1: Accuracy of measurement is related to both trueness of measurement
68 and precision of measurement.
69 NOTE 2: Accuracy cannot be given a numerical value in terms of the measurand,
70 only descriptions such as 'sufficient' or 'insufficient' for a stated purpose.
72 Arrhenius plot: Mathematical function that describes the approximate relationship between the
73 rate constant of a chemical reaction and the temperature and energy of activation.
74 Source: [2]
75 Batch/Lot: Defined amount of material that is uniform in its properties and has been
76 produced in one process or series of processes.
78 Component: Part of a finished, packaged and labelled IVD medical device.
80 NOTE 1: Typical kit components include antibody solutions, buffer solutions,
81 calibrators and/or control materials. Source: [5].
82 Constituent Raw materials used to make a component.
83 Source: WHO
84
85 Design input: The physical and performance requirements of an IVD that are used as a basis for
86 IVD design.
87 Source: [6], definition (f)
88 Drift: Characteristic slow change of a metrological value from a measuring instrument.
89 Source: [7]

90 Environmental factors: Variables that might affect the performance or efficacy of IVD reagents e.g.
91 temperature, airflow, humidity, light.
92 Source: [2]
93 WHO note: For WHO purposes, this also includes dust and micro-organisms.
94 Evidence: Information which can be proved true, based on facts obtained through
95 observation, measurement, test or other means
96 Source: Modified from [8], definition 3.8.1
97 Instructions for Use (IFU): Information supplied by the manufacturer to enable the safe and proper
98 use of an IVD
99 NOTE: Includes the directions supplied by the manufacturer for the use,
100 maintenance, troubleshooting and disposal of an IVD, as well as warnings and
101 precautions.
103 In vitro diagnostic (IVD): A medical device, whether used alone or in combination, intended by the
104 manufacturer for the in vitro examination of specimens derived from the human
105 body solely or principally to provide information for diagnostic, monitoring or
106 compatibility purposes.
107 NOTE 1: IVDs include reagents, calibrators, control materials, specimen
108 receptacles, software, and related instruments or apparatus or other articles and
109 are used, for example, for the following test purposes: diagnosis, aid to diagnosis,
110 screening, monitoring, predisposition, prognosis, prediction, determination of
111 physiological status.
112 NOTE 2: In some jurisdictions, certain IVDs may be covered by other regulations.
113 Source: [9]
114 IVD reagent: Chemical, biological or immunological components, solutions, or preparations
115 intended by the manufacturer to be used as an IVD
117 WHO note: This document uses the terms IVD and IVD reagent interchangeably.
118
119 Metrological traceability: Property of the result of a measurement or the value of a standard
120 whereby it can be related to stated references, usually national or international
121 standards, through an unbroken chain of comparisons all having stated
122 uncertainties.
123 NOTE 1: Each comparison is affected by a (reference) measurement procedure
124 defined in a calibration transfer protocol.
125 Source: [4]
126 Performance claim: Specification of a performance characteristic of an IVD as documented in the
127 information supplied by the manufacturer
128 NOTE 1: This can be based upon prospective performance studies, available
129 performance data or studies published in the scientific literature.
130 “Information supplied by the manufacturer” includes but is not limited to: statements in
131 the IFU, in the dossier supplied to WHO and /or other regulatory authorities, in advertising,
132 on the internet.
133 Referred to simply as “claim” or” claimed” in this document

135
136 Real-time stability evaluation: Study designed to establish or verify the shelf-life of the IVD reagent
137 when exposed to the conditions specified by the manufacturer
138 NOTE 1: Conditions that can affect stability of an IVD reagent include temperature,
139 transport conditions, vibration, light, humidity.
141 Risk management: The systematic application of management policies, procedures and practices to
142 the tasks of analysing, evaluating, controlling and monitoring risk.
143 Source: [10]
144 Risk management plan: For the particular IVD being considered, the manufacturer shall establish
145 and document a risk management plan in accordance with the risk management
146 process.
147 Source: [10], para 3.4
148 Shelf-life: Period of time until the expiry date, during which an IVD reagent, in its original
149 packaging, maintains its stability under the storage conditions specified by the
150 manufacturer
151 NOTE 1: Stability and expiry date are related concepts.
152
154 WHO NOTE: In this document “Labelled life” is considered as the time up to the expiry date
155 printed on the label of an IVD or a component of the IVD.
156 Stability: The ability of an IVD reagent to maintain its performance characteristics within
157 the limits specified by the manufacturer
158 NOTE 1: Stability applies to
159 - IVD reagents, calibrators, and controls, when stored, transported and used in the
160 conditions specified by the manufacturer
161 - reconstituted lyophilized materials, working solutions, and materials removed
162 from sealed containers, when prepared, used and stored according to the
163 manufacturer’s instructions for use
164 - and measuring instrument or measuring systems after calibration.
165 NOTE 2 to entry: Stability of an IVD reagent or measuring system is normally
166 quantified with respect to time:
167 - in terms of the duration of a time interval over which a metrological property
168 changes by a stated amount,
169 - in terms of the change of a property over a stated time interval.
171 In-use stability: Duration of time over which the performance of an IVD reagent within its
172 expiration date remains within specified limits after opening the container system
173 supplied by the manufacturer, and put into use under standard operation
174 conditions (e.g. storage on the instrument)
175 For the purpose of this guidance, WHO considers that it also includes the number of times
176 the reagents can be removed and returned to the storage condition without impact on
177 test kit performance. It shall reflect the routine conditions of use e.g. On-board stability,
178 reconstitution, and open-vial/bottle stability.
179 Source: [2]
180 Stability monitoring: Real-time stability testing at certain points in time during shelf-life (or in-use)
181 to assure that an IVD reagent performs within specified claims.
182 Source: [2]

183 NOTE: A continuing stability monitoring program (ongoing stability monitoring)

184 is required to verify that the stability claim is maintained over the commercial life
185 of the product. Data on stability should be obtained at end of shelf life
186 (Reference [1] para. 4.1) and additionally at half assigned-life to so that if
187 problems occur they can be dealt with in a timely fashion.
188 WHO note: WHO expect that file samples are used for each component and lot of
189 product.
190
191 Trueness of measurement: Closeness of agreement between the average values obtained from a
192 large series of results of measurements and a true value.
194 2.2 Abbreviations

CE Conformité Européenne (European Conformity)
CV Coefficient of variation
CD4 Cluster of differentiation 4
CLSI Clinical and Laboratory Standards Institute
EIA Enzyme-linked immunoassay
GHTF Global Harmonization Task Force
HBsAg Hepatitis B surface antigen
HBV Hepatitis B virus
HCV Hepatitis C virus
HIV Human immunodeficiency virus
IFU Instructions for Use
IgM Immunoglobulin M
IMDRF International Medical Devices Regulators Forum
ISO International Organization for Standardization
IVD In vitro diagnostic
NAT Nucleic acid test
OD Optical density
PEI Paul Ehrlich Institute
QA Quality assurance
QC Quality control
QMS Quality management system
RDT Rapid diagnostic test
RPM Revolutions per minute
R&D Research and development
TP Treponema pallidum
USP U.S. Pharmacopoeial convention
195

196 3 WHO prequalification requirements

197 WHO requires that reports of studies used in establishing the stability claims for
198 the product are submitted as part of the prequalification application 2. In the
199 submission, manufacturers should describe the rationale, the study methods, the
200 stability monitoring program followed and the testing algorithms used, with
201 references to the relevant standard operating procedures. The information
202 provided should demonstrate the link to the predetermined user requirements
203 and product development.
204 3.1 Manufacturer responsibility

205 It is a manufacturer’s responsibility to ensure that all claims made regarding the
206 stability of the IVD performance are supported by objective, scientifically-sound
207 evidence.
208 3.2 Standards

209 WHO recommends the following standards for the use in establishment of
210 stability claims: ISO 23640:2013 [1], CLSI EP25-A [2] and ASTM:D4169-14 [11].
211 It is recommended that manufacturers be familiar with these standards and
212 consider them when designing and planning their stability studies.
213 3.3 Suitability for use in Member States

214 The stability studies submitted to WHO should accurately reflect the expected
215 environmental conditions and the normal usage conditions/methods encountered
216 by the users in WHO Member States, such as:
217 • Extremes of temperature for in-use conditions and during transportation
218 • Extremes of humidity encountered during in-use conditions, transportation
219 and storage
220 • Dust
221 • Light, both the amount required for accurate testing/results interpretation and
222 any affects that light may have on the IVD functionality
223 • Micro-organisms
224 3.4 Meeting customer requirements

225 By undertaking well-designed stability studies including periodic verification
226 activities, the manufacturer can demonstrate that the product meets input (i.e.
227 customer) requirements, as required by ISO 13485:2003 (see [12] under 7.2,
228 Customer-related processes). Meeting predetermined user expectations, not
229 merely evaluating the capability of an IVD, is a fundamental aspect of
230 development of IVDs (see [6] definition (f) and [12] para 7.3.4). It is a proactive
231 means for the manufacturer to prevent quality problems at lot release and in the
232 post-production and marketing phase.
2
PQDx_049 Product dossier checklist and PQDx_018 Instructions for compilation of a product dossier
Both available on the WHO Prequalification of in vitro diagnostics website
http://www.who.int/diagnostics_laboratory/evaluations/en/

233 4 Basic principles for stability testing
234 4.1 Critical characteristics of the IVD

235 A well-designed stability study must generate evidence of stability of each of the
236 critical constituents of the IVD (risk-evaluated critical constituents), each of the
237 claimed analytes, and any particular level of performance including precision,
238 sensitivity and specificity of the kit.
239 Examples:
240 1) A hepatitis C virus (HCV) assay containing the critical constituents related to
241 detection of NS3 or core proteins should have the stability of all such constituents
242 proven.
243 2) For an assay designed to detect both IgG and IgM by use of protein A and protein
244 L, the stability of both protein A and protein L should be proven.
245 3) For CD4, all the antibodies involved (e.g. anti-CD3 and anti-CD4) must be shown
246 to be stable.
247 4) For an IVD claimed to detect particular seroconversion specimens, or genotypes,
248 or to have specified precision at particular analyte concentrations, or a particular
249 specificity, each of these claims must be proven over the stated shelf-life.
250 4.2 Finalised product presentation

251 During stability testing, all IVD components (including the device, calibrator
252 and/or control material, etc.) must be made and tested to the finalised
253 manufacturing documentation and in the finalised packaging including intended
254 labels and containers. All presentations (e.g. different buffer volumes used for
255 different kit sizes) must be used during stability testing.
256 4.3 Environmental conditions

257 The study should subject the IVD to a combination of conditions which define the
258 limits of stability for all lots made during its commercial life. The combinations of
259 conditions, durations of exposure and the number of lots to be used will be driven
260 by a manufacturer’s risk assessment for the IVD and data from R&D. The risk
261 assessment should take into account at least:
262 • the variability of the constituent materials (identifying the most important
263 sources of variability);
264 • the nature of the users’ environments; and
265 • extreme conditions potentially occurring during transportation to those
266 users (see also 3.3).
267 Boundary conditions for stability studies should reflect realistic extreme
268 conditions that are consistent with the design input requirements for the IVD. The
269 consequent stability studies will prove the IVD capable of meeting performance
270 requirements at the end of its stated shelf-life, after transport to the users.

271 4.4 Minimum number of lots

272 Similarly to clinical performance validation, the design of stability studies should
273 take into consideration lot-to-lot variation, with a risk assessment to identify the
274 most important sources of variability. Lot variability is caused usually more by the
275 biological reagents than by the actual manufacturing process. Although existing
276 standards recommend the use of one lot for certain stability studies, the impact of
277 lot-to-lot variability must be taken into consideration and use of additional lots
278 may be necessary. To ensure the potential of lot-to-lot variability is addressed,
279 optimally lots containing different batches of critical constituents such as
280 nitrocellulose membranes, recombinant antigens, peptides, nucleic acids and
281 enzymes used in nucleic acid testing (NAT), etc. that are as different as possible
282 should be used.
283 Example: For NAT assays, it is critical to use unique enzyme lots for stability
284 studies. Other components including primer, probe and buffer can also be
285 affected by the manufacturing process (purity, pH, DNase & RNase
286 contamination, etc.). For these, different lots are also highly desired that
287 represent both material and process variability.
288 4.5 Assessment of liquid components

289 It is standard best practice in stability studies to ensure that liquid components
290 are in contact with all the parts of their container – vial, sachet or bottle, such as
291 the stopper, the seal and the body of the container. This is sometimes called
292 “inverted container stability” but is probably best studied by ensuring all
293 containers are on their sides and disturbed by movement during the stability
294 study. This aspect needs particular attention for in-use stability studies of those
295 components that are diluted or reconstituted from freeze-dried before use.
296 4.6 Specimens for the stability testing panel

297 The specimens used in the stability testing panels must reflect all the performance
298 claims related to the IVD. The specimen types most likely to be utilised with an
299 IVD in WHO Member States should be considered and included in the specimen
300 panels used throughout the stability studies (see Appendix 2). If a variety of
301 specimen types (e.g. serum, plasma, whole blood, saliva) is claimed as being
302 suitable for use in the instructions for use (IFU), the stability plan must be
303 designed to provide evidence that the IVD will maintain each of the claims (e.g.
304 sensitivity, specificity, proportion of valid runs, precision) for each of the specimen
305 types for the whole of the claimed shelf-life including transport to the final users.
306 Evidence should be statistically valid (see section 10).The stability testing panel
307 must be validated accordingly and rejection and replacement criteria should be
308 established. Regulatory requirements may also dictate the addition of panel
309 members.
310 A stored validated stability testing panel is not always feasible. For example, this is
311 often the case for assays requiring fresh and/or whole blood specimens e.g. CD4,
312 assays to detect RNA. When replacing panel members, the accuracy of results
313 generated with the replacement material must be confirmed using an appropriate
314 reference comparator method. Replacement criteria for unstable panel members
315 will include the duration for which a critical member will give valid results.

316 4.7 Validation of stability testing panel

317 The validation of the stability testing panel members used is critical. Stability
318 testing panel members themselves must be stable, and they must monitor
319 parameters that are useful to control the component involved.
320 4.8 Assigning criteria to panel members

321 Stability testing panel members are chosen deliberately to ensure each member
322 has an attribute pertinent to the intended use. As with lot release testing, the goal
323 of stability testing is to ensure that the test method appropriately monitors the
324 functionality of the antigens, epitopes, and antibodies that are relevant to the
325 intended use at the end of the assigned (shelf/in use) life.
326 For instance, the intended use claim may be that early seroconversion specimens
327 are detected. To show that this claim is true at the end of the product’s life, a very
328 early seroconversion specimen is included in the stability panel. This specimen
329 may be a weakly reactive IgM specimen.
330 An expected value is then assigned to each panel member and this is used to
331 assign the acceptance criteria for that panel member. The value for each member
332 is assigned in a measurable manner relevant to the outputs of the particular
333 methodology. For instance, the acceptance criteria for each panel member may
334 be assigned in terms of sample-to-cut-off ratio, cycle time (CT) values, and band
335 intensity measured semi-quantitatively/quantitatively.
336 In the example of a weakly reactive IgM seroconversion specimen, the specimen
337 at the start of shelf life may have a reading score on an RDT of 1+ out of 4,
338 assigned by using a semiquantitative value based on band intensity. The
339 acceptance criteria may be that all reactive specimens remain reactive, and all
340 non-reactive specimens do not react in the assay.
341 As such, panel members must be chosen that not only will be relevant to
342 demonstrate the intended use, but have values that will appropriately detect and
343 therefore monitor any deleterious effects of storage. A strong positive specimen,
344 which has a 4+ out of 4 semi-quantitative reading value, may remain giving this
345 reading despite decay in the assay, whereas a specimen with a reading of 1+ out
346 of 4 (with an assigned acceptance criteria of remaining positive) is more likely to
347 give an indication of the ongoing stability of the assay.
348 Thus it is essential to know that where a panel member meets acceptance criteria,
349 this is a true reflection of the stability of the product and not due to the inability
350 of the specimen result to reflect this change.
351 4.9 Time points

352 A simple study design requires three testing intervals:
353 • an initial baseline test and
354 • a test at the time point beyond the claimed stability limit
355 • and one point in between.
356 However, this is a high risk approach that has the potential for wastage of time
357 and resources. If the IVD does not meet the acceptance criteria at the end of
358 testing there is little information about the deterioration of the component or IVD
359 (or lack of deterioration) in the interim period.

360 A more effective approach is to test at predetermined time point intervals. The
361 manufacturer should decide on practical intermediate test points. The number
362 and length of testing intervals should be determined in advance and form part of
363 the stability plan/protocol. This planning will help to understand the resources
364 required to execute the experiment.
365 Testing of all panel members is not required at all test/time points. However
366 testing with all panel members is required at the initial, the second last and the
367 last test/time point of any of the study specimen types. The manufacturer should
368 decide on practical intermediate test points at which a smaller minimal number of
369 panel members are tested. There should be a documented rationale for the
370 choice of the panels used at the intermediate test points (e.g. representative
371 members, specimens that are close to the medical decision points and at the
372 extremes of the assay range tested).
373 4.9.1 Duration of testing

374 Testing conducted in stability studies should extend beyond the shelf-life
375 determined from the user needs. The shelf-life should be assigned based on a risk
376 assessment of the lot-to-lot variability in signal change at the end of shelf life. At a
377 minimum, testing should extend at least one time point (one testing interval)
378 beyond the determined user requirement. This provides a safeguard in the event
379 of unexpected IVD failure at the end of the testing period, in which event
380 extrapolation from an earlier time point would not be considered acceptable.
381
382 It is recommended to utilize standardized units of measure for the entire study
383 (e.g. Unopened kit shelf life are always measured in months; opened kit /reagent
384 stability in days or weeks )

385 5 Shelf-life studies
386 5.1 Requirements for determination of shelf life

387 The stated shelf-life of an IVD must be based on real-time experimental results.
388 Accelerated stability studies are usually not sufficient to support a claimed shelf-
389 life. They might be used in situations where experience already exists with similar
390 products (see para 4.1 in Reference [1]) or when the stability of very similar
391 products is already known (see para 7.3.1 in Reference [2]).
392 WHO prequalification requirements: If at the time of dossier submission
393 the real-time study outcome is not available, accelerated studies might
394 be considered. The manufacturer must justify why the accelerated study
395 is acceptable as supportive evidence until real-time experimental results
396 become available. In these cases, the results of real-time stability studies
397 will be requested as a condition of WHO prequalification. The shelf-life of
398 the IVD could be extended as the real-time data accumulates.
399 5.1.1 Real-time stability studies

400 Real-time stability is determined using storage temperatures derived from user
401 requirements, over a period longer than the required life of the IVD.
402 Where a range of storage temperature is claimed (e.g. “Store at 4–40°C”), WHO
403 expects the studies will provide evidence for stability over the whole of the
404 temperature range for at least the length of the claimed shelf-life. Exceptionally,
405 where claimed stability is restricted to a limited range e.g. “Store at 2-8°C”, it is
406 acceptable that stability studies are conducted at a single temperature within this
407 range.
408 A sequential approach should be used [2], in which IVDs are first submitted to
409 stresses simulating transport before they are placed into a shelf-life or in-use
410 study. This approach best simulates the real-life situation, where products will
411 first be transported to the end-user and then stored under the recommended
412 conditions before use, possibly almost until the end of their labelled shelf-life.
413 5.1.2 Accelerated stability studies

414 Accelerated stability studies are designed to predict the shelf-life of an IVD from
415 the increased rates of chemical and/or physical degradation caused by extreme
416 environmental conditions (e.g. elevated temperature at higher humidity).
417 If the Arrhenius equation is used to calculate the expected life at temperatures
418 other than those actually used, then the parameters of the equation must be
419 derived from the data and not assumed [2].
420 Accelerated stability studies provide results in a relatively short time. However the
421 results of these studies are made using assumptions about the degradation of
422 reagents and IVD components that may not reflect their performance under
423 normal conditions of storage and use.

424 6 Component stability studies
425 6.1 General principles
426 6.1.1 Testing on final specifications

427 Component stability studies, including antimicrobial and desiccant studies, must
428 be performed using components made according to finalized and approved
429 manufacturing specifications – ideally to validated manufacturing scale – on
430 qualified manufacturing equipment and meeting finalized and approved in-
431 process quality control (QC) specifications.
432 6.1.2 Considering component stability

433 Sometimes components of IVDs are prepared in bulk and stored before being
434 used in several different lots of a completed IVD. The design input documentation
435 should define how long components are likely to be stored before use. With that
436 information, component stability studies should be planned to give evidence that
437 component labelled lives will not restrict IVD labelled lives: an IVD cannot have a
438 labelled life beyond that of any of its dependent components.
439 Shelf-lives of components manufactured in bulk and used in several different lots
440 of an IVD can be verified as for the IVD itself – three lots of the component as a
441 minimum for shelf-life studies and, depending on documented risk assessment
442 related to variability, one or more lots subsequent to change. The evaluated lots
443 of the component must differ in batches of critical constituents but, again subject
444 to documented risk assessment, may all be tested in their final presentation with
445 a single set of the other components which will be used together to constitute the
446 IVD.
447 Examples of stored components: Wash solutions and substrates for
448 enzyme immunoassay (EIA), amplification reagents for nucleic acid
449 testing, calibrators for quantitative tests, manufactured and stored in
450 their final labelled vials ready to be put into a kit
451 Component stability can be assessed from the functionality of the lot and also by
452 factors related to the component itself, such as turbidity, colour change, microbial
453 contamination and pH of liquid components changes over time.
454 Depending on the IVD and the conditions it is subjected to it may be necessary to
455 distinguish between turbidity that arises from heat/cold denaturation and
456 turbidity that arises from microbial contamination.
457 6.1.3 Considering constituent stability

458 The plan should also consider whether components made from new constituents
459 (antigens, recombinant antigens, enzymes, antibodies, membranes) will have the
460 same lives as components made from stored raw materials. Although this aspect
461 is difficult to study, some evidence should be provided supporting the use of
462 stored constituents, as well as a plan to evaluate lot-to-lot variance from different
463 critical constituents.
464 The choice of the reagents to be used to measure the performance of the
465 constituent under study (either materials of proven shelf-life or freshly made)
466 needs substantial consideration.

467 Examples of stored constituent: Purified recombinant antigens and

468 monoclonal antibodies stored in aliquots ready for dilution and striping
469 onto RDT membranes or other supports
470 6.2 Stability of control materials

471 Assay specific control materials provided by the manufacturer are to show that an
472 IVD has performed as intended during use. The manufacturer must be able to
473 demonstrate that the loss of signal of a control does not occur at a different rate
474 from the loss of signal from a validated stability testing panel member or from
475 genuine, critical specimens; otherwise a failed IVD might be regarded as still
476 functional. Thus the stability of the control material must accurately reflect the
477 stability of the assay. A control that is more stable than the IVD and other
478 components, or incorrectly set values for the control material, must be avoided
479 [13].
480 Example: It is frequently seen in dossiers relating to IVDs submitted to
481 the WHO Prequalification Programme that a positive run control will
482 produce a signal of >2.0 optical density (OD) in a freshly manufactured
483 lot, and the IFU will state that an OD > 0.8 for the same control qualifies
484 a run. Thus the IVD could have lost more than half its activity and still
485 appear functional, although some critical specimens are shown in the
486 dossier to have very weak signals on freshly made IVDs. This is not
487 considered appropriate unless data can be provided that demonstrate
488 that the critical specimens will still be detected at the end of shelf life.
489 6.3 Antimicrobial stability and efficacy
490 6.3.1 Rationale

491 IVDs are used in areas which are not necessarily clean and sterile, and
492 antimicrobials are not stable under some circumstances. Bacterial and fungal
493 organisms relevant to the environment of use should be identified in the design
494 input risk assessment, and antimicrobial preservatives should be chosen to avoid
495 contamination of the product. The manufacturer must obtain evidence that the
496 antimicrobial preservative and concentration chosen is stable and effective
497 against the micro-organisms of concern throughout the claimed shelf-life and in-
498 use shelf life.
499 6.3.2 Study conditions

500 The studies should reflect expected in-use conditions in opened containers: clean,
501 particle-free laboratories do not usually reflect universal user environment.
502 See Reference [14] Sections <51> ,<61> and <62>; and Reference [15] Appendix XI
503 for suggested methods. Examples of bacterial groups to consider are spore-
504 forming bacteria, fungus, indigenous bacteria, bacteria found in the environment
505 of the country of manufacture, as well as use of a negative control. Specific
506 examples outlined in References [14] and [15] include Aspergillus niger, Bacillus
507 subtilis, Candida albicans, Escherichia coli, Salmonella species, Pseudomonas
508 aeruginosa, Clostridium sporogenes and Staphylococcus aureus.

509 6.3.3 Time of testing

510 Antimicrobial preservative effectiveness, as measured by the viable microbial
511 species load present in kit components, should be demonstrated during
512 development, during scale-up, and throughout the shelf-life.
513 Testing for antimicrobial preservative content should normally be performed at
514 release. Under certain circumstances, in-process testing may suffice in lieu of
515 release testing. The acceptance criteria for in-process testing should remain part
516 of the specification [16].
517 6.4 Desiccant functionality

518 Desiccants affect the stability of the entire IVD. Stability studies must show that
519 the desiccant will support the product over the whole claimed shelf-life within the
520 predetermined extremes of transport, storage and in-use conditions.
521 WHO Note:
522 1) WHO recommends that a self-indicator (a humidity indicator that changes colour
523 upon saturation) be part of the desiccant design. However, WHO strongly
524 recommends against the use of cobalt dichloride, the most commonly used
525 humidity indicator, as it is a carcinogenic substance.
526 2) Sachets are preferable over tablets, since labelling such as “Do not eat” is more
527 visible. There have been anecdotal reports of desiccants in a tablet formulation
528 being mistaken for antimalarial medicine.

529 7 Stability during transport
530 7.1 Rationale

531 Transport stability studies evaluate the tolerance of an IVD to the kinds of
532 environmental conditions (e.g. temperature, humidity, dust) and physical
533 conditions (inversion, vibration, physical handling, stacking) to which it is likely to
534 be subjected in the time between shipping from the manufacturer to its final user.
535 They should provide evidence that there will be no impact on the IVD
536 performance over the whole of its stated shelf-life after recommended
537 transportation methods for the IVD. The manufacturer should assess the potential
538 impact of multiple factors and justify and document whether or not to include
539 them in the evaluation.
540 WHO expects that a transportation challenge should precede the real-time
541 determination of shelf-life This serves to determine that transportation conditions
542 do not reduce the shelf-life of the IVD (see also 5.1) .
543 In some cases it might be acceptable to test the product only over the transport
544 simulation duration, without a subsequent long-term study under normal storage
545 conditions. If that is done, shelf-life must be established under specified storage
546 conditions along with a stringent, evidence-based risk assessment of the
547 probabilities of extreme transport stress affecting the performance at the end of
548 the claimed life (see para. 4.2.3 in Reference [2]).
549 7.2 Challenge conditions

550 Determination of the stability during transportation of an IVD should take into
551 consideration the local routes, transport means and transit used to supply the IVD,
552 usually defined in the design input risk assessment. It is not necessary to test the
553 IVDs to the point where it is no longer usable, but merely to validate the window
554 of transport conditions within which the IVD will retain its claimed performance to
555 the end of its stated shelf-life. However, knowledge of the possible limitations of
556 an IVD and at what point the IVD becomes unusable is useful to a manufacturer
557 when trouble shooting post-market problems. WHO expects the manufacturer to
558 consider that the product might continue to be subjected to sub-optimal storage
559 conditions at the end-user.
560 Example: While a static challenge of 45°C for 3 days might represent
561 conditions seen during actual transport of a IVD, a more stringent
562 challenge of cyclical higher and low temperatures (including freezing) for
563 a longer period of time and under vibration might better cover a ‘worst
564 case scenario’ of shipment, storage and subsequent transportation to
565 the end-user.
566 7.3 Number of lots

567 Where transport stability studies are incorporated into studies to establish shelf
568 life, as recommended in this guidance, a minimum of three lots of the IVD should
569 be used. For transport studies alone, at least one lot of the IVD can be used,
570 however, as with shelf life studies, more may be required depending on lot
571 variability (see 9.1).

572 7.4 Multiple stress test sequences

573 Mere proof of performance after actual shipment is generally insufficient
574 evidence of stability under all conditions and with the hazards of delays. Multiple
575 stress test sequences are typically needed to address the range of transport
576 conditions used for global product delivery, including some extreme conditions
577 expected to be evaluated according to relevant guidance [11].
578 Appropriate sequences may be developed on the basis of data from actual
579 product transport studies. Testing multiple stress sequences allow a manufacturer
580 to identify the most cost- and/or resource effective transport conditions from a
581 set of alternatives while ensuring adequate product stability protection
582 (Reference [2] para 4.2.3).
583 WHO requirement: Environmental conditions investigated as part of a
584 stability study must reflect those likely to be encountered in resource-
585 limited Member States. Temperatures at some airport tarmacs in Sub-
586 Saharan Africa can exceed 40°C while temperatures encountered during
587 air transport fall below 0°C. Significant delays can be encountered at all
588 times and especially during wet season transport to remote health
589 centres.
590 7.5 Physical conditions

591 Physical handing can be both manual and mechanical. The relevant user and
592 commercial factors should be identified as part of the design input risk
593 assessment and the packaging and shipping methods developed accordingly.
594 Reference [11] defines a number of factors to be considered, and their evaluation:
595 drop, impact, compression, vibration, repetitive shock, longitudinal shock, cyclic
596 exposure, vacuum, impact, inversion; along with the size, weight, and composition
597 of the packaging.
598 7.6 Simulated versus actual challenge

599 An actual shipping challenge can be used to verify the conditions found in the
600 simulated transportation challenges. However it should only replace a simulated
601 shipping challenge when there is an appropriate risk evaluation and with
602 experience and data already actively collected from similar products and
603 documented in detail (for example it is insufficient to note “no complaints”).
604 In the R&D phase, actual data from shipping can be used to define the conditions
605 needed for an appropriate simulation of extremes. However in the post-
606 production phase actual shipping challenges often do not explore the full range of
607 shipping conditions that could be encountered, including extreme values.

608 8 In-use stability studies
609 8.1 Rationale

610 In-use stability of an IVD is the period of time over which components retain
611 adequate performance, after transport to the users, once they are opened,
612 reconstituted and/or diluted and exposed to the environmental conditions in
613 which they will be used.
614 If a range of conditions for use is stated in the IFU (e.g. use at 15–40°C) evidence
615 should be provided to prove the stability over that range with all the specimen
616 types (e.g. serum, whole blood, oral fluid) claimed. It is considered best practice
617 that the manufacturer extends the stability range by 5°C at the lower and upper
618 end of the proposed acceptable range on the labelling for all components to
619 ensure that that the claimed stability ranges is acceptable.
620 8.2 Conditions of use

621 Determination of the in-use stability of an IVD and/or its components should
622 reflect routine conditions of use of the IVD. Freeze-thaw stability should be
623 considered to address reagents which are exposed to multiple freeze-thaw during
624 use.
625 WHO recommendation: In-use stability studies must take into account
626 environmental conditions and usage conditions encountered by WHO
627 users and Member States, such as exposure to extreme temperatures,
628 humidity, dust, light and micro-organisms.
629 8.3 Multiple in-use stability claims

630 Depending on the way in which the IVD is used it may be necessary to have
631 several in-use stability claims. In situations where multiple stability claims are
632 made, a manufacturer must provide evidence from testing that investigates
633 routine use supporting each of the claims.
634 Examples:
635 1) A reagent may have a stated period of stability once it has been placed on-board
636 an instrument and another period of stability once it is in active use (i.e. during
637 actual use/testing).
638 2) Multiple use reagents (e.g. buffers) may repeatedly be exposed to high
639 temperatures during the day while in use and exposed to lower temperatures
640 when not in use and stored in the refrigerator. The actual use of the multiple use
641 reagent – squeezing of bottles, exposure of the lid and tip to working surfaces,
642 hands, exposure to dust and light – also affect stability. Stability studies should
643 take into account all of these factors.

644 9 Production lots used in stability studies

645 Comments in this section apply equally to all types of studies described in this
646 guidance.
647 9.1 Considering variability

648 As noted in 11.3_Consider_variability, planning for stability studies must take into
649 consideration all possible sources of variation within and between manufactured
650 lots. For most IVDs it is likely that differences between batches of the biological
651 reagents will cause the most variance. Factors to consider include apparently
652 minor, technically-uncontrollable differences in culture and purification for
653 recombinant antigens and antibodies; synthesis and purification for primers,
654 probes and peptides; undocumented production changes of an outsourced buffer
655 component and the lot of nitrocellulose membrane used in lateral-flow IVDs.
656 At a minimum, lots chosen for stability studies should be different in the critical
657 constituents, e.g. different purification and/or culture batches for all recombinant
658 antigens and monoclonal antibodies. If pilot or small scale lots are chosen, special
659 attention must be paid to the potential for variability (see also 11.3). However,
660 the sources of variation will depend on the particular process, product and
661 component, and should be identified during product development risk analyses.
662 Use of different batches of critical components ensures that the stability evidence
663 obtained is more likely to be representative of long-term manufacture. Any
664 variability found can be taken into consideration when assessing the outcome of
665 the studies against the design input requirements and when making claims. This
666 minimizes user problems and hence complaints.
667 9.2 Testing the final configuration

668 Shelf-life, in-use and transport stability must be determined for the finalized,
669 approved product in terms of:
670 • manufacturing specifications;
671 • release-to-market QA criteria;
672 • packaging and labelling; and
673 • validated manufacturing scale on qualified manufacturing equipment.
674 Testing methods should be as included in the IFU of the commercialized IVD.
675 WHO prequalification requirements: It is important that it can be
676 established that the stability studies were conducted on the IVD as
677 submitted to WHO for prequalification. Even changes perceived as small
678 (e.g. change in production scale, bulk container materials, supplier of a
679 critical biological, change in vial stopper) can have unexpected effects on
680 stability and other performance characteristics. After such changes, a
681 stability plan and study is needed again. Manufacturers should have
682 change control procedures in place compliant with ISO 13485:2003 [12].
683 Stability studies undertaken in the R&D phase of the product lifecycle
684 are important to understand how to design the product so it will meet
685 the final stability requirements in the input documentation. However,
686 these studies are not sufficient for submission to WHO since they might
687 not reflect the final design and manufacture of the IVD.

688 9.2.1 Exceptions

689 If any of the above criteria are not met (for example if “pilot lots” or small scale
690 lots are used, or if the IFU is not finalized), strong evidence must be provided that
691 the evaluated materials will perform exactly the same as the final commercial
692 product.
693 WHO prequalification requirements: In some exceptional circumstances,
694 where it is not possible to sample from actual production lots, samples
695 from pre-production or development lots might be used. If this is the case,
696 manufacturers should justify why production lots were not used, and
697 they should provide robust evidence that the lots chosen are expected to
698 behave identically to the production lots. Data concerning lot-to-lot
699 variability must still be submitted. Although WHO will consider the
700 available evidence on its merits, this preliminary information must be
701 followed by stability claims conducted on production lots. A post-
702 prequalification commitment may be required to amend this situation
703 when the manufacturer is able to produce fully qualified production lots.
704 9.3 Number of lots required for testing

705 Existing guidance [1,2] recommends that three product lots at a minimum must
706 be used to verify shelf-life; in-use claims require testing on a minimum of one lot.
707 The actual minimum number of lots to be used should be determined by a
708 stringent risk assessment based on evidence of variability obtained during R&D,
709 (see section 9.1). However, the minimum will never be less than three lots for
710 shelf life verification.
711 Note from WHO prequalification experience: It is not acceptable to
712 sample IVDs from a single production lot but label them so that they
713 appear to have been taken from three separately manufactured
714 production lots. This is true for all performance evaluation and
715 regulatory submission purposes. This aspect will be investigated during
716 an onsite inspection by WHO. Non-compliance with this requirement
717 may result in a critical non-conformity grading.
718 9.4 Components of lots required for testing

719 Existing guidance [1,2] requires that stability work is performed using materials in
720 their final packaging, with intended labelling. If there is more than one variant of
721 the IVD (e.g. pack size differences, CE marked and non-CE marked) any potential
722 effects on performance, including stability, must be assessed.
723 In particular, if different reagent-container sizes are used in packs intended for
724 different numbers of use, stability evidence should be obtained on all variants,
725 even if the contents of the containers are identical.
726 Once component shelf-lives are assigned use relatively fresh components and
727 components which have progressed into their assigned shelf-life in the different
728 production lots used in the establishment of the product shelf-life.

729 10 Stability plan

730 Stability studies should be well designed, scientifically sound, well implemented,
731 well recorded and able to deliver meaningful conclusions about IVD performance.
732 This will minimize the time and resources taken by the manufacturer to generate
733 appropriate evidence and by the regulatory authority to assess it.
734 It is good practice to prepare, within the mechanisms of a quality management
735 system (QMS), a plan for the investigation of each aspect of IVD stability. A well-
736 developed study plan, with clearly defined objectives, responsibilities and
737 pass/fail criteria should be developed, reviewed and internally approved in
738 advance of testing. The plan should be associated with the design input
739 requirements.
740 It is essential that the study plan takes into account the intended use of the
741 product to ensure that these elements are covered within the stability studies.
742 The results of the stability studies support the claims in the instructions for use.
743 Careful forward planning will make a significant contribution to ensuring that
744 sufficient resources are made available, effective experiments are performed and
745 both experimental results and associated documentation are recorded in an
746 appropriate manner.
747 10.1 Responsibilities

748 The study plan should outline responsibilities and applicable training for said
749 responsibilities of all staff involved in the study. The R&D department is usually
750 responsible for set up of the study and testing of newly developed IVDs,
751 monitoring, and any equipment validation if required, and for the documentation
752 of the testing plan and sample selection.
753 The R&D department should nominate a responsible person for investigating
754 failures. The QA department should nominate a responsible person for conducting
755 risk assessments if the IVD fails to meet the requirements of the design inputs.
756 Performance evaluation is not to characterize an IVD but to show that it meets (or
757 exceeds) predetermined qualities.
758 10.2 Preparing the testing plan

759 A complete, detailed description should be prepared that fully documents
760 everything to be done and the expected outcomes. Authorization of the plan
761 should be obtained internally in advance of starting work. The plan should include
762 the following details.
763 • Qualification and training of technical staff performing the work

764 • Biohazard issues identified with reagents
765 • The instrumentation, including storage facilities or rooms, validation,
766 calibration, monitoring, servicing
767 • The batch numbers of kits to be used with justification for any
768 manufacturing anomalies or excursions from documented procedures
769 • The expected life of the kit from the input documentation
770 • Any proposal, with justification to launch a kit with a life based on
771 accelerated data, or to launch with a shorter life than in the input

772 documentation while awaiting the conclusion of real-time testing

773 documented.
774 • The documented nature and extent of in-use testing
775 • The justification for the choice of lots and components taking into account
776 lot-to-lot variation and the critical characteristics
777 • The number of units (cassettes, bottles, tablets, etc.) of each component
778 to be collected and stored under each condition
779 • The nature of the stability testing panel to be used, justifying each panel
780 member’s inclusion and defining the volume and characterization of the
781 bulk specimen to be used and the aliquot size and number to be stored
782 for the testing (See section 10.5)
783 • The expected criteria for each stability testing panel member at the
784 beginning and end of the product’s proposed shelf-life
785 • The statistical methods to be used for data analysis
786 • Graphs (paper or electronic) to visualize the performance of each stability
787 testing panel member over the course of testing
788 • Methods of approval and justification of any deviations from the plan
789 10.3 Product storage

790 A sufficient number of product components from the identified lots should be
791 reserved and stored separately to ensure that the study will be completed with
792 identified products. Sufficient volumes should be retained to allow for the
793 predetermined invalid rate.
794 10.4 Documentation

795 The plan should make reference to a study report which will be used to
796 summarize interim, and ultimately, final study findings and conclusions. The study
797 plan, the testing protocol and study report and all associated documentation
798 (worksheets, etc.) should be controlled within the manufacturer’s QMS. At the
799 end of the study, the manufacturer should be able to confirm that design input
800 requirements have been met.
801 Any changes in method must be recorded and undergo risk assessment. It should
802 refer to the development of a detailed and valid testing protocol which includes
803 all information and material relevant to testing.
804 10.5 Statistical methods

805 Statistical methods are used to support stability claims by providing estimates of
806 the probability of results being as stated. For example: prior to the stability
807 studies on an EIA it has been documented that if a stability testing panel member
808 has at least a particular optical density (OD) then that device will meet a particular
809 claim. Given the results of the stability study using that stability testing panel
810 member and showing the variability within and between lots of the IVD, the
811 probability of future similar production of the device meeting claims at the
812 assigned life can be estimated. The derivation of valid criteria and the probability
813 of maintenance of all claims can be estimated by appropriate statistical methods.
814 There is a wealth of information on the statistical methods used in R&D of IVD
815 from both ISO [17, 18, 19] and CLSI [2, 20, 21, 22]. Most of these methods apply

816 only to quantitative assays but information on statistic methods for qualitative
817 assays is available also in Reference [23].
818 A fundamental problem is that of how many replicates should be used at each
819 time point and from how many different production lots to produce acceptable
820 overall probability estimates of the likelihood of all future production of similar
821 devices and lots meeting claims (and hence user input requirements) at the end of
822 the assigned life. There are two aspects to this – what is “acceptable” and “how
823 many replicates?” “Acceptability” is a decision critical to quality and must be
824 decided in advance from the user requirements – for example 80% confidence
825 that 95% of all lots will meet the claims. This is in fact a tolerance interval as
826 described in ISO 16269-6:2014 [19]. “How many replicates” can then be derived
827 from the tolerance interval required but advice from a professional statistician is
828 strongly advised – after defining the quality critical requirement but before
829 beginning any experimental work.
830 The statistical methods to be used will be documented in the plans and protocols
831 of any stability study and consideration given to treatment of unexpected and
832 atypical results. In general all results must be used unless there is a documented
833 physical reason (e.g. known operator error, too little volume, incorrect timing, use
834 of an unqualified instrument such as lacking maintenance or calibration) why a
835 result can be ignored – but even then that result must be recorded and included
836 in the report of the stability work.
837 10.6 Stability testing protocol

838 As part of an approved study plan for the determination of IVD stability, a detailed
839 testing protocol should be prepared (examples of stability protocols are provided
840 in Appendix 1:
841 Example stability protocols) including the following as a minimum, as appropriate.
842 • QMS identifiers (e.g. experiment name, document references, etc.) that
843 allow traceability to both the overarching study plan and to subsequently
844 generated records/documents such as result worksheets
845 • The name(s) of operator(s)
846 • The dates and times when the experiment was performed
847 • Signatures of the operator and supervisor(s)
848 • The objectives of the study (i.e. determination of shelf-life, determination
849 of in-use stability of a component, etc.)
850 • The name and lot number of the IVD and/or components being
851 investigated
852 • How the components will be sampled from the production department
853 • Stability testing panel members and their characterization to be used,
854 including valid test methods which reflect the IFU claims
855 • The experimental method that will be used for testing. This must follow
856 the finalized testing method from the IFU. It must describe clearly how
857 the experiment was performed in terms of:
858 − required storage and/or challenge conditions;
859 − the duration of storage/challenge;
860 − the schedule of testing intervals (see Reference [2] section 4.3);
861 − the stability testing panel; and
862 − the numbers of replicate tests performed for each stability testing
863 panel member.

864 • How and where results are to be recorded

865 • Acceptance criteria
866 • How aberrant, discordant or invalid results will be dealt with
867 • How storage/challenge conditions are to be applied
868 Example: For determination of stability during transportation it should be
869 made clear that each IVD will be subjected to a sequence of stated
870 temperatures.
871 • How actual storage/challenge conditions are recorded
872 Example: Recording of temperature not as “room temperature” but as an
873 actual numerical value obtained from calibrated instrumentation
874
875 Note: It can be unclear to a regulatory or WHO reviewer from a general

876 statement such as “… Sample buffer was stored at the required
877 temperature and tested each month…” whether (1) the bottles of sample
878 buffer were stored open at the required temperature for the entire testing
879 period, or (2) the bottles were stored capped and refrigerated, and only
880 reopened briefly at the required temperature at each schedule test point.
881
882 10.7 Reading and recording results
883 10.7.1 Avoiding reader bias

884 It is good practice to use approaches to make the reading more objective, such as
885 a scoring system. For IVDs where a subjective element forms part of the result, e.g.
886 reading the intensity of an RDT band within a specified time frame, the results
887 should always be reviewed by a first and second reader to avoid operator bias.
888 Both readers must be blinded to the expected results; the second reader must be
889 blinded to the first reader’s results. If a validated band intensity scoring tool is to
890 be included in the final RDT kit, this should be used to record results.
891 10.7.2 Recording actual individual results

892 The results of a test, not only the test interpretation, should be recorded. An
893 interpretation on its own has insufficient resolving power to allow degradation of
894 a signal over time to be observed.
895 Some IVDs, e.g. line-blots, may require particular band patterns to allow an
896 interpretation to be reached, and several different patterns may yield the same
897 final result. Recording only the final interpretation of a test specimen may cause
898 the failure of particular bands to go unnoticed while allowing the IVD to otherwise
899 “pass”. Photographic records of qualitative tests are recommended, as
900 appropriate.
901 This is particularly important when testing a panel of like specimens, e.g. “20 HIV
902 antibody positive specimens” for which the acceptance criterion is “all 20
903 specimens must be positive”. It is not sufficient to simply record “all 20 positive”
904 or “pass” without first recording the individual test result directly from the IVD for
905 each specimen in the panel.
906 Example 1: For most enzyme-linked immunoassays (EIAs) if the sample-
907 to-cut-off ratio is > 1 then the result is interpreted as “positive” or
908 “reactive”. In this case three pieces of information should be recorded: (1)

909 the numerical value of the assay sample-to-cut-off ratio, (2) the
910 numerical value of the signal for the specimen and (3) the final
911 interpretation.
912 Example 2: Some rapid diagnostic tests (RDTs) may stipulate that the
913 strength of test band is not correlated with the strength of antibody titre.
914 Nevertheless, the following should be recorded: (1) the intensity of
915 observed patterns according to a predetermined, validated intensity
916 scoring system with as fine a gradation as possible, and (2) the final
917 result interpretation.
918 Example 3: A qualitative NAT assay may report “positive” and “negative”
919 for a particular analyte, but the underlying decisional parameter is often
920 quantitative (e.g., a PCR signal-based cycle number). The quantitative
921 parameter should be recorded.
922
923 10.7.3 Retention of records

924 WHO encourages retention of photographic records, machine printouts,
925 electronic data or physical retention of membranes from opened cassettes, as
926 appropriate. Records should be retained for the period of time equivalent to the
927 commercial lifetime of the IVD but not less than two years. (Modified from
928 Reference [12] para 4.2.4)
929 10.8 Degradation vs deterioration

930 Testing at more than two time points can be important to avoid confusion
931 between imprecision and stability. For example, if the end testing shows 10%
932 decrease, one may not judge if the difference was due to imprecision or
933 degradation. If tested one or more times in between are used, fluctuation caused
934 by imprecision can be distinguished from drift due to instability. This can be
935 ameliorated by increasing the number of replicates and runs.
936 All studies should support precisely defined periods of in-use stability claims.
937 Example: An RDT test cassette – may be labelled “Use immediately on
938 opening”. In such cases it is still necessary to determine the interval (one
939 hour, one day, etc.) over which the IVD performance remains stable after
940 the component is opened.
941 10.9 Testing schedule

942 Testing intervals should be selected to detect any trending activity over the
943 testing period. Concurrent testing of separate types of components may be
944 approached with different intervals. For example, it may be appropriate to test an
945 IVD test cassette against a stability testing panel on a monthly or quarterly basis.
946 10.9.1 Acceptance criteria for results

947 The acceptance criteria to establish what is acceptable or not acceptable should
948 be defined according to the stability testing panel criteria for both qualitative and
949 quantitative test methods. Results from failed (invalid) test runs must not be used
950 in the determination of the stability claim. However the invalid results should also
951 be recorded.

952 11 Stability report
953 11.1 General

954 After testing has been completed, the findings should be summarized in a stability
955 study report. The report should clearly identify the IVD that was tested, the
956 objectives of the study, the conditions under which the IVD was tested and
957 conclusions that were drawn from findings. The report should be traceable to the
958 study plan, testing protocol and user needs. It should make clear references to
959 other supporting documentation (e.g. result worksheets).
960 11.2 Link to claims

961 The results and conclusions of stability studies presented in the study report must
962 support the claims of IVD stability reported in the IFU and elsewhere in the
963 dossier.
964 11.3 Consider variability

965 An overall stability claim (whether for shelf-life, in-use stability, or stability during
966 transportation) must be based on the expected stability when taking into account
967 inter-lot variability.
968 Example: The manufacturer should evaluate the variability between the
969 different lots studied (see 9.1) and assume that any differences in shelf-
970 life are inherent to the manufacturing process. The claimed life should be
971 calculated so that a known and stated proportion of all lots
972 (usually >95%) will meet the claimed shelf-life. Frequently more than
973 three lots are needed to obtain a realistic idea of the variability of the
974 results.
975 11.4 IVD stability versus component stability

976 A claim of stability for an IVD as a whole must not exceed any individual
977 component stability.
978 Example: For an IVD claimed to detect HIV-1 and HIV-2 antibodies – if
979 detection of HIV-1 antibodies is stable to 24 months but that of HIV-2 to
980 only 18 months, then the shelf-life must be based on the shorter time.

981 12 Changes to a Prequalified IVD
982 12.1 Dealing with change

983 Any critical or major modification to a prequalified IVD or to its process of
984 manufacturing will require provision of direct evidence of stability. An appropriate
985 risk analysis and an accelerated stability study comparing the original product and
986 the modified product for usability, performance and lot-to-lot variation may serve
987 to assess the impact of the changes to a product formulation or manufacture. It
988 would be necessary to validate the stability of the modified IVD in at least one lot
989 of the IVD (subject to risk analysis) in order to demonstrate equivalence between
990 the original and modified IVDs. More lots may be appropriate depending on the
991 product nature, variability of components and failure risk . (Reference [2] section
992 7.1.2). WHO expects results of accelerated testing to be confirmed by real-time
993 studies.
994 If there are different presentations, the stability of each one must be assured (see
995 also 9.4).
996 The following examples seek to illustrate the scope for considering the
997 performance evidence from one IVD as support for performance in another:
998 Examples:
999 1) For an HIV RDT which uses an identical cassette and physical components of a
1000 manufacturer’s existing, fully validated HCV RDT, the reagent formulations are
1001 different (antigen/antibodies, buffers, conjugates, etc.). Evidence of stability of
1002 the HCV RDT would not suffice for the HIV RDT.
1003 Even if the manufacturer claims that both IVDs have been sold in a number of
1004 countries for several years and no adverse feedback has been reported, this
1005 would not constitute evidence in support of the stability of either IVD.
1006 2) For an HIV RDT that has been fully validated for detection of HIV-1 antibodies; a
1007 new product is developed which includes detection of HIV-2 antibodies. The
1008 stability of any sample buffers that are identical between the two IVDs would
1009 probably not need to be validated. However, other components (conjugates,
1010 antigens, antibodies) that are different between the two IVDs would need to be
1011 tested; it would not be sufficient to assume that HIV-1 reagents will have the
1012 same stability in the new IVD. A modification of this nature is likely to require
1013 substantial validation of stability.
1014 3) An HIV RDT IVD previously intended for testing serum/plasma has added to it a
1015 claim for detection of HIV-1 in whole blood. The only substantive design change
1016 associated with the new claim is the addition of a small pad of some suitable
1017 material near the sample port which acts as filter for whole blood specimens.
1018 Depending on the nature of the material it may be reasonable to argue that the
1019 material would not be expected to age; that it is not, in any practical sense,
1020 chemically labile. Consequently, shelf-life and in-use stability may not necessarily
1021 need to be retested in full. However, stability during transportation may need to
1022 be determined to provide confidence that the modification is able to withstand
1023 likely shipping conditions (e.g. that the extra square of filter paper doesn’t
1024 dislodge when packages are jostled and bumped in transit).
1025

1026 4) Based on an HIV RDT that has been fully validated for detection of HIV-1
1027 antibodies, a new IVD is developed which includes detection of antibodies to
1028 Treponema pallidum (TP). Detection of TP specific antibodies occurs on a
1029 completely separate membrane (and associated architecture) to that of HIV
1030 antibody detection. Additional handling steps may have an impact on the
1031 stability of the HIV-1 antibodies and it may be required to retest. It may be
1032 necessary to review evidence of stability during transportation to ensure that
1033 new components are not affected by transport (for example a new packaging
1034 concept is used).
1035 If a new machine is used for striping of the HIV-1/TP IVD,
1036 validation of the new machine (installation qualification, operational
1037 qualification and performance qualification) would be required to show that the
1038 stability studies are still valid.
1039 If the IVD is designed in a way that HIV and TP detection occurs
1040 either on the same membrane and/or using most of the same architecture (and
1041 assuming that sample buffers are identical between IVDs) it is likely that this new
1042 IVD would need to be fully validated.
1043
1044 It should be noted that these observations pertain specifically to IVD stability.
1045 Other aspects of IVD performance should still be validated as appropriate.
1046

1047 13 Authors and acknowledgements

1048 The document TGS-2 Establishing stability of an in vitro diagnostic for WHO
1049 prequalification was prepared by Julian Duncan (consultant, London, UK), Sue
1050 Best, Susie Jane Braniff, Mark Lanigan (National Serology Reference Laboratory,
1051 Australia), Deirdre Healy (WHO/HIS/EMP) and Robyn Meurant (WHO/HIS/EMP).
1052 WHO wishes to thank Sally Hojvat (consultant, USA), Luc Kestens (ITM), Debbie
1053 Lepine (Health Canada), Luann Ochs, CLSI and members of the CLSI Consensus
1054 Committe, the ISO TC212 WG3 committee and Monika Zweygarth (editor) for
1055 their contributions. This document was developed as part of the Bill and Melinda
1056 Gates Foundation Umbrella grant. This document was produced under the
1057 coordination and supervision of Robyn Meurant and Irena Prat (WHO/HIS/EMP),
1058 Geneva, Switzerland.
1059 The draft guidance has been posted on the WHO website for public consultation
1060 from the 14 December 2015 to 31 January 2016.

1061 14 References
1062
1 ISO 23640:2011. In vitro diagnostic medical IVDs - Evaluation of stability of in vitro diagnostic reagents.
Geneva, Switzerland: International Organization for Standardization; 2011.
2 CLSI. Evaluation of Stability of In Vitro Diagnostic Reagents; Approved Guideline. CLSI document EP25-A.
Wayne, PA: Clinical and Laboratory Standards Institute; 2009.
3 ICH Harmonised Tripartite Guideline Specifications: Test Procedures and Acceptance Criteria for
Biotechnological/Biological Products Q6B. International Conference on Harmonization of Technical
Requirements for Registration of Pharmaceuticals for Human Use. Current Step 4 version. 10 March
1999.
4 ISO 17511:2003. In vitro diagnostic medical IVDs – Measurement of quantities in biological samples –
Metrological traceability of values assigned to calibrators and control materials. Geneva, Switzerland:
International Organization for Standardization; 2003.
5 ISO 18113-1:2009. In vitro diagnostic medical IVDs – Information supplied by the manufacturer (labelling) –
Part 1: Terms, definitions and general requirements. Geneva, Switzerland: International Organization
for Standardization; 2009.
6 United States CFR - Code of Federal Regulations Title 21. Sec. 820.3 Definitions.
7 ISO/IEC Guide 99:2007. International vocabulary of metrology -- Basic and general concepts and associated
terms (VIM). Geneva, Switzerland: International Organization for Standardization; 1993.
8 ISO 9000:2005. Quality management systems – Fundamentals and vocabulary. Geneva, Switzerland:
International Organization for Standardization; 2005.
9 GHTF/SC/N4:2012 (Edition 2). Glossary and Definitions of Terms Used in GHTF Documents. Global
Harmonization Task Force (GHTF) Steering Committee; 2012.
10 ISO 14971:2007. Medical IVDs – Application of risk management to medical IVDs. International Organization
for Standardization; Geneva, Switzerland: 2007.
11 American Society for Testing and Materials (ASTM). ASTM D4169-14. Standard Practice for Performance
Testing of Shipping Containers and Systems. ASTM International, West Conshohocken, PA; 2014.
12 ISO 13485:2003. Medical IVDs – Quality management systems – Requirements for regulatory purposes.
Geneva, Switzerland: International Organization for Standardization; 2003.

13 ISO 15198:2004. Clinical laboratory medicine – In vitro diagnostic medical IVDs – Validation of user quality
control procedures by the manufacturer. Geneva, Switzerland: International Organization for
Standardization; 2004.
14 United States Pharmacopeia and National Formulary (USP 31-NF 26). Rockville, MD, United States:
Pharmacopeia Convention; 2008.
15 Pharmacopoeia of the People’s Republic of China. English edition. Beijing, China: The State Pharmacopoeia
Commission of the People's Republic of China; 2000.
16 ICH Harmonised Tripartite Guideline. Specifications: Test procedures and acceptance criteria for new drug
substances and new drug products: chemical substances. Q6A. International Conference on
Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use; 1999.
17 ISO 5725-1,2,3,4,6:1994, ISO 5725-5:1998 Accuracy (trueness and precision) of measurement methods and
results- Parts 1-6. Geneva, Switzerland: International Organization for Standardization; 1994 and 1998
18 ISO 3534-1,2:2006, ISO 3534-3:2013. Statistics -- Vocabulary and symbols – Part 1-3. Geneva, Switzerland:
International Organization for Standardization; 2006 and 2013
19 ISO 16269-4:2010, ISO 16269-6:2014, ISO 16269-7:2001, ISO 16269-8:2004. Statistical interpretation of data.
Geneva, Switzerland: International Organization for Standardization; 2001, 2004, 2010, 2014.
20 CLSI. Evaluation of the Linearity of Quantitative Measurement Procedures: A Statistical Approach; Approved
Guideline. CLSI document EP06-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2003.
21 CLSI. Interference Testing in Clinical Chemistry; Approved Guideline - Second Edition. CLSI document EP07-
A2. Wayne, PA: Clinical and Laboratory Standards Institute; 2005.
22 CLSI. Evaluation of Detection Capability for Clinical Laboratory Measurement Procedures; Approved
Guideline - Second Edition. CLSI document EP17-A2. Wayne, PA: Clinical and Laboratory Standards
Institute; 2012.
23 Valcárcel, M., Cárdenas, S., Barceló, D. et al. Metrology of qualitative chemical analysis, KI-NA-20-605-EN-C,
ISBN 92-894-5194-7; 2002 Available free of charge from: http://bookshop.europa.eu/en/metrology-of-
qualitative-chemical-analysis-pbKINA20605/
24 CLSI. Evaluation of Precision of Quantitative Measurement Procedures; Approved Guideline—Third Edition.

CLSI document EP05-A3. Wayne, PA: Clinical and Laboratory Standards Institute; 2014.

1063 Appendix 1:
1064 Example stability protocols
1065
1066 This appendix contains examples for a wholly fictitious IVD, illustrating the kinds
1067 of experimental design to determine the following:
1068 1. Stability of whole kit during transport
1069 2. Stability of whole kits during shelf-life, and
1070 3. In-use stability of whole kits including reagents
1071 The information provided in these examples should not be taken as a checklist of
1072 sufficient conditions, but should be used as a guide on possible approaches to
1073 generate evidence of a standard sufficient to satisfy the requirements of the WHO
1074 Prequalification Programme. Additional examples can be found in the WHO
1075 sample CD4 dossier available on the WHO prequalification website.
1076 It is recommended that transportation stress studies are undertaken prior to the
1077 shelf-life studies.
1078 Description of fictitious IVD

1079 The fictitious IVD used for the purpose of the examples is a RDT for the detection
1080 of antibodies to HIV-1, HIV-2 and Treponema pallidum in serum, plasma and
1081 whole blood. It is recommended that the kit is stored at 8–40°C, but components
1082 of the kit must be used at 15–30°C. The product is supplied as a kit with each test
1083 cassette sealed in a foil pouch (with desiccant). The pouch must be brought to 15–
1084 30°C. Once opened, it is recommended that the cassette is used immediately.
1085 The IVD includes a bottle of specimen buffer/diluent for use with all three
1086 specimen types. The specimen buffer is expected to have similar stability as the
1087 test cassette in its unopened form. The stability of the opened bottle of specimen
1088 buffer is determined below (see Example 3: In-use stability protocol).
1089 The manufacturer of this product proposes to determine the stability of its product
1090 and has written a stability plan. As part of this plan a preliminary determination of
1091 accelerated stability has been conducted at several extremes of temperature and
1092 suggests that the IVD would be stable to an equivalent of 12 months following
1093 manufacture. The plan now calls for the development of real-time stability
1094 protocols that will form the basis of subsequent testing of the IVD.
1095 Preliminary work has shown that the variability between lots is minimal so that
1096 three independent lots (no critical constituents in common) will suffice to enable a
1097 reasonable estimation of shelf-life taking lot variation into account.

1098 Example 1: Evaluation of stability during transportation

1099 Objective
1100 To determine the stability during transportation of the HIV RDT in real-time using
1101 simulated shipping conditions and to generate stressed components to be used in
1102 real-time shelf-life studies as proposed in Stability Study Plan XZY00001.
1103 Preparation
1104 Acquire sufficient numbers of kits from three independent production lots using a
1105 predetermined sampling protocol (e.g. random, first X kits in first box, every 100th
1106 kit, etc.). Allow at least 10% for unexpected requirements and re-testing
1107 Note 1: To provide security against unforeseen events, duplicate tests should be
1108 performed as a minimum. Testing in triplicate as a minimum provides a level of
1109 statistical confidence in the observed test result.
1110 Testing will be conducted at 0, 3, 6, 9, 12 and 13 months.
1111 Note 2: Testing beyond 13 months will allow an understanding of when, in real-
1112 time, the IVD is likely to ‘fail’ and may allow an extension of the proposed shelf-life.
1113 Note 3: For determination of shelf-life a fresh bottle of specimen buffer must be
1114 opened at each testing point – although there may be circumstances in which
1115 multiple sampling could be taken from the same bottle after it has been opened.
1116 Acquire sufficient volume of each stability testing panel member for the duration
1117 of the testing schedule.
1118 The protocol for these studies specifies the number of devices to be picked, the
1119 statistical sampling plan to be used and the required stability testing panel
1120 members and their volumes.
1121 In Worksheet XYZ00001 record the following:
1122 • The lot numbers from which kits were sampled
1123 • The number of kits sampled from each lot
1124 • Details (including manufacturing/lot information) for each of the kit
1125 components that will be tested as part of this protocol:
1126 Test cassette:..
1127 Bottle of Sample Buffer:…
1128 The product kits chosen to be tested are in their final packaging including all
1129 labelling.
1130 The IVDs are stored so that the reagents are in contact with all elements of the
1131 packaging (e.g. the bottles in the product kits are stored horizontal lying flat on
1132 their sides).
1133 Kits will be divided into two groups. One group will be stored at 42±2°C, the other
1134 at 4±2°C. Kits from each group will then be subjected to the following conditions.
1135 Testing schedule: for transport simulation
1136 Condition 1, Temperature and humidity sequence: all kits will be taken through a
1137 temperature and humidity sequence consisting of:
1138 Ambient humidity (X% RH)
1139 Put at IFU storage temperature for 24±4 hours followed by
1140 30 ± 5°C for 24±4 hours followed by

1141 45 ± 5°C for 24±4 hours, followed by

1142 8 ± 5°C for 24±4 hours , followed by
1143 IFU storage temperature for 24±4 hours
1144 Followed by
1145 Desert humidity (30% RH)
1147 30 ± 5°C for 24±4 hours), followed by
1151 Followed by
1152 Tropical humidity (85% RH)
1158 Followed by
1159 Ambient humidity (X% RH)
1165 Note 1: It is important to make clear that the above complete sequence of
1166 temperatures will be used as opposed to separate kits held at individual
1167 temperatures. The actual temperatures, durations and the nature of the sequence,
1168 will depend on the IVD and the kinds of conditions expected to be encountered
1169 during shipping
1170 Note 2: Freezing temperatures are not considered in this example but would
1171 need to be if the kits could be exposed to freezing temperatures during transport.
1172 Note 3: If transport by air is anticipated the effect of reduced pressure must be
1173 included in this protocol (ASTM D4169 section 16) and should be for at least 10%
1174 longer than the longest anticipated flight at a pressure expected in aircraft holds.
1175 Note 4: The protocol will call for testing at least five individual devices after each
1176 stress condition with the stability panel members giving the most informative
1177 results. This will verify that the devices are sufficiently stable to progress to the
1178 next condition but that should already be certain from preliminary experiments
1179 and R&D work.
1180 Condition 2, Shaking. Each kit will be placed on a shaking table at X rpm for
1181 X hours/days at 42 ± 5°C as defined by ASTM D4169 section 12 (Reference [11]).
1182 After the simulated shipping challenge each kit will be returned to its
1183 corresponding storage temperature (42 ± 5°C or 8 ± 5°C).

1184 Testing will be conducted at 0, 3, 6, 9, 12 and 13 months. At each scheduled time

1185 point the allotted number of IVDs will be brought to room temperature and used
1186 to test each member of the panel in triplicate.
1187 Note 1: the test at 0 months will provide evidence that the device is stable under
1188 extreme (but quite likely) conditions of shipping, the testing at later time points
1189 will give evidence to support the claimed shelf-life after transport, that beyond the
1190 claimed shelf-life will provide evidence that the device is stable and not close to a
1191 failure point.
1192 Documentation for transport stress conditions
1193 In Worksheet XYZ00001 record:
1194 • The lot numbers of the IVD used to conduct the test
1195 • The Operator(s) name(s)
1196 • The dates of testing
1197 • Identifying details for each member of the stability testing panel being tested
1198 • The temperature that kits are stored at
1199 • The values of temperature and humidity for each of the challenge conditions
1200 • Instrument settings for the shaking apparatus and duration of operation
1201 • The ambient temperature and humidity during testing
1202 • Each test result as an interpretation according to the IFU
1203 • Each test result as a band intensity. Band intensity should be scored using the
1204 calibrated scale described in ProtocolZXY0001 (e.g. 0, faint/trace, +1, +2,
1205 +3 … +10) (even though the IFU does not give scores to results)
1206 • Any aberrations or deviations from the protocol, the reason for the deviation
1207 and any remedial action undertaken. Results from invalid assays must be
1208 recorded but not included in calculations of shelf-life. Apparently aberrant
1209 results, unless the underlying cause can be positively identified as not related
1210 to a problem with the device, must be included in the calculations of life.
1211 Stability testing panel

1212 See the suggestions in Appendix 2: Specimens for the stability testing panel.
1213 Acceptance criteria
1214 Each stability testing panel member should exhibit a band intensity at each time
1215 which matches its expected result. The expected result must be validated so that
1216 if the device would fail to meet claims (e.g. fail to detect critical specimens, have
1217 unacceptable performance at medical decision concentrations, have unacceptable
1218 specificity) the stability testing panel member would also fail to meet its specified
1219 result
1220 The stability after transportation of the IVD will be taken as the time point before
1221 the last time point to have met the acceptance criteria, e.g. if the IVD is stable to
1222 13 months, the stability during transportation will be deemed to be 12 months.
1223 The stability after transportation should be identical to the claimed shelf-life of
1224 the IVD, i.e. the extremes of possible conditions to which the IVD is likely to
1225 subjected during transport must not affect the shelf-life of the IVD.
1226 Calculation of results
1227 It is not the intent of this guide to provide detailed statistical instruction: that
1228 must be obtained from a professional statistician with an understanding of the
1229 requirements expressed herein. Professional statistical guidance is especially

1230 recommended when calculating confidence limits for discrete data such as
1231 readings from a graduated scale
1232 The following applies separately at each time point.
1233 The variance of the results for all replicates within and between all the lots must
1234 be calculated for each stability testing panel member. From the overall variance
1235 between lots the confidence with which future lots of the device will detect the
1236 panel member at that time point after manufacture and transport can be
1237 calculated. If the confidence of the panel member meeting its specification is less
1238 than some pre-defined value (normally 95%) then it must be deemed to have
1239 failed at that time point and the life of the device restricted accordingly.
1240 If regression analysis is used to define the time point at which a panel member
1241 would not meet its criterion then lot-to-lot variation must be included when
1242 setting the confidence limits around the regression line. However, real time data
1243 must extend beyond the claimed shelf-life so the intercept of the regression
1244 confidence limit and the expected value must be at a time longer than the claim.
1245 It is usually more appropriate to calculate as in the previous paragraph particularly
1246 if the regression cannot be proven to be linear.
1247 The stability of a device is not governed by the least stable lot that happens to
1248 have been tested – shelf-life must be supported by statistical evidence that all lots
1249 manufactured in that way will achieve the claimed life. This, of course, is true of
1250 all performance claims.
1251 Example 2: Shelf-life protocol

1252 Objective
1253 To determine the shelf-life of the HIV/TP RDT in real-time when stored at 8–40°C
1254 as proposed in Stability Study Plan XZY00001.
1255 Acquire sufficient numbers of kits from three separate production lots using a
1256 predetermined sampling protocol (e.g. random, first X kits in first box, every 100th
1257 kit, etc.).
1259 of the testing schedule. Establish a method for randomising the panel members
1260 for IVD testing. Stability data of the panel members should already be established
1261 and recorded.
1262 Documentation
1264 • The lot numbers kits were sampled from
1268 Test cassette: …
1269 Bottle of Specimen Buffer: …
1270 Note: The stability of an opened bottle of specimen buffer must be determined
1271 (see Example 3: In-use stability). For determination of shelf-life a bottle of
1272 specimen buffer should be unopened (i.e. fresh) at each testing point – there may

1273 be circumstances in which multiple sampling could be taken from the same bottle
1274 after it has been opened.
1275 Preparation
1276 The product kits chosen to be tested are in their final packaging including labelling.
1279 their sides).
1280 Kits will be divided into two groups. One group will be stored at 42 ± 5°C, the
1281 other at 8 ± 5°C. Kits from each group will then be subjected to the following
1282 conditions.
1283 Testing schedule
1284 At each scheduled time point the allotted number of IVDs will be brought to room
1285 temperature (20 ± 2°C) and used to test each member of the stability testing
1286 panel (see below) in triplicate.
1287 Note: To provide surety against unforeseen events, duplicate tests should be
1288 performed as a minimum. Testing in triplicate as a minimum provides a level of
1289 statistical confidence in the observed test result.
1290 Testing will be conducted at 0, 3, 6, 9, 12 and 13 months.
1291 Note: Testing beyond 13 months would allow an understanding of when, in real-
1292 time, the IVD is likely to ‘fail’ and may allow an extension of the proposed shelf-life.
1293 It may be useful to know the “fail” point of an assay as it is the best measure of
1294 stability. However if it is obvious that the kit performance is decreasing over time,
1295 it can also be estimated visually and statistically when it will fail.
1296 Documentation
1298 • The lot number(s) of the IVD(s) used to conduct the test
1303 calibrated scale described in Protocol ZXY0001 (e.g. 0, faint/trace, +1, +2, +3 …
1304 +10)
1307 and any remedial action undertaken
1308 • The temperature that kits are stored at
1309 • The ambient/room temperature during testing
1310 Stability Testing Panel

1312 Acceptance Criteria
1314 point which matches its expected result.
1315 The shelf-life of the IVD will be taken as the time point before the last time point
1316 to have met the acceptance criteria.

1317 Example: If the IVD is stable to 13 months, the shelf-life will be deemed
1318 to be 12 months.
1319 Example 3: In-use stability protocol

1320 Objective
1321 To determine the stability of opened bottles of HIV RDT/TP Sample Buffer in real-
1322 time when stored at 15–30°C as proposed in Stability Study Plan XZY00001.
1323 [In this example the manufacturer recommends that the test cassette be used
1324 immediately upon opening; this claim should also be validated in a separate
1325 experiment, such that it can be established that the IVD will still perform
1326 satisfactorily after the test cassette has been removed from its pouch and open at
1327 room temperature for 1, 2, 6, 24 hours, etc., as appropriate.]
1328 Acquire sufficient numbers of kits from one production lot using a predetermined
1329 sampling protocol (e.g. random, first X kits in first box, every 100th kit, etc.).
1331 of the testing schedule. Establish a method for randomising the stability testing
1332 panel for IVD testing.
1334 • The lot numbers kits were sampled from
1338 Test cassette: …
1339 Bottle of Sample Buffer: …
1340 Preparation
1341 Two sets of sample buffer are to be tested. One set of the component must be
1342 freshly made, the other towards the end of the assigned shelf-life of the device.
1343 The component is to be tested are in its final packaging including labelling.
1346 their sides).
1347 Half of each set will be stored at 30± 5°C, the other half at 15± 5°C. At the start of
1348 testing each bottle will be brought to room temperature (20 ± 2°C), opened, used
1349 for testing and then recapped and returned to the stated storage temperature.
1350 Note 1: It is important that the components under test are opened and used
1351 under circumstances likely to occur in users’ laboratories (i.e. not in rooms with
1352 HEPA filtered air) mimicking as far as possible genuine use.
1353 Testing schedule
1354 At each subsequent scheduled time point the allotted number of bottles will be
1355 brought to room temperature and used to test each panel member in triplicate.
1356 Testing will be conducted at 0, 1, 2, 3, 4 weeks up to the end of the claimed in-use
1357 life

1358 Documentation
1360 • The lot number of the IVD used to conduct the test
1365 calibrated scale described in ProtocolZXY0001 (e.g. 0, faint/trace, +1, +2, +3 …
1366 +10)
1369 and any remedial action undertaken
1370 • The temperature at which kits are stored
1371 • The ambient temperature during testing
1372 Stability Testing Panel

1374
1375 Acceptance Criteria

1377 which matches its expected result.
1378 The in-use stability of the sample buffer will be taken as the time point before the
1379 last time point to have met the acceptance criteria.
1380 Example: If the IVD is observed to be stable to 5 weeks, the in-use
1381 stability will be deemed to be 4 weeks.

1382 Appendix 2: Specimens for the stability testing panel
1383 Examples in this section

1384 Not all of the specimens in the examples that follow will be necessary for all IVDs,
1385 nor is the list exhaustive. Panels must be composed according to strict risk
1386 management principles, and all decisions must be documented and traceable.
1387 The minimum specimens to be included in a testing panel for the different
1388 products are outlined below.
1389 1 Nucleic acid test (NAT)

1390 If a proprietary nucleic acid preparation /extraction system is provided, the recovery must be shown to
1391 meet claims for each genotype from each of the specimen types claimed (e.g. dried blood spots, whole
1392 blood, plasma). Successful removal of inhibitory substances, if intended, must be demonstrated for
1393 appropriate specimen types. Unless potentially variable biological reagents are involved this system
1394 would be expected to be verified in manufacture and not necessarily tested at release.
Specimens Remarks
Specimens to demonstrate Traceability is probably required to one of the WHO
maintenance of sensitivity and/or international standards (e.g. 3rd HIV-1 International Standard
limit of detection, and/or NIBSC code: 10/152; 4th International Standard for hepatitis C
accuracy, and precision virus for Nucleic Acid Amplification Techniques NIBSC code:
06/102; 3rd International Standard for HBsAg NIBSC code:
12/226).
More than one genotype may be required to validate these
claims: see 1st WHO International Reference Panel for HBV
Genotypes for NAT-Based Assays, PEI code 5086/08.
This may be required on each of the claimed specimen types.
Specimens to demonstrate Sufficient negative specimens should be included to ensure that
specificity and validity of runs the claims will be met at end of shelf life.
Specimens (or reagents) to If more than one part of the genome is to be detected, both
demonstrate stability of each of systems must be shown to be stable.
the critical components of the IVD If both DNA and RNA are measured the complete system must
be shown to be stable.

1395 2 Stability testing panel for CD4 measuring IVDs

1396 Rationale
1397 CD4 measurements are quantitative, and accuracy at the clinical decision points is important. The
1398 design input should have information on the accuracy and other parameters required, and the panel
1399 must be designed to provide evidence that these parameters are maintained over the assigned-life of
1400 the reagent and measuring IVD.
1401 Parameters
1402 The stability testing panel used in stability work must be able to demonstrate the following.
1403 • Stability of all the antibodies used in the IVD (frequently anti-CD4 and anti-CD3 antibodies; any
1404 other critical components must be covered).
1405 • Accuracy and trueness of measurement maintained at the critical level (at least five specimens
1406 required)
1407 • Claimed linearity over the required range of CD4 count (at least five specimens required)
1408 • Measure drift
1409 Specimens
1410 Artificial specimens, such as stabilized blood specimens, can be used if a risk assessment based on R&D
1411 work indicates that they are effective. Fresh specimens are usually required. Measurements should be
1412 compared to an approved reference system.
1413 Examples of approaches

1414 Aged or in-use lots may be compared with a reference, e.g. a new lot. Precision studies can be
1415 performed as described in Reference [24].
1416 More information is found in the “Sample Product Dossier for WHO Prequalification Simu POC CD4
1417 System”, which is available on the WHO Prequalification Team’s website
1418 http://www.who.int/diagnostics_laboratory/evaluations/140314_simu_poc_cd4_dossier_web.pdf?ua=1.
1419 3 Specimens to monitor tests for HIV antibodies

Specimens Remarks
IgM first seroconversion Possible approaches to obtain samples :
specimens and IgG first • Study the early data from commercial seroconversion panels
seroconversion specimens where the seroconversion was frequently monitored by IgM
and IgG blots
• Study the responses to second and third generation assays or
protein A and protein L assays (this approach is less useful).
All other parts of the HIV
proteome included, e.g. reverse
transcriptase (RT)
Late stage specimens – usually a This might serve to monitor any kit run control.
high dilution set near the sample- HIV type serology is not particularly genotype dependent. It is
to-cut-off ratio usually not necessary to include controls for genotype detection
unless risk or experiment shows that it is for a particular IVD.
HIV-2, diluted to near the sample- Seroconversion specimens are very rare
to-cut-off ratio
HIV-1 (0), if claimed
Difficult specimens to monitor 100 negatives at release subject to risk analysis and statistical
specificity and invalid rates analysis of the allowable (relative to the claimed) false reactive
rate and invalidity rate

1420 4 Specimens to monitor tests for antibodies for HIV-1/2 and

1421 Treponema pallidum (TP)
Specimens Remarks
Specimens to detect HIV See 3 Specimens to monitor tests for HIV antibodies
Specimens to detect all the critical Note: Each of these epitopes play a role in detecting syphilis in
epitopes in the IVD, for example different stages of the infection. It is necessary to have a stability
TpN47, TpN17 and TpN15 testing panel member to monitor each epitope system present
(and possibly each stage of infection), even if poly-fusion
proteins are used. This can be avoided if the manufacturer can
demonstrate that each epitope system is equally stable.
Specimens able to show that the Note: It would not be sufficient for WHO prequalification to
invalidity and specificity rates do extrapolate to the stability of HIV-2/TP detection by testing only
not fall outside the claims, HIV-1 positive specimens.
particularly if whole blood is a
claimed specimen type
1422
1423 5 Specimens to monitor tests for hepatitis C (HCV) virus

1424 antibodies
Specimens Remarks
NS3 first seroconversion
specimens and core first
seroconversion specimens
Specimens to monitor any other Results can be obtained from line immunoassays that
antibodies claimed (frequently differentiate antibody responses to the different proteins.
against NS5 and NS4)
A late stage dilution near the Note: HCV serology is not particularly genotype-dependent in
sample-to-cut-off ratio terms of anti-core and anti-NS3, but it is possible to make
serotyping assays based on NS4 that mimic genotyping
reasonably well. It is usually not necessary to include controls for
genotype detection, unless risk assessment or experiment for a
particular IVD show otherwise
Difficult specimens to monitor 100 negatives at release subject to risk analysis and statistical
specificity and invalid rates analysis of the allowable false reactive rate and invalidity rate
(relative to the claimed rates)

1425 6 Specimens to monitor for tests for hepatitis B surface antigen

1426 (HBsAg)
Specimens Remarks
Specimens to define sensitivity This will almost certainly be one or more specimens with
relative to the claim traceability to the HBsAg international standards and probably
also to the ad and ay standards available from PEI.
Seroconversion specimens commercially available are almost all
rd
of the adw2 serotype, different from the 3 international
standard – so claims of critical threshold specimen detection
must be proven by specimens in the stability testing panel.
Specimens to monitor the These will almost certainly be traceable to the “1st International
maintenance of the claims of a Reference Panel for HBV genotypes for HBsAg—based assays"
variety of serotypes / genotypes (WHO/BS/2011.2180).
and mutant forms
Specimens to control against
prozone effect if found or if
theoretically an issue
If detection of HBsAg in the
presence of anti-HBsAg is claimed
(current best practise) proof of
maintenance of the claim
Specimens to monitor the critical If the monoclonal antibodies used have particular function or
components of the IVD bias, such as against the ayr or adr serotypes (not controlled by
the standards) or to detect mutant forms of the antigen, each
must be monitored to ensure viability at end of life. These might
well be the same specimens as in the previous paragraphs.
If there are critical dissociation chemicals or red-cell capture or
rupture agents used these must be monitored.
Difficult specimens to monitor 100 negatives at release, subject to risk analysis and statistical
specificity and invalid rates analysis of the allowable (relative to the claimed) false reactive
rate and invalidity rate.
1427
1428

1429 Appendix 3: Summary table of standards relevant for stability studies

Expectation Comment Standard
Studies must be compliant with CLSI EP 025A and The minimum expected standards CLSI EP25A
ISO 23640:2011 ISO 23640:2011
Studies must be fully documented with risk Risk assessment must be specific to the analyte, type of physical device and assay CLSI EP25A (many
evaluations, plans and protocols prior to initiation format, and previous manufacturing experiences, not generic nor by rote section),
ISO 23640:2011 Section 2
ISO 14971:2007
Studies and risk management must take into This is particularly important for transport stress where extreme conditions must
consideration conditions likely to be encountered be evaluated
in the geographies and healthcare settings in
which the device is intended to be used
Devices must be subjected to simulation of This is particularly important to WHO-PQ as transport will always be involved CLSI EP25A paragraph
transport stress before being used to establish any before use of a device and transport conditions cannot be guaranteed nor 4.2.3 & 5.2 [1]
form of stability predicted
Transport simulation must cover the extremes of It is most unlikely that actual transport will involve all extreme conditions that CLSI EP25A Section 4.2.3
environmental conditions ascertained during risk might occur during the marketing life of the device, nor that the conditions during
evaluations actual transport can be adequately documented
Devices used in any stability studies must be made If devices are not made to final validated and documented manufacturing scales a Good manufacturing
to finalised manufacturing specifications, to final stringent proof that scale change will not affect any parameters of the device, nor practice (GMP)
scale and in the packaging, including all labelling, any of the manufacturer’s claims, must be presented. Pre-production lots can CLSI EP25A
in which the devices will be made available only be used for stability work if these conditions are met
If several presentations of the device are to be If, for example two pack sizes are to be provided, even though the contents are CLSI EP25A
presented all aspects of stability must be shown identical except for vial size, each pack size must be evaluated completely
for each
47
Expectation Comment Standard
Sufficient numbers of independent lots of the “Independent lots” means lots with different critical reagents (e.g. biological CLSI EP25A Section 4.4
device must be evaluated to enable each form of reagents prepared in different syntheses, growths or purifications; other risk-
stability to be evaluated in terms of inter-lot defined critical reagents from different manufactured lots, or different suppliers if
variability applicable).
CLSI EP25A and ISO 23640 specify minimum numbers of lots to be used but give no
guidance to recommended numbers beyond documented risk evaluation
If critical components of the device are assigned It must be documented that stored materials, e.g. freeze thawed biological CLSI EP25A Section 4.4
lives independently of the life of the device the reagents operate as expected during the whole of the assigned lives
various forms of stability of the device must be
proven with those reagents at different stages of
their lives
Each form of stability must be defined statistically If any lot-to-lot variability is found the manufacturer must provide evidence that
with respect to any inter-independent lot subsequent lots will not have worse stability than that claimed
variability, not just assigned to the minimum
stability found among the lots that happened to
be evaluated experimentally
If any control material with a claim to prove the If the analytic function of the device is out of specification from any cause,
functionality of the device is provided to users that including stability failure, the control material must be demonstrated to be able to
claim must be justified in stability studies in alert the user to that fact
addition to any other studies
Use of accelerated stability, even to provide Accelerated stability is acceptable to provide interim life if the parameters of the CLSI EP25A Section 7.3 &
interim life assignments, must justified Arrhenius equation, or any other method used, are adequately proven and Appendix B
scientifically documented ISO 23640:2011 para 5.3.1
notes 1 & 2
1430

tgs2 Stability

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

tgs2 Stability

Uploaded by

Copyright:

Available Formats

Technical Guidance Series (TGS)

Draft for public comment 14 December 2015

Working document 14 December 2015

Contact: Irena Prat, EMP Prequalification Team Diagnostics

WHO - 20 Avenue Appia - 1211 Geneva 27 Switzerland

Working document 14 December 2015

Working document 14 December 2015 3

WHO Prequalification Programme: IVD Technical Guidance Series

Prequalification In vitro diagnostics (IVDs) prequalified by WHO are expected to be accurate,

4 Working document 14 December 2015

20 1.1 Key concepts

25 1.2 Rationale of stability studies

32 1.3 Purpose of this document

36 1.4 Limitations of this guidance

Working document 14 December 2015 5

48 2 Definitions and abbreviations

6 Working document 14 December 2015

Working document 14 December 2015 7

8 Working document 14 December 2015

183 NOTE: A continuing stability monitoring program (ongoing stability monitoring)

194 2.2 Abbreviations

Working document 14 December 2015 9

196 3 WHO prequalification requirements

204 3.1 Manufacturer responsibility

208 3.2 Standards

213 3.3 Suitability for use in Member States

224 3.4 Meeting customer requirements

10 Working document 14 December 2015

233 4 Basic principles for stability testing

234 4.1 Critical characteristics of the IVD

250 4.2 Finalised product presentation

256 4.3 Environmental conditions

Working document 14 December 2015 11

271 4.4 Minimum number of lots

288 4.5 Assessment of liquid components

296 4.6 Specimens for the stability testing panel

12 Working document 14 December 2015

316 4.7 Validation of stability testing panel

320 4.8 Assigning criteria to panel members

351 4.9 Time points

Working document 14 December 2015 13

373 4.9.1 Duration of testing

14 Working document 14 December 2015

385 5 Shelf-life studies

386 5.1 Requirements for determination of shelf life

399 5.1.1 Real-time stability studies

413 5.1.2 Accelerated stability studies

Working document 14 December 2015 15

424 6 Component stability studies

425 6.1 General principles

426 6.1.1 Testing on final specifications

432 6.1.2 Considering component stability

457 6.1.3 Considering constituent stability

16 Working document 14 December 2015

467 Examples of stored constituent: Purified recombinant antigens and

470 6.2 Stability of control materials

489 6.3 Antimicrobial stability and efficacy

490 6.3.1 Rationale

499 6.3.2 Study conditions

Working document 14 December 2015 17

509 6.3.3 Time of testing

517 6.4 Desiccant functionality

18 Working document 14 December 2015