1 Draft May 27, 2010 2
ENGLISH ONLY 3
4
Recommendations to Assure the Quality, Safety and Efficacy of Recombinant 5
Hepatitis B Vaccines 6
7
Proposed replacement of: TRS 786, Annex 2 and TRS 889, Annex 4. 8
9
NOTE: 10 This document has been prepared for the purpose of inviting comments and suggestions on the proposals 11 contained therein, which will then be considered by the Expert Committee on Biological Standardization 12 (ECBS). Publication of this early draft is to provide information about the proposed revision of the WHO 13 recommendations TRS 786, Annex 2 and TRS 889, Annex 4 to a broad audience and to improve transparency 14 of the consultation process. 15 16 These recommendations were developed based on the outcomes and consensus of the WHO consultations 17 convened in 2008 and 2009 with participants from national regulatory authorities, national control 18 laboratories and vaccine manufacturers. 19 20 The text in its present form does not necessarily represent an agreed formulation of the Expert 21 Committee. Comments proposing modifications to this text MUST be received by 30 June 2010 and 22 should be addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention: Quality Safety 23 and Standards (QSS). Comments may also be submitted electronically to the Responsible Officer: Dr Maria 24 Baca-Estrada, email: [email protected] 25 26 The outcome of the deliberations of the Expert Committee will be published in the WHO Technical Report 27 Series. The final agreed formulation of the document will be edited to be in conformity with the "WHO style 28 guide" (WHO/IMD/PUB/04.1). 29
© World Health Organization 2010 30
All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, World Health 31 Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: 32 [email protected]). Requests for permission to reproduce or translate WHO publications – whether for sale or for 33 noncommercial distribution – should be addressed to WHO Press, at the above address (fax: +41 22 791 4806; e-mail: 34 [email protected]). 35
The designations employed and the presentation of the material in this publication do not imply the expression of any 36 opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city 37 or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent 38 approximate border lines for which there may not yet be full agreement. 39 40 The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or 41 recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors 42 and omissions excepted, the names of proprietary products are distinguished by initial capital letters. 43 44 All reasonable precautions have been taken by the World Health Organization to verify the information contained in this 45 publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. 46 The responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health 47 Organization be liable for damages arising from its use. 48
49 The named authors [or editors as appropriate] alone are responsible for the views expressed in this publication. 50
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51
52
Recommendations published by the WHO are intended to be scientific and advisory. Each of the
following sections constitutes guidance for national regulatory authorities (NRAs) and for
manufacturers of biological products. If a NRA so desires, these Recommendations may be adopted
as definitive national requirements, or modifications may be justified and made by the NRA. It is
recommended that modifications to these Recommendations be made only on condition that
modifications ensure that the vaccine is at least as safe and efficacious as that prepared in
accordance with the recommendations set out below. The parts of each section printed in small type
are comments for additional guidance intended for manufacturers and NRAs, which may benefit
from those details.
53
Table of Content 54
Introduction ........................................................................................................................................ 3 55
General considerations ...................................................................................................................... 4 56
Part A. Manufacturing recommendations ....................................................................................... 5 57
A.1. Definitions...............................................................................................................................................................5 58
A.2 General manufacturing recommendations .............................................................................................................8 59
A.3 Control of source materials .....................................................................................................................................8 60
A.4 Fermentation..........................................................................................................................................................11 61
A.5 Single harvests ......................................................................................................................................................12 62
A.6 Control of aqueous bulk (purified antigen bulk) .................................................................................................13 63
A.7 Final vaccine bulk ................................................................................................................................................16 64
A.8 Filling and containers ..........................................................................................................................................18 65
A.9 Control tests on final vaccine lot ...........................................................................................................................18 66
A.10 Records.................................................................................................................................................................21 67
A.11 Retained samples .................................................................................................................................................21 68
A.12 Labelling ..............................................................................................................................................................21 69
A.13 Distribution and shipping ...................................................................................................................................22 70
A.14 Stability testing, storage and expiry date.............................................................................................................22 71
Part B. Nonclinical evaluation......................................................................................................... 23 72
B.1 Strategy for cloning and expressing the gene product .........................................................................................23 73
B.2 Characterization of purified HBsAg for new vaccines ........................................................................................23 74
B.3 Animal models .......................................................................................................................................................24 75
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B.4 Nonclinical safety studies .....................................................................................................................................25 76
B.5 Toxicology studies.................................................................................................................................................25 77
Part C. Clinical evaluation .............................................................................................................. 26 78
C.1 Consideration for clinical studies..........................................................................................................................26 79
C.2 Assessment of immune responses..........................................................................................................................27 80
C.3 Clinical studies.......................................................................................................................................................27 81
C.4 Post-marketing studies...........................................................................................................................................32 82
Part D. Guidelines for national regulatory authorities................................................................. 32 83
D.1 General...................................................................................................................................................................32 84
D.2 Release and certification .......................................................................................................................................32 85
Authors and Acknowledgements .................................................................................................... 33 86
References ......................................................................................................................................... 35 87
Methodological considerations. Potency tests for recombinant hepatitis B vaccines. ...............................................37 88
Appendix 2 ........................................................................................................................................ 44 89
Summary protocol for manufacturing and control of hepatitis B vaccines. ..............................................................44 90
Appendix 3 ....................................................................................................................................... 56 91
Model certificate for the release of recombinant hepatitis B vaccine by a national regulatory authority ................56 92 93
Introduction 94
These Recommendations are intended to provide national regulatory authorities (NRAs) and vaccine 95
manufacturers with background and guidance on the production, quality control and evaluation of 96
the safety and efficacy of recombinant hepatitis B vaccines for prophylactic use. 97
98
The first document outlining the requirements for the production and control of hepatitis B vaccines 99
containing hepatitis B surface antigen (HBsAg) purified from the plasma of chronically infected 100
individuals was adopted by the Expert Committee on Biological Standardization (ECBS) in 1980 (1) 101
and later revised in 1987 (2). 102
103
Following the development of hepatitis B vaccines containing HBsAg produced by recombinant 104
DNA techniques in yeast, a new set of requirements were developed following a meeting of experts 105
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in 1985 (3) and adopted by the ECBS in 1986 (4). These were revised to include vaccines produced 106
by recombinant techniques in mammalian cells as well as yeast cells in 1988 (5). 107
108
With the development and implementation of new in vitro assays to determine antigen content, an 109
amendment was published to include the use of the in vitro assay in the quality control of 110
recombinant hepatitis B vaccines (6). 111
112
This present document applies to vaccines containing HBsAg only and will replace the WHO 113
Requirements for hepatitis B vaccine made by recombinant DNA techniques in Technical Report 114
Series No. 786, Annex 2 and the corresponding amendment TRS No. 889, Annex 4 and should be 115
read in conjunction with all other relevant WHO guidelines including those on nonclinical (7) and 116
clinical evaluation (8) of vaccines. 117
118
119
General considerations 120
Hepatitis B virus has several characteristics that distinguish it from the other families of DNA 121
viruses. It has an outer coat (more substantial than a membrane or envelope) consisting of protein, 122
lipid, and carbohydrate and bearing a unique antigen complex, HBsAg. Its nucleic acid consists of a 123
circular DNA genome of relative molecular mass about 2 million, part of which is double-stranded 124
and part single-stranded, an unusual feature among viruses. Virus recovered from the plasma of a 125
hepatitis B carrier was used to clone the HBsAg gene. 126
127
The HBsAg gene has been inserted into yeast and mammalian cells by means of appropriate 128
expression vectors. Antigen expressed in several species of yeast, namely Saccharomyces cerevisiae, 129
Pichia pastoris and Hansenula polymorpha, and Chinese hamster ovary (CHO) cells has been used 130
to produce hepatitis B vaccines for over 20 years. Electron microscopy revealed that purified 131
HBsAg obtained from transfected cultures exists as particles 15-30 nm in diameter, with the 132
morphological characteristics of free surface antigen in plasma. Purified antigen has been shown to 133
induce antibodies in mice and guinea-pigs and to protect chimpanzees from infection with hepatitis 134
B virus. 135
136
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All hepatitis B vaccines currently in the market require formulation with adjuvants. Preservatives 137
are used for multidose presentations but there are some single dose presentations available without 138
preservative. Recombinant hepatitis B vaccines are available as monovalent products or included in 139
combination vaccines together with other antigens such as hepatitis A virus, diphtheria toxoid, 140
tetanus toxoid, whole-cell or acellular pertussis components and inactivated poliomyelitis viruses. 141
142
The requirements that follow apply to the manufacture, quality control, nonclinical and clinical 143
evaluation of hepatitis B vaccines containing HBsAg made by recombinant DNA methods. It is 144
expected that new or significantly modified recombinant hepatitis B vaccine formulations will be 145
characterized according to the recommendations made in Part A and B of this document and 146
assessed in clinical studies as described in Part C. Specific issues relevant to the combination 147
vaccines containing recombinant HBsAg will be addressed in a separate document. 148
149
Particular emphasis is placed on the introduction of “in-process’’ controls to monitor consistency of 150
production, rather than relying on tests on the final product. Certain tests will be required on every 151
batch of vaccine, whereas others will be required only to support licensure or significant 152
manufacturing changes. 153
154
The vaccine lots used in clinical trials should be adequately representative of the formulation and 155
manufacturing scale intended for marketing. 156
157
158
Part A. Manufacturing recommendations 159
A.1. Definitions 160
A.1.1 International name and proper name 161
The international name should be Vaccinum hepatitidis B recombinatum. The proper name should 162
be the equivalent of the international name in the language of the country of origin. The use of the 163
international name should be limited to vaccines that satisfy the requirements elaborated below. 164
165
A.1.2 Descriptive definition 166
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Vaccinum hepatitidis B recombinatum is a preparation of purified HBsAg that has been produced by 167
recombinant DNA techniques. The antigen may be formulated with a suitable adjuvant. Such 168
vaccines are for prophylactic use. 169
170
A.1.3 International reference materials 171
International standards and reference reagents for the control of hepatitis B vaccine potency are not 172
available. Therefore, product-specific reference preparations may be used. 173
174
The Second International Standard (IS) for Hepatitis B Surface Antigen (non-adjuvanted HBsAg) 175
subtype adw2, genotype A contains 33 IU per vial. This material is is intended as a quantitative 176
reference standard for HBsAg subtype adw2, genotype A and the use of this standard will give an 177
indication of the analytical sensitivity of an assay for the detection of HBsAg. IS for HBsAg should 178
not be used as a vaccine reference. 179
180
An international standard for hepatitis B immunoglobulin is available for use in assays designed to 181
quantify antibody to HBsAg (anti-HBs) in human serum. Antibody responses to hepatitis B vaccines 182
should be expressed in IU. The second International Standard for Hepatitis B Immunoglobulin 183
(2008) was prepared from fractionated human plasma and freeze dried in ampoules. It has an 184
assigned potency of 100 IU/ampoule. This preparation is in the custody of the NIBSC, Potters Bar, 185
UK. 186
187
A.1.4 Terminology 188
Adjuvant: A vaccine adjuvant is a component that potentiates the immune response to an antigen 189
and/or modulates it towards the desired immune responses. 190
191
Adventitious agents: Contaminating microorganisms of the cell substrate or source materials used in 192
their cultures, that may include bacteria, fungi, mycoplasmas, and endogenous and exogenous 193
viruses that have been unintentionally introduced. 194
195
Aqueous bulk: Purified antigen bulk before the addition of an adjuvant. 196
197
Anti-HBs: Antibodies to hepatitis B surface antigen 198
199
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Cell bank: A collection of containers (e.g. ampoules, vials) containing aliquots of a suspension of 200
cells from a single pool of cells of uniform composition, stored frozen under defined conditions 201
(typically <−60 °C for yeast, and in liquid nitrogen for mammalian cell lines). 202
203
End of production cells: A cell suspension containing the cells harvested at the end of 204
culture/fermentation. 205
206
Final vaccine bulk: The formulated bulk prepared from one or more batches of aqueous bulk 207
(purified antigen) to which adjuvant has been added, present in the container from which the final 208
containers are filled. 209
210
Final vaccine lot: A collection of sealed final containers of vaccine that is homogeneous with 211
respect to the risk of contamination during the filling process. A final vaccine lot must therefore 212
have been filled from a single vessel of final bulk in one working session. 213
214
Hepatitis B virus: a 42-nm double-shelled virus particle, originally known as the Dane particle, 215
which contains the DNA genome of the virus. 216
217
HBsAg: hepatitis B surface antigen, comprising a complex of antigens associated with the virus 218
envelope and subviral forms (22-nm spherical and tubular particles). Native HBsAg is encoded by 219
envelope gene sequences (S plus pre-S) in the viral DNA. Recombinant DNA-derived hepatitis B 220
vaccines may contain the S gene product or products of the S/pre-S combination. 221
222
Production cell culture: A cell culture derived from one or more containers of the WCB used for the 223
production of vaccines. 224
225
Master cell bank (MCB): A collection of containers containing aliquots of a suspension of cells from 226
a single pool of cells of uniform composition, stored frozen under defined conditions (typically < 227
−60 °C for yeast, and in liquid nitrogen for mammalian cell lines). The MCB is used to derive all 228
working cell banks for the anticipated lifetime of the vaccine production. 229
230
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Single harvest: the biological material prepared from a single production run. 231
232
Working cell bank (WCB): A collection of containers containing aliquots of a suspension of cells 233
from a single pool of cells of uniform composition, derived from the MCB, stored frozen under 234
defined conditions (typically <−60 °C for yeast, and in liquid nitrogen for mammalian cell lines). 235
One or more aliquots of the WCB are used for routine production of the vaccine. Multiple WCBs 236
are made and used during the lifetime of the vaccine production. 237
238
A.2 General manufacturing recommendations 239
The general manufacturing requirements contained in the WHO Guidelines on good manufacturing 240
practices for pharmaceuticals (9) and biological products (10) should apply to the establishment of 241
manufacturing facilities manufacturing hepatitis B vaccine, with the addition of the following: 242
• Production areas should be decontaminated before they are used for the manufacture of 243
hepatitis B vaccine. 244
• Hepatitis B vaccine should be produced by staff who have not handled animals or infectious 245
microorganisms in the same working day. The staff should consist of persons who have been 246
examined medically and have been found to be healthy. 247
• No cultures of microorganisms or eukaryotic cells other than those approved by the NRA 248
should be introduced into or handled in the production area at any time during manufacture 249
of the vaccine. 250
251
A.3 Control of source materials 252
A.3.1 Cell substrates for antigen production 253
The use of any cell culture should be based on a cell bank system. Only cells that have been 254
approved and registered with the NRA should be used to produce HBsAg protein. The NRA should 255
be responsible for approving the cell bank. Appropriate history of the cell bank should be provided. 256
257
A.3.1.1 Yeast cells 258
The characteristics of the recombinant production strain (host cell in combination with the 259
expression vector system) should be fully described and information given on the absence of 260
adventitious agents and on gene homogeneity for the master and working cell banks. A full 261
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description of the biological characteristics of the host cell and expression vectors should be given. 262
The physiological measures used to promote and control the expression of the cloned gene in the 263
host cell should be described in detail. This should include genetic markers of the host cell, the 264
construction, genetics and structure of the expression vector and the origin and identification of the 265
gene that is being cloned. 266
267
The nucleotide sequence of the gene insert and of adjacent segments of the vector and restriction-268
enzyme mapping of the vector containing the gene insert should be provided as required by the 269
NRA. Characterization of gene product (HBsAg) should be provided in support of licensure (see 270
Part B). 271
272
Master and working cell banks should be tested for the absence of adventitious bacteria, fungi 273
according to Part A section of the WHO General requirements for the sterility of biological 274
substances (11) or by a method approved by the NRA. Cells must be maintained in a frozen state 275
that allows recovery of viable cells without alteration of genotype. The cells should be recovered 276
from the frozen state, if necessary in selective media such that the genotype and phenotype 277
consistent with the unmodified host and unmodified recombinant DNA vector are maintained and 278
clearly identifiable. Cell banks should be identified and fully characterized by means of appropriate 279
tests. 280
281
Where appropriate, plasmid retention in the cell bank should be monitored at regular intervals. Data 282
that demonstrate the stability of the expression system during storage of the recombinant WCB up to 283
or beyond the passage level used for production should be provided and approved by the NRA. Any 284
instability of the expression system occurring in the seed culture, or after a production-scale run 285
(end of production cells), should be documented. 286
287
A.3.1.2 Mammalian cells 288
If mammalian cells are used, the cell substrate and cell banks should conform with the WHO 289
Requirements for the use of animal cells as in vitro substrates for the production of biologicals (12); 290
other relevant guidelines provide additional information (13). Cell substrates and cell banks should 291
be approved by the NRA. 292
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293
The maximum population doublings (or number of passages) allowable between the MCB, the 294
WCB and the production cells should be approved by the NRA. The MCB is made in sufficient 295
quantities and stored in a secure environment and is used as the source material to make 296
manufacturers WCB. In normal practice a MCB is expanded by serial subculture up to a population 297
doubling (or passage number, as appropriate) selected by the manufacturer and approved by the 298
NRA, at which point the cells are combined to give a single pool, distributed into containers (e.g. 299
ampoules, vials) and preserved cryogenically to form the WCB. 300
301
Tests on the master and working cell banks are performed in accordance with WHO Requirements 302
for use of animal cells as in vitro substrates for the production of biologicals (12) and WHO 303
guidelines for assuring the quality of pharmaceutical and biological products prepared by 304
recombinant DNA technology for recombinant cells (14) and should be approved by the NRA. 305
306
A.3.2 Cell culture medium 307
If serum is used for the propagation of mammalian cells, it should be tested to demonstrate freedom 308
from bacteria, fungi and mycoplasmas, according to WHO requirements (11). Suitable tests for 309
detecting viruses in bovine serum are given in Appendix 1 of WHO Recommendations for 310
production and control of poliomyelitis vaccine (oral) (15). 311
312
Validated molecular tests for bovine viruses may replace the cell culture tests of bovine sera. As an 313
additional monitor of quality, sera may be examined for freedom from phage and endotoxin. 314
Gamma-irradiation may be used to inactivate potential contaminant viruses. 315
316
The acceptability of the source(s) of any components of bovine, porcine, sheep or goat origin used 317
should be approved by the NRA. These components should comply with current WHO guidelines in 318
relation to animal transmissible spongiform encephalopathies (16). 319
320
If trypsin is used for preparing cell cultures, it should be tested and found free of bacteria, fungi, 321
mycoplasmas and infectious viruses, especially bovine or porcine parvoviruses, as appropriate. The 322
methods used to ensure this should be approved by the NRA. The trypsin should be gamma 323
irradiated. 324
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325
Human serum should not be used. However, human serum albumin may be used only if it complies 326
with the WHO Requirements for the collection, processing and quality control of blood, blood 327
components and plasma derivatives (17). In addition, human albumin and materials of animal origin 328
should comply with current WHO guidelines regarding to animal transmissible spongiform 329
encephalopathies (16). 330
331
Penicillin and other beta-lactams should not be used at any stage of the manufacture because of their 332
nature as highly sensitizing substances. 333
334
Other antibiotics may be used in the manufacture provided that the quantity present in the final 335
product is acceptable to the NRA. 336
337
Non-toxic pH indicators may be added, e.g. phenol red at a concentration of 0.002%. Only 338
substances that have been approved by the NRA may be added. 339
340
A.4 Fermentation 341
A.4.1 Production of cell cultures 342
Only cell cultures derived from the WCB should be used for production. All processing of cells 343
should be done in an area in which no cells or organisms are handled other than those directly 344
required for the process. The medium used should comply with the requirements given in section 345
A.3.2. 346
347
A.4.1.1 Control of HBsAg production up to single harvest in yeast expression system 348
Microbial purity in each fermentation vessel should be monitored at the end of the production run by 349
methods approved by the NRA. 350
351
Any agent added to fermentor or bioreactor in purpose to feed cells or to induce /increase cell 352
density should be approved by the NRA. 353
354
A.4.1.2 Control of HBsAg production up to single harvest in mammalian cells 355
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Production of cell cultures should be carried out under conditions agreed with the NRA. These 356
conditions should include details of the culture system used, the cell doubling time, the number of 357
subcultures or the duration of the period of subcultivation permitted, and the incubation temperature. 358
Cell culture vessels should be monitored for potential microbial contamination during and at the end 359
of the production runs by methods approved by the NRA. 360
361
A.5 Single harvests 362
A.5.1 Storage and intermediate hold times 363
During the purification process, all intermediates should be maintained under conditions shown by 364
the manufacturer to retain the desired biological activity. Hold times should be approved by the 365
NRA. 366
367
A.5.2 Tests on single harvest 368
A.5.2.1 Sampling 369
Samples required for the testing of single harvests should be taken immediately on harvesting prior 370
to further processing. For mammalian cell cultures, if the tests for adventitious agents are not 371
performed immediately, the samples taken for these tests should be kept at a temperature of -60ºC or 372
below and subjected to no more than one freeze-thaw cycle. 373
374
A.5.2.2 Test for bacteria, fungi and mycoplasma contamination 375
Bacterial and fungal contamination in the cell culture vessels should be monitored during and at the 376
end of the production runs by methods approved by the NRA. 377
If mammalian cells are used in production each single harvest or single harvests pool should be 378
shown to be free from bacteria, fungi and mycoplasma contamination by appropriate tests (11). 379
Nucleic Acid Amplification Techniques (NAT) alone or in combination with cell culture, with an 380
appropriate detection method, might be used as an alternative to one or both of the compendial 381
mycoplasma detection methods after suitable validation and agreement from NRA. 382
383
A.5.3 Consistency of yield 384
Data on the consistency of yield between runs and during individual production runs should be 385
provided, and the NRA should approve the criteria for an acceptable production run. 386
387
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A.5.4 Plasmid retention 388
A sample of cells that are representative of each harvest must be tested to confirm that the 389
recombinant phenotype has been retained. The method used should be approved by the NRA. 390
391
When the production method has shown to yield consistently harvests that comply with the 392
requirement for plasmid retention, the test may be omitted on the harvest after approval by the NRA. 393
However, the stability of the vector should be monitored on a regular basis on the working cell bank. 394
395
Where the plasmid is integrated in the host cell genome, presence of the integrated HBsAg gene 396
insert should be confirmed 397
398
A.5.5 Tests for adventitious agents if mammalian cells are used in production 399
Single harvest or single harvests pool should be tested for adventitious agents it cell cultures in 400
accordance with WHO Requirements for use of animal cells as in vitro substrates for the production 401
of biologicals (12, note that this guideline is under revision and the reference will be changed 402
once the guideline is approved). Additional testing may be performed using nucleic acid 403
amplification methods. 404
405
A.6 Control of aqueous bulk (purified antigen bulk) 406
The purification procedure can be applied to a single harvest, to a part of a single harvest, or to a 407
pool of single harvests. The maximum number of single harvests that may be pooled should be 408
approved by the NRA. Adequate purification may require several purification steps based on 409
different principles. This will minimize the possibility of co-purification of extraneous cellular 410
materials. The methods used for the purification of the HBsAg should be appropriately validated and 411
approved by the NRA. Any agent added during the purification process, should be documented and 412
its removal adequately validated and tested for as appropriate (see A.6.1.8). 413
414
The monovalent purified antigen bulk can be stored under conditions shown by the manufacturer to 415
retain the desired biological activity. Intermediate hold times should be approved by the NRA. 416
Additional tests on intermediates during purification process may be used to monitor the 417
consistency/ yields. 418
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419
A.6.1 Tests on the aqueous bulk (purified antigen) 420
The aqueous bulk should be tested according to the tests listed below. All quality control release 421
tests and specifications for aqueous bulk, should be validated and approved by the NRA. 422
423
A.6.1.1 Purity 424
The degree of purity of each aqueous bulk should be assessed by suitable methods. Examples of 425
suitable methods of analysing the proportion of potential contaminating proteins in the total protein 426
of the preparation are polyacrylamide gel electrophoresis (PAGE), optionally followed by 427
densitometric analysis, or high-performance liquid chromatography (HPLC). The aqueous bulk 428
should be not less than 95% pure. 429
430
A.6.1.2 Protein content 431
The protein content should be determined using a suitable method such as the micro-Kjeldahl 432
method, the Lowry technique or another suitable method. 433
434
A.6.1.3 HBsAg content 435
The HBsAg content of the aqueous bulk should be determined by an appropriate immunochemical 436
method. An appropriate reference material should be included in these assays so that consistency of 437
production is monitored. This reference material could be a representative bulk of known HBsAg 438
and protein content or a highly purified preparation of HBsAg of known HBsAg and protein content 439
stored in single use aliquots. It is important to note that the reference materials based on adjuvanted 440
product is not suitable for use in assays of non-adjuvanted intermediate bulks of HBsAg. 441
442
The ratio of HBsAg content to protein content should be determined. The antigen/protein ratio 443
should be within the limits approved by the NRA. 444
445
A.6.1.4 Identity 446
The test for antigen content will generally serve as confirmation of the identity of the protein in the 447
bulk. Alternatively, immunoblots using HBsAg specific antibodies in the assessment of purity could 448
also serve to confirm the molecular identity of the product. Such tests should be approved by the 449
NRA. 450
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451
A.6.1.5 Lipids 452
The lipid content of each aqueous bulk should be determined by an appropriate method. The 453
methods used and the permitted concentrations of lipid should be approved by the NRA. This test 454
may be omitted for routine lot release upon demonstration of consistency of the purification process 455
to the satisfaction of the NRA. 456
457
A.6.1.6 Carbohydrates 458
The carbohydrate content of each aqueous bulk should be determined by an appropriate method. The 459
methods used and the permitted concentrations of carbohydrates should be approved by the NRA. 460
This test may be omitted for routine lot release upon demonstration of consistency of the 461
purification process to the satisfaction of the NRA. 462
463
A.6.1.7 Sterility tests for bacteria and fungi 464
Each aqueous bulk should be tested for freedom from bacteria and fungi according to WHO 465
Requirements (11), or by a method approved by the NRA. 466
467
A.6.1.8 Tests for agents used during purification or other phases of manufacture 468
The aqueous bulk should be tested for the presence of any potentially hazardous agents used during 469
manufacture. The method used and the concentration limits should be approved by the NRA. This 470
test may be omitted for routine lot release upon demonstration that the purification process 471
consistently eliminates the agent from the purified bulks. 472
473
Where a monoclonal antibody is used in vaccine preparation, for example for immunological 474
affinity chromatography to purify HBsAg, the antibody used should be characterized and its purity 475
determined. The product should be tested for residual antibody. The methods used and the permitted 476
concentrations of antibody should be approved by the NRA. 477
478
Several NRAs have drafted guidelines for the control of monoclonal antibody preparations used for 479
the manufacture of biological products for human use. 480
481
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If the HBsAg has been treated with formaldehyde and/or other agents, then the material should be 482
tested for the presence of free formaldehyde and/or the other agents. The method used and the 483
permitted concentration should be approved by the NRA. 484
485
A.6.1.9 Tests for residuals derived from the antigen expression system 486
The amount of residuals derived from the antigen expression system (e.g. DNA or host cell proteins) 487
should be determined in each monovalent antigen purified bulk by sensitive methods. In the case of 488
yeast-derived products, these tests may be omitted for routine lot release upon demonstration that 489
the purification process consistently eliminates the residual components from the monovalent bulks 490
to the satisfaction of the NRA. In the case of HBsAg produced in mammalian cells substrates, the 491
level of host cell DNA should not exceed the maximum level cited in the WHO Requirements for 492
use of animal cells as in vitro substrates for the production of biologicals (12). 493
494
A.6.1.10 Bacterial endotoxins 495
Each final aqueous bulk should be tested for bacterial endotoxins. 496
497
A.6.1.11 Adjuvants 498
Hepatitis B vaccines may contain the immunostimulant monophosphoryl lipid A (MPL) adsorbed 499
onto aluminium compounds (e.g. aluminium phosphate), when the adsorption of the MPL is 500
performed prior to the final formulation step, the degree of adsorption of MPL should be determined 501
using a suitable method (e.g. gas chromatography). 502
503
A.6.2 Additional tests when mammalian cells are used 504
A.6.2.1 Albumin content 505
If animal serum is used in mammalian cell cultures, or at any stage in the manufacturing process, 506
testing should be performed to assess the residual serum in the purified bulk. The concentration of 507
animal serum in the vaccine should be not more than 50ng per human dose of vaccine. 508
509
A.7 Final vaccine bulk 510
The final vaccine bulk may consist of one or more purified aqueous bulks. Only aqueous bulks that 511
have satisfied the requirements outlined in previous sections should be formulated into the final 512
vaccine bulk. The antigen concentration in the final formulation should be sufficient to ensure the 513
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dose which is consistent with that shown to be safe and effective in human clinical trials. 514
Formulation is generally based on protein content, but HBsAg content may be used. 515
It should be noted that formulation based on HBsAg may be affected by changes in the kits used to 516
determine antigen content. This should be considered and included in validation/bridging studies 517
when kit changes occur. 518
519
The operations necessary for preparing the final vaccine bulk should be conducted in such a manner 520
as to avoid contamination of the product. In preparing the final vaccine bulk, any substances such as 521
diluents, stabilizers or adjuvants that are added to the product should have been shown to the 522
satisfaction of the NRA not to impair the safety and efficacy of the vaccine in the concentration 523
used. Until the bulk is filled into containers, the final vaccine bulk suspension should be stored 524
under conditions shown by the manufacturer to retain the desired biological activity. 525
526
A.7.1 Tests on the final vaccine bulk 527
All tests and specifications for the final vaccine bulk, unless otherwise specified, should be 528
approved by the NRA. 529
530
The HBsAg may be formulated with other vaccine antigens into a combined vaccine (e.g. HAV, 531
DTwP-HBsAg, DTaP-HBsAg etc). Specific issues related to the formulation and quality control of 532
final vaccine bulk of combination vaccines will be addressed in a separate document. 533
534
A.7.1.1 Sterility tests 535
Each final vaccine bulk should be tested for bacterial and fungal sterility according to the WHO 536
Requirements (11). 537
538
A.7.1.2 Adjuvants 539
Each final vaccine bulk should be assayed for the content of adjuvants. Where aluminium 540
compounds are used, the content of aluminium should not be greater than 1.25 mg per ml. Other 541
adjuvants included in the vaccine should be assessed using suitable tests. For example, gas 542
chromatography to quantify the immunostimulant MPL. 543
544
A.7.1.3 Degree of adsorption 545
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The degree of adsorption of HBsAg antigen present in the final vaccine bulk should be assessed. 546
This test may be omitted upon demonstration of the process consistency to the satisfaction of the 547
NRA or if performed on the final vaccine lot. 548
549
A.7.1.4 Preservative content 550
The final vaccine bulk should be assayed for preservative content, if added. This test may be omitted 551
if performed on the final vaccine lot. 552
553
A.7.1.5 Potency 554
If an in vivo potency test (i.e. immunogenicity) is used, this test may be performed on the final 555
vaccine bulk. The method for detection of antibodies to HBsAg and the analysis of data should be 556
approved by the NRA. The vaccine potency should be compared with that of a reference preparation 557
and the NRA should determine limits of potency and approve the reference preparation used. If an 558
in vitro potency test is performed, it should be performed on every lot of final vaccine lot. 559
Methodological considerations regarding potency assays are outlined in Appendix 1. 560
561
A.8 Filling and containers 562
The requirements concerning filling and containers given in the WHO Guidelines on good 563
manufacturing practices for biological products (10) should apply to vaccine filled in the final form. 564
565
Care should be taken to ensure that the materials of which the container and, if applicable, 566
transference devices and closure are made do not adversely affect the quality of vaccine. The 567
manufacturers should provide the NRA with adequate data to prove the stability of the product 568
under appropriate conditions of storage and shipping. 569
570
A.9 Control tests on final vaccine lot 571
Samples should be taken from each final vaccine lot to be tested and fulfill requirements of this 572
section. All the tests and specifications including methods used and the permissible limits for the 573
different parameters listed under this section, unless otherwise specified, should be approved by the 574
NRA. 575
576
A.9.1 Inspection of containers 577
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Each container of each final vaccine lot should be inspected visually and those showing 578
abnormalities should be discarded. 579
580
A.9.2 Appearance 581
Visual inspection of the appearance of the vaccine should be described with respect to the form and 582
color. 583
584
A.9.3 Identity 585
The vaccine should be identified as HBsAg by appropriate methods. The potency test may serve as 586
the identity test. 587
588
A.9.4 Sterility tests 589
Each final vaccine lot should be tested for bacterial and fungal sterility according to the WHO 590
requirements (11), or by acceptable methods. 591
592
A.9.5 General safety (innocuity) test 593
Each final lot should be tested for the absence of abnormal toxicity in mice and guinea pigs using a 594
general safety (innocuity) test approved by the NRA and should pass the test. This test may be 595
omitted for routine lot release once consistency of production has been established to the satisfaction 596
of the NRA. 597
598
A.9.6 pH and osmolality 599
The pH and osmolality of a pool of final containers should be tested. The test for osmolality may be 600
omitted until consistency of production is demonstrated to the satisfaction of the NRA. 601
602
A.9.7 Preservatives 603
Each final vaccine lot should be tested for preservative content, if added. This test may be omitted if 604
performed on the final vaccine bulk. 605
606
A.9.8 Pyrogen / endotoxin content 607
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The vaccine in the final container should be tested for pyrogenic activity by intravenous injection 608
into rabbits or by a Limulus amoebocyte lysate test. Endotoxin content or pyrogenic activity should 609
be consistent with levels found to be acceptable in vaccine lots used in clinical trials and approved 610
by the NRA. The test is conducted until consistency of production is demonstrated to the satisfaction 611
of the NRA. 612
613
A.9.9 Assay for adjuvant 614
Each final vaccine lot should be assayed for adjuvant content. Where aluminium compounds are 615
used, the amount of aluminium should not be greater than 1.25 mg per ml. Should an 616
immunostimulant (e.g. monophospohoryl lipid A) be present, each final vaccine lot should be 617
assayed for the immunostimulant content. This test may be omitted if performed on the final vaccine 618
bulk. 619
620
621
A.9.10 Degree of adsorption 622
The degree of adsorption of the antigen and, where applicable, MPL to the aluminium compounds 623
(aluminium hydroxide or hydrated aluminium phosphate) in each final vaccine lot should be 624
assessed if not performed on the MPL bulk or final bulk. This test may be omitted for routine lot 625
release upon demonstration of the product consistency to the satisfaction of the NRA. 626
627
A.9.11 Potency tests 628
An appropriate quantitative test for potency by an in vivo or in vitro method should be performed on 629
samples representative of the final vaccine lot. The method and the analysis of data should be 630
approved by the NRA. If an in vivo potency test is performed on the final bulk, the test on final 631
container may be omitted. Methodological considerations regarding potency assays are outlined in 632
Appendix 1. 633
634
Because of the diversity in the reactivity of vaccines produced by different manufacturing 635
techniques and differences in the adjuvants used for the formulation, it is unlikely that an 636
International Standard will be suitable for the standardization of assays of vaccines from all 637
manufacturers. Manufacturers should therefore establish a product specific reference preparation 638
which is traceable to a lot of vaccine shown to be efficacious in clinical trials. The NRA should 639
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approve the reference preparation used and agree with the potency limits applied. The performance 640
of this reference vaccine should be monitored by trend analysis using relevant test parameters and it 641
should be replaced when necessary. 642
643
The stability of the product-specific reference vaccine may also be monitored by routine 644
assay against a stable HBsAg preparation. The inclusion of such a preparation in assays at 645
regular intervals would monitor stability of the product-specific reference and monitor test/kit 646
performance. However, this preparation would not be intended for use in establishing potency 647
values. 648
649
A.10 Records 650
The requirements given in the WHO Guidelines on good manufacturing practices for biological 651
products (10) should apply. 652
653
A.11 Retained samples 654
The requirements given in the WHO Guidelines on good manufacturing practices for biological 655
products (10) should apply. 656
657
A.12 Labelling 658
The requirements given in the WHO Guidelines on good manufacturing practices for biological 659
products (10) should apply, with the addition of the following information. 660
661
The label on the carton, the container or the leaflet accompanying the container should state: 662
• the nature of the cells used to produce the antigen; 663
• the nature of any preservative and amount of adjuvant, present in the vaccine; 664
• the volume of one recommended human dose, the immunization schedules, and the 665
recommended routes of administration (this information should be given for newborn babies, 666
children, adults, and immunosuppressed individuals, and should be the same for a given 667
vaccine for all regions of the world); 668
• the amount of HBsAg protein contained in one recommended human dose. 669
670
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A.13 Distribution and shipping 671
The requirements given in the WHO Guidelines on good manufacturing practices for biological 672
products (10) should apply. 673
674
In addition, the conditions of shipping should be such as to ensure that the adjuvanted vaccine does 675
not freeze. Temperature indicators should be packaged with each vaccine shipment to indicate 676
whether freezing occurs. If freezing has occurred, the vaccine should not be used. 677
678
A.14 Stability testing, storage and expiry date 679
A.14.1 Stability testing 680
Adequate stability studies form an essential part of vaccine development. Current guidance on 681
evaluation of vaccine stability is provided in the WHO guidelines on stability evaluation of vaccines 682
(18). Stability testing should be performed at different stages of production, namely on single 683
harvests, aqueous bulk, final vaccine bulk and final vaccine lot to validate their claimed shelf life. 684
The stability of the vaccine in its final form and at the recommended storage temperatures should be 685
demonstrated to the satisfaction of the NRA on final containers from at least three lots of final 686
product. The formulation of HBsAg antigens and adjuvant must be stable throughout its shelf-life. 687
Acceptable limits for stability should be agreed with national authorities. 688
689
A.14.2 Storage conditions 690
The final container vaccine should be kept at 2-8°C. If other storage conditions are used, they should 691
be fully validated by appropriate stability studies and approved by the NRA. The vaccine should 692
have been shown to maintain its potency for a period equal to that between the date of release and 693
the expiry date. During storage, liquid adsorbed vaccines should not be frozen. 694
695
A.14.3 Expiry date 696
The expiry date should be fixed upon the approval of the NRA, and should take account of the 697
experimental data on stability of the vaccine. 698
Some manufacturers base the expiry date on the date of formulation of the final bulk. Others base 699
the expiry date on the date of the last satisfactory potency test, i.e., the date on which the animals 700
were inoculated with the vaccine in an in vivo test, or from the date of the in vitro potency test 701
performed on the final container. 702
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Part B. Nonclinical evaluation 703
Nonclinical evaluation of hepatitis B vaccines should be based on WHO guidelines on nonclinical 704
evaluation of vaccines (7). The following specific issues should be considered in the context of the 705
development of new recombinant hepatitis B vaccines. Prior to clinical testing of any new or 706
modified hepatitis B vaccine in humans there should be extensive product characterization, proof of 707
concept/immunogenicity studies and safety testing in animals. 708
709
B.1 Strategy for cloning and expressing the gene product 710
A full description of the biological characteristics of the host cell and expression vectors used in 711
production should be given. This should include details of: (a) the construction, genetics, and 712
structure of the expression vector; and (b) the origin and identification of the gene that is being 713
cloned (c) potential retrovirus-like particles in and genetic markers for mammalian cell based 714
expression systems. The physiological measures used to promote and control the expression of the 715
cloned gene in the host cell should be described in detail. 716
717
Data that demonstrate the stability of the expression system during storage of the WCB and beyond 718
the passage level used for production should be provided. Any instability of the expression system 719
occurring in the seed culture or after a production-scale run, for example involving rearrangements, 720
deletions, or insertions of nucleotides, must be documented. The NRA should approve the system 721
used. 722
723
B.2 Characterization of purified HBsAg for new vaccines 724
Rigorous identification and characterization of recombinant DNA-derived vaccines is required as 725
part of the Marketing Authorization application. The ways in which these products differ chemically, 726
structurally, biologically, or immunologically from the naturally occurring antigen must be fully 727
documented. Such differences could arise during processing at the genetic or post-translational level, 728
or during purification. 729
730
B.2.1 Characterization of gene products 731
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The molecular size of the expressed protein and its composition should be established by techniques 732
such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver 733
staining under reducing and non-reducing conditions or N-terminal sequencing by Edman 734
degradation method. 735
736
The identity of the protein should be established by peptide mapping and/or terminal amino acid 737
sequence analysis. Following SDS PAGE, the protein bands should be identified in immunoblots 738
using specific antibodies, e.g monoclonal antibodies, to confirm the presence of the expected 739
products of the hepatitis B virus surface antigen gene. The primary structure of the protein should be 740
further characterized by suitable methods such as partial amino-acid sequence analysis and by 741
peptide mapping. Mass spectrometry may be used to confirm the average molecular mass and the 742
presence of the protein in the preparation. 743
744
Since it is known that conformational epitopes are essential for efficacy, it is essential that the 745
morphological characteristics of the HBsAg particles and degree of aggregation should be 746
determined to ensure vaccine efficacy. In addition, the protein, lipid, nucleic acid and carbohydrate 747
content should be measured when applicable. Particle characterization may be done by atomic force 748
and transmission electron microscopy, dynamic light scattering. 749
750
The gene products from lots produced during vaccine development should be shown to possess 751
antigenic determinants characteristic of HBsAg by means of tests with monoclonal antibodies or 752
polyclonal antibodies of defined specificity directed against epitopes of HBsAg known to be 753
relevant to the protective efficacy of the vaccine. 754
755
B.3 Animal models 756
There is no adequate, relevant animal model for hepatitis B infection other than the chimpanzee. The 757
efficacy of recombinant HBsAg vaccines has been demonstrated in challenge studies in this model 758
by several manufacturers and therefore, such studies are no longer required for new vaccines based 759
on the HBsAg protein. 760
761
The immunogenicity of new HBsAg vaccines and existing vaccines for which there has been a 762
significant manufacturing change should be evaluated in non-clinical (e.g. in rabbits, guinea pigs, 763
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mice and possibly non-human primates). The non-clinical program should take into account the 764
following: 765
• The titres of anti- HBsAg should be directly compared between the candidate vaccine and at 766
least one licensed comparator, preferably one for which there has been extensive clinical use 767
and generation of data supporting its effectiveness in routine use. If testing is performed due 768
to a significant change in manufacturing then the candidate vaccine should be compared with 769
the corresponding licensed vaccine. 770
• If it is proposed that a new candidate vaccine contains an adjuvant its inclusion should be 771
supported by adequate immunogenicity data that, in addition to measuring humoral antibody, 772
may include an assessment of the cellular immune response. Studies should compare the 773
adjuvanted candidate vaccine with the HBsAg alone and/or with HBsAg administered in 774
conjunction with a well established adjuvant (such as an aluminium salt). 775
• The potential need to evaluate other antibody responses and/or cellular immune responses, to 776
characterize the immune response more in depth. 777
778
B.4 Nonclinical safety studies 779
As no effects other than on the immune system are expected with hepatitis B vaccines based on the 780
absence of specific toxins, safety pharmacological studies are not required. 781
782
B.5 Toxicology studies 783
Toxicology studies should be undertaken in accordance with the WHO guidelines for nonclinical 784
evaluation of vaccines (7). Such studies should reflect the intended clinical use of the vaccine in 785
babies and young children. 786
787
If the vaccine is formulated with a novel adjuvant, nonclinical toxicology studies should be 788
conducted as appropriate for the adjuvant concerned. Repeated dose toxicity study may be used to 789
compare the safety profile of the novel adjuvant with the safety profile of an established vaccine 790
formulation taking into account existing guidelines. 791
792
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If a novel cell substrate (i.e. a substrate that had not been previously licensed or used in humans) is 793
used for the production of a hepatitis B vaccine, safety aspects, such as potential immune responses 794
elicited by residual host cell proteins, should be investigated in a suitable animal model. 795
796
Variations to the route of administration or to the vaccine formulation require evaluation of 797
immunogenicity of the hepatitis B vaccine together with adequate animal safety/toxicological 798
studies taking into account existing guidelines (7, 8). 799
800
Part C. Clinical evaluation 801
This section addresses the clinical evaluation of new hepatitis B vaccines and of existing vaccines 802
for which a significant change to the manufacturing process is proposed. The content and extent of 803
the clinical program will vary according to the each possible scenario and it is recommended that 804
vaccine-specific requirements for clinical studies are discussed with the appropriate NRAs. 805
806
C.1 Consideration for clinical studies 807
In general, clinical trials should adhere to the principles described in the WHO guidelines on good 808
clinical practice (19). General principles described in the WHO guideline on regulatory expectations 809
for clinical evaluation of vaccines (8) apply to hepatitis B vaccines and should be followed. Some of 810
the issues that are specific to the clinical development program for hepatitis B vaccines are 811
discussed in the following sections and should be read in conjunction with the general guidance 812
mentioned above. These recommendations should be viewed in the light of further data on the safety 813
and immunogenicity of hepatitis B vaccines and any relevant data on similar type of vaccines that 814
may become available in the near future. 815
816
With more than 20 years of clinical use of recombinant HBsAg vaccines in addition to the 817
experience gained with use of the early plasma-derived vaccines there is sufficient experience to 818
support the approval of new candidate vaccines (including those that may contain a novel adjuvant) 819
and major changes to manufacturing of existing vaccines based on clinical studies that assess safety 820
and immunogenicity in seronegative subjects. 821
822
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Infant immunization is the most effective strategy to prevent hepatitis B infection and this approach 823
has been incorporated in the immunization programs in over 177 countries (20). However, catch-up 824
strategies, adult vaccination and vaccination of special populations are common and studies that 825
address different types of usage are considered in the following sections. 826
827
C.2 Assessment of immune responses 828
The assessment of the immune response should be based on measurement of the anti-HBsAg 829
antibody concentration in serum using a validated and standardized assay. An International Standard 830
for hepatitis B immunoglobulin (anti-HBs) has been established and should be used in the assays to 831
determine antibody responses in immunogenicity clinical studies. 832
833
The use of validated quantitative assays is critical for the evaluation of immune responses, testing 834
should be conducted by laboratories that implement quality assurance of testing procedures. Assay 835
validation data should be reviewed and approved by the NRA. Assay validation involves 836
demonstration that the performance characteristics of the method meet the requirements for its 837
intended use (21). The protocols for assay validation studies should identify and justify the choice of 838
the parameters to be studied along with the pre-defined acceptance criteria. The validation report 839
should include a detailed description of the processing and storage of samples, reference standards 840
and reagents and generation of the calibration curve. 841
842
See section C3.3.1 regarding analysis of the immunogenicity data. 843
844
C.3 Clinical studies 845
New hepatitis B vaccines should be compared directly with at least one licensed vaccine for which 846
there is considerable clinical experience in routine use. The selection of the comparator should be 847
discussed with NRAs and should be selected taking into account the total antigen content of the 848
candidate vaccine and the study population. Where possible it is preferred that a candidate vaccine 849
(whether or not monovalent) should be compared with a monovalent licensed vaccine. However, 850
this may not be feasible in studies in infants due to the need to deliver numerous antigens 851
concomitantly using multivalent HBsAg-containing vaccines. More information regarding the 852
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clinical evaluation of hepatitis B containing combination vaccines is discussed in a separate 853
document. 854
855
In studies performed to support major changes to manufacture of a licensed vaccine the candidate 856
vaccine should be compared with the existing vaccine (i.e. manufactured according to the licensed 857
process). 858
859
New HBsAg vaccines should usually be tested initially in healthy adult volunteers. In this regard it 860
is important to take into account that the immune response to HBsAg is age dependent and 861
decreases in magnitude with increasing age of adults. After the age of 30-40 years antibody levels 862
indicative of protection are achieved after a primary vaccinations series in less than 90% of subjects 863
and in only 65–75% of vaccinees aged more than 60 years (20). Therefore studies my restrict 864
enrolment of adults by age or may employ age stratification. 865
866
Once immunogenicity is demonstrated in adults further studies may be conducted in younger target 867
populations according to the intended use (e.g. newborns, infants etc). However, this may not be 868
feasible if the recombinant HBsAg is only one component of a multivalent vaccine of content that is 869
unsuitable for adults (e.g. it contains infant doses of diphtheria and tetanus toxoids). If the latter 870
situation applies then the possible need to first assess the new recombinant HBsAg alone in adults 871
before progressing to studies with the combination vaccine in infants should be discussed with 872
NRAs. After carefully taking into account the results of non-clinical studies and similarities and 873
differences between the manufacturing process for the candidate and licensed vaccines it may not 874
always be necessary to perform such studies and it may be acceptable to go straight to the target 875
population. 876
877
The amount of recombinant HBsAg administered per dose requires justification based on non-878
clinical studies and, if necessary, formal dose-ranging studies in adults. However, these may not 879
always be necessary if the non-clinical data and mode of manufacturing are considered to support 880
the dose appropriate at least for the initial clinical studies. In this regard, it should be noted that it is 881
usual that lower doses of HBsAg are administered to subjects aged less than approximately 12-15 882
years compared to adults (e.g. half the adult dose is commonly used in infants and children up to a 883
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selected age). Therefore any new recombinant HBsAg-containing vaccine should be evaluated for 884
dose-related immunogenicity according to age. 885
886
The primary series schedule(s) that are examined will likely follow those already approved for other 887
recombinant HBsAg-containing vaccines according to specific target populations. However, if a 888
candidate vaccine is proposed to contain an antigen dose and/or an adjuvant that is considerable 889
different to licensed vaccines then a formal evaluation of schedule may be necessary according to 890
specific populations (e.g. by age and/or other host factors). 891
892
Enrolment should usually be limited to subjects who have no history of hepatitis B vaccination or 893
disease. It is preferred that studies should screen participants prior to enrolment for the presence of 894
HBsAg or anti-HBc antibody. If results of these tests are available only after the first vaccination is 895
given, any subjects with a positive result should be eliminated from the primary analysis of 896
immunogenicity. 897
898
Studies in neonates may include those born to HBsAg positive and/or negative mothers depending 899
on the study objectives and may stratify neonates accordingly. Studies in infants may be limited to 900
those born to HBsAg negative mothers (with no birth dose of hepatitis B-specific immunoglobulin) 901
with or without a prior birth dose of vaccine according to the objectives and may employ 902
stratification accordingly. 903
904
One other scenario that requires a specific clinical development program concerns vaccines that 905
contain high doses of antigen and/or an adjuvant that are intended for populations known to respond 906
poorly or not at all to standard primary courses. In such cases the studies need to be tailored 907
according to the properties of the vaccine (e.g. to justify the dose of antigen and the adjuvant content 908
in specific target populations). It may be appropriate to enroll subjects who have shown no 909
detectable antibody response to a complete primary series in order to evaluate the benefit of a higher 910
dose and/or adjuvanted vaccine. See also section C.3.3. 911
912
C.3.1 Immunogenicity endpoints 913
914
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C.3.1.1 Primary analysis 915
The protective efficacy of hepatitis B vaccines has been shown to be directly related to the induction 916
of anti-HBs antibody. An anti-HBs antibody concentration of ≥10 mIU is generally considered to be 917
a marker of protection against hepatitis B (20) and studies should determine the percentage of 918
seronegative individuals who achieve this antibody level at approximately 4 weeks after completion 919
of a primary series. In addition, studies should compare percentages that reach ≥100 mIU post-920
primary series, which may correlate with the likelihood of very long-term protection. 921
922
For the comparison between the candidate and reference vaccine the protocol and analysis plan 923
should pre-define a well-justified non-inferiority margin (22) for the comparison of the percentage 924
of subjects with ≥ 10mIU/ml anti-HBs. 925
926
C.3.1.2 Secondary analysis 927
� In all studies it is appropriate that protocols should plan at least for a secondary analysis of 928
percentages that achieve ≥ 100 mIU/ml anti-HBs and to present reverse cumulative distributions 929
(23). In addition, it is recommended that secondary analyses should compare the Geometric 930
mean titres (GMTs) between vaccines and studies may plan for a formal comparison of GMT 931
ratios. 932
933
It is expected that at least some of the clinical studies, including those in the primary target 934
population(s), should be conducted with different lots manufactured using the same process as for 935
the vaccine intended for the market. However, as indicated in the WHO Guidelines on clinical 936
evaluation of vaccines: regulatory expectations (8) a formal clinical trial to demonstrate lot-to-lot 937
consistency is not normally required unless there is a particular concern with respect to the 938
manufacturing consistency of the product and the potential impact that this may have on the efficacy 939
and safety of the vaccine. If performed, lot-to-lot consistency should be designed in accordance with 940
the principles outlined in section B.3.3.3 of the WHO Guidelines on clinical evaluation of vaccines: 941
regulatory expectations (8). 942
943
C.3.2 Persistence of anti-HBs antibody 944
Long-term observations of efficacy in various age groups have indicated that the loss of detectable 945
anti-HBs antibody in subjects who had responded satisfactorily to a primary series does not 946
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necessarily indicate lack of protection due to the effect of persistence of immune memory. That is, a 947
number of long-term follow-up studies from various epidemiological settings have confirmed that 948
HBsAg-carrier status or clinical HBV-disease rarely occurs in subjects who responded to a primary 949
series even when the anti-HBsAg concentrations decline to ≤10 mIU/ml over time (20). However, 950
the persistence of ≥ 10mIU/ml anti-HBsAg should be evaluated for any new hepatitis B vaccine. 951
The total duration of serological follow-up should be discussed and planned in advance in 952
conjunction with NRAs. 953
954
Since it is current opinion that booster doses of HBsAg may not be needed after completion of a 955
primary series (the occasional exception being routine use of a booster dose with some specific 956
vaccines in toddlers who received a primary series during infancy) it may be difficult to justify 957
studies that plan for administration of a booster dose to study subjects solely to examine immune 958
memory. However, it is of interest and potential benefit to subjects to administer a booster dose with 959
the same vaccine used for priming or with a licensed monovalent vaccine (if the original vaccine 960
used is no longer appropriate due to its total antigen content) to those who have failed to maintain ≥ 961
10mIU/ml anti-HBsAg. In these instances the titres obtained after boosting should be compared with 962
the post-primary titres. Careful attention should be paid to the documentation of safety associated 963
with booster doses. 964
965
C.3.3 Studies in special populations 966
There are several host factors that have been described in association with lack of response or poor 967
responses to hepatitis B vaccines (e.g. male gender, age over 40 years, smoking, obesity and several 968
underlying diseases that include advanced HIV infection, chronic renal failure, chronic hepatic 969
disease and diabetes). Clinical studies may be conducted to specifically assess the safety and 970
immunogenicity of new recombinant HBsAg-containing vaccines in populations at risk of not 971
responding adequately to vaccination. The design of such studies should take into consideration the 972
potential need for a higher antigen dose and/or adjuvant. 973
974
C.3.4 Concomitant administration with other vaccines 975
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The potential for immune interference between hepatitis B vaccines and other routine vaccines that 976
might need to be given at the same time for convenience should be investigated in order to make 977
recommendations regarding concomitant use. 978
979
C.4 Post-marketing studies 980
The manufacturer has a responsibility to assess safety and effectiveness following initial approval of 981
a new hepatitis B vaccine, in particular when formulated with other components as part of a 982
combination vaccine. NRAs should ensure that adequate pharmacovigilance plans are in place 983
regarding these activities at the time of first licensure. There should be specific commitments made 984
by manufacturers to provide data to NRAs on a regular basis and in accordance with national 985
regulations. The data that are collected and submitted to the responsible NRAs should be assessed 986
rapidly so that action can be taken if there are implications for the marketing authorization. 987
988
The collection of reliable and comprehensive data on effectiveness involves close co-operation 989
between manufacturers and public health authorities. Therefore, pre- and post-approval discussions 990
between vaccine manufacturers responsible for placing the product on the market and national and 991
international public health bodies are essential for ensuring that reliable effectiveness data are 992
collected in the post-marketing period in selected countries/regions. 993
994
Part D. Guidelines for national regulatory authorities 995
D.1 General 996
The general recommendations for control laboratories given in Guidelines for national authorities on 997
quality assurance for biological products (24) should apply. These guidelines specify that no new 998
biological substance should be released until consistency of manufacturing and quality as 999
demonstrated by a consistent release of batches has been established. The detailed production and 1000
control procedures and any significant changes in them should be discussed with and approved by 1001
the NRA. For control purposes, the NRA should obtain the working reference from manufacturers 1002
1003
D.2 Release and certification 1004
A vaccine lot should be released only if it fulfils the national requirements and/or Part A of the 1005
present Recommendations. A protocol based on the model given in Appendix 1, signed by the 1006
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responsible official of the manufacturing establishment, should be prepared and submitted to the 1007
NRA in support of a request for release of vaccine for use. 1008
1009
A statement signed by the appropriate official of the national control laboratory should be provided 1010
if requested by a manufacturing establishment and should certify whether or not the lot of vaccine in 1011
question meets all national requirements, as well as Part A of these Recommendations. The 1012
certificate should also state the lot number, the number under which the lot was released, and the 1013
number appearing on the labels of the containers. In addition, the date of the last satisfactory HBsAg 1014
potency test as well as assigned expiry date on the basis of shelf life should be stated. A copy of the 1015
official national release document should be attached. The certificate should be based on the model 1016
given in Appendix 2. The purpose of the certificate is to facilitate the exchange of recombinant 1017
hepatitis B vaccines between countries. 1018
1019
Authors and Acknowledgements 1020
The first draft of this document was prepared by Dr Morag Ferguson, National Institute of 1021
Biological Standards and Control, Potters Bar, England; Dr Maria Baca-Estrada, Immunization, 1022
Vaccines and Biologicals, World Health Organization, Avenue Appia 20, Geneva 27, CH-1211, 1023
Switzerland taking into considerations the discussions of the Working Group WHO Informal 1024
Consultation on hepatitis B vaccines made by recombinant DNA techniques 3~4 April 2008, and 1025
Informal Consultation on the potency testing of Hepatitis B vaccines Bangkok, Thailand, 18-20 1026
August 2008, and attended by the following participants: 1027
1028
April 2008 meeting. 1029
Temporary Advisers: Dr Morag Ferguson, National Institute of Biological Standards and Control, 1030
UK; Dr Christoph Conrad, Paul-Ehrlich-Institut, Paul-Ehrlich, Germany; Dr Dokeun Kim, Korea 1031
Food & Drug Administration, Republic of Korea; Mrs. Dede Kusmiaty, National Quality Control 1032
Laboratory of Drug and Food, Indonesia; Dr Mair Powell, Medicines and Healthcare Products 1033
Regulatory Agency, UK; Dr Catherine Milne, European Directorate for the Quality of Medicines 1034
and HealthCare, France; Dr Ana Lara Sterling, Centro para el Control Estatal de la Calidad de los 1035
Medicamentos, Habana, Cuba; Dr Richard Gibert, Agence Française de Sécurité Sanitaire des 1036
Produits de Santé, France; Representatives from Developing Country Vaccine Manufacturer's 1037
Network (DCVMN): Mr. Li Jin, Beijing Tiantan Biological Product Co. Ltd. China; Dr Yong Park, 1038
Berna Biotech Korea Corp, Republic of Korea; Dr K. Suresh, Serum Institute of India Ltd. India; 1039
Representatives from International Federation of Pharmaceutical Manufacturer's Association 1040
(IFPMA): Dr Prakash Bhuyan, Merck & Co., Inc. USA; Dr Michel Duchêne, GSK Biologicals, 1041
Belgium; Dr Diana Felnerova, Berna Biotech Crucell Company, Switzerland; WHO Secretariat: Dr 1042
Ivana Knezevic, Dr Nora Dellepiane, Dr Carmen Rodriguez Hernandez, Dr Jeewon Joung. 1043
1044
August 2008 meeting. 1045
Draft
Page 34
Temporary Advisers: Ms Elizabeth Ika Prawahju Arisetianingsih, National Quality Control 1046
Laboratory of Drug and Food, Indonesia; Dr Maria Baca-Estrada, Health Canada, Canada; Dr 1047
Nguyen Huy Cuong, National Institute of Quality Control for Vaccines and Biologicals, Viet Nam; 1048
Dr Roland Dobbelaer, WHO Advisor, Belgium; Dr Morag Ferguson, National Institute of Biological 1049
Standards and Control, UK; Mrs Teeranart Jivapaisarnpong, Ministry of Public Health, Thailand; 1050
Mr Dokeun Kim, Korea Food & Drug Administration, Republic of Korea; Prof. Summana 1051
Khomvilai, Queen Saovabha Memorial Institute, Thailand; Ms Corné Kleyn, South African NCL; 1052
South Africa; Dr Zhenglun Liang, National Institute for the Control of Pharmaceutical & Biological 1053
Products, China; Dr Alexandrine Maes, Scientific Institute of Public Health, Belgium; Dr Catherine 1054
Milne, European Directorate for the Quality of Medicines, Council of Europe, France; Dr Sylvie 1055
Morgeaux, Agence Française de Sécurité Sanitaire des Produits de Santé, France; Dr Puntawit 1056
Natakul, Ministry of Public Health, Thailand; Dr Saeed-Reza Pakzad, Food and Drugs Control 1057
Laboratory, Ministry of Health and Medical Education, Iran; Dr Ana Lara Sterling, Centro para el 1058
Control Estatal de la Calidad de los Medicamentos, Cuba; Professor Nguyen Thu Van, National 1059
Institute of Hygiene and Epidemiology, Viet Nam; Representatives from Developing Country 1060
Vaccine Manufacturer's Network (DCVMN): Ms Iin Susanti Budiharto, Biofarma, Indonesia; Dr 1061
Revathi J. Chaganti, Shantha Biotechnics Limited, India; Mr K. Gopinathan, Bharat Biotech 1062
International Limited, India; Lic. Mabel Izquierdo Lopez, Centro de Ingenieria Genetica y 1063
Biotecnologia, Cuba; Dr Jung Jin, Lee, LG Life Sciences, Ltd., Republic of Korea; Mr Jin Yong 1064
Park, Berna Biotech Korea Corp., Republic of Korea; Munukutla Subramanya Rama Sarma, Indian 1065
Immunologicals Limited, India; Dr Jaspreet Singh, Biological E. Ltd., India; Mr Anil Sood, Panacea 1066
Biotec Ltd., India; Dr K. Suresh, Serum Institute of India Ltd., India; Elizabeth Wunsch, The Biovac 1067
Institute, South Africa; WHO Secretariat: Dr Nora Dellepiane, Dr Jeewon Joung, Dr Alireza 1068
Khadem, Mr Dinesh Kumar, Ms Carmen Amelia Rodriguez. 1069
1070
The Second draft of these recommendations was prepared by Dr Maria Baca-Estrada, Dr Morag 1071
Ferguson and Dr Mair Powell taking into consideration comments received on the first draft and 1072
during the Hepatitis B vaccine section of the WHO Informal Consultation on Acellular Pertussis, 1073
DTwP, Hepatitis B and Combination Vaccines held in Geneva on 13 November 2009 attended by 1074
the following participants: 1075
1076
Temporary Advisers: Dr Christoph Conrad, Paul-Ehrlich-Institut, Paul-Ehrlich, Germany; Dr Roland 1077
Dobbelaer, WHO Adviser, Belgium; Dr Morag Ferguson, WHO Adviser, UK; Dr Richard Gibert, 1078
Agence Française de Sécurité Sanitaire des Produits de Santé, France; Dr Peter Hubrechts, Staten 1079
Serum Institut, Denmark; Mrs Teeranart Jivapaisarnpong, Department of Medical Sciences, 1080
Ministry of Public Health, Thailand; Dr Dede Kusmiaty, National Quality Control Laboratory of 1081
Drug and Food, Indonesia; Dr Alexandrine Maes, Scientific Institute of Public Health, Belgium; Dr 1082
Bruce Meade, WHO Adviser, USA; Dr Saeed-Reza Pakzad, Food and Drug Control Laboratory, 1083
Iran; Dr Hyun Chul Song, Korea Food & Drug Administration, Republic of Korea; Dr Ana Lara 1084
Sterling, Centro para el Control Estatal de la Calidad de los Medicamentos, Cuba; Dr Dianna 1085
Wilkinson, National Institute for Biological Standards and Control, UK; Representatives from 1086
Developing Country Vaccine Manufacturer's Network (DCVMN): Dr Penmetsa V.V. S. Murthy, 1087
Biological E Limited, India; Dr V.K. Srinivas Bharat Biotech International Ltd, India; Prof 1088
Xiaoming Yang, Wuhan Institute of Biological Products, China; Representatives from International 1089
Federation of Pharmaceutical Manufacturer's Association (IFPMA): Dr Jeanne-Marie Jacquet, 1090
GlaxoSmithKline Biologicals, Belgium; Mr Luciano Nencioni, Crucell, Switzerland; Ms Isabelle 1091
Pierard, GlaxoSmithKline Biologicals, Belgium; Ms Sylvie Uhlrich, Sanofi Pasteur, France; WHO 1092
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Secretariat: Dr Maria Baca-Estrada, Dr Nora Dellepiane, Dr Ivana Knezevic, Dr Dianliang Lei, Dr 1093
Sergio Nishioka, Dr Carmen Rodriguez, Dr David Wood. 1094
1095
Acknowledgements are also due to the following experts for their helpful comments: 1096
Dr Stephen Feinstone, US Food and Drug Administration; Dr Steven Wiersma, Expanded Program 1097
on Immunization, WHO. 1098
1099
1100
References 1101
1. WHO Requirements for hepatitis B vaccines WHO Technical Report Series No. 658, Annex 4. 1102
1103
2. WHO Requirements for hepatitis B vaccines prepared from plasma. WHO Technical Report 1104
Series No. 771, Annex 8. 1105
1106
3. Report of a WHO meeting on hepatitis B vaccines produced by DNA techniques. WHO 1107
Technical Report Series No. 760, Annex 5. 1108
1109
4. WHO Requirements for hepatitis B vaccines made by recombinant DNA techniques in yeast. 1110
WHO Technical Report Series No. 760, Annex 6. 1111
1112
5. WHO Requirements for hepatitis B vaccines made by recombinant DNA techniques. WHO 1113
Technical Report Series No. 786, Annex 2. 1114
1115
6. WHO Requirements for hepatitis B vaccines made by recombinant DNA techniques 1116
(amendment). WHO Technical Report Series No. 889, Annex 4. 1117
1118
7. WHO Guidelines on nonclinical evaluation of vaccines. WHO Technical Report Series No. 1119
927, Annex 1. 1120
1121
8. WHO Guidelines on clinical evaluation of vaccines: regulatory expectations. WHO 1122
Technical Report Series No. 924, Annex 1. 1123
1124
9. WHO Guidelines on good manufacturing practices for pharmaceutical products. WHO 1125
Technical Report Series, No. 823. 1126
1127
10. WHO Guidelines on good manufacturing practices for biological products. WHO Technical 1128
Report Series, No. 822. 1129
1130
11. WHO General requirements for the sterility of biological substances. WHO Technical 1131
Report Series, No. 530, Annex 4. 1132
1133
12. WHO Requirements for the use of animal cells as in vitro substrates for the production of 1134
biologicals. WHO Technical Report Series No. 878, Annex 1 1135
1136
13. Guideline on viral safety evaluation of biotechnology products derived form cell lines of 1137
human or animal origin. International Conference on Harmonization (ICH) Guideline Q5A. 1138
Draft
Page 36
1139
14. WHO Guidelines for assuring the quality of pharmaceutical and biological products prepared 1140
by recombinant DNA technology for recombinant cells. WHO Technical Report Series 1141
No.814, Annex 3. 1142
1143
15. WHO Recommendations for production and control of poliomyelitis vaccine (oral). WHO 1144
Technical Report Series No.904, Annex 1, Appendix 1. 1145
1146
16. WHO Guidelines on transmissible spongiform encephalopathies in relation to biological and 1147
pharmaceutical products. World Health Organization, 2003. 1148
1149
17. WHO Requirements for the collection, processing and quality control of blood, blood 1150
components and plasma derivatives. WHO Technical Report Series No.840, Annex 2. 1151
1152
18. WHO Guidelines on stability evaluation of vaccines. WHO Technical Report Series (in press) 1153
1154
19. WHO Guidelines for good clinical practice (GCP) for trials on pharmaceutical products. 1155
WHO Technical Report Series, No. 850, Annex 3. 1156
1157 20. Hepatitis B vaccines – WHO position paper Weekly epidemiological record No. 40, 2009, 1158
84, 405–420 (http://www.who.int/wer). 1159
1160 21. Guideline on validation of analytical procedures: Text and Methodology. International 1161
Conference on Harmonization (ICH) Guideline Q2 (R1). 1162
1163 22. European Medicines Agency Guideline on the choice of the non-inferiority margin. 1164
EMEA/CPMP/EWP/2158/99. 1165
1166
23. George F. Reed GF, Meade BD, Steinhoff MC. The reverse cumulative distribution plot: a 1167 graphic method for exploratory analysis of antibody data. Pediatrics, 1995, 96: 600-603. 1168
1169 24. WHO Guidelines for national authorities on quality assurance for biological products. WHO 1170
Technical Report Series No. 822, Annex 2. 1171
1172 25. Chovel, ML; Fernandez de Castro, A. Comparison between in vitro potency tests for Cuban 1173
Hepatitis B vaccine: contribution to the standardization process. Biologicals, 2004, 32:171-1174
176. 1175
1176
26. Giffroy D, Mazy C, Duchene M. Validation of a new ELISA method for in vitro potency 1177
assay of Hepatitis B-containing vaccines. Pharmeuropa Bio, 2006, 1:7-13. 1178
1179
27. Chovel, ML, Sterling, AL, Nicot, IA, Rodriguez, MG, Garcia OR. Validation of a new 1180
alternative for determining in vitro potency in vaccines containing Hepatitis B from two 1181
different manufacturers Biologicals, 2008, 36:375 -382. 1182
1183
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Appendix 1 1184
1185
Methodological considerations. Potency tests for recombinant hepatitis B 1186
vaccines. 1187
1188
Background 1189
Recombinant hepatitis B vaccines were licensed in the mid-1980s. WHO published requirements for 1190
hepatitis B vaccines produced by recombinant DNA techniques in yeast and mammalian cells in 1191
1989 (5) and these were revised to include the use of in vitro potency tests in 1997 (6). 1192
1193
Because of the diversity in the reactivity of vaccines containing HBsAg produced by different 1194
manufacturing processes and to which different adjuvants or immunostimulants have been added, 1195
recombinant hepatitis B vaccines produced by different manufacturers must be considered as 1196
different products. In view of such differences, the establishment of an International Standard that 1197
would be suitable for the standardization of assays of vaccines from all manufacturers is unlikely to 1198
succeed. Furthermore, the stability of such a vaccine is unlikely to be adequate for long term use and 1199
in the calibration of secondary standards. Manufacturers therefore establish a product specific 1200
reference preparation which is traceable to a lot of vaccine shown to be efficacious in clinical trials. 1201
This vaccine will serve as a working standard and be included in all potency tests. The NRA 1202
approves the reference preparation used and the potency limits applied. The performance of this 1203
reference vaccine should be monitored by trend analysis using control charts of relevant test 1204
parameters (e.g. ED50 for in-vivo assays,) and it should be replaced when necessary. 1205
1206
Potency tests 1207
Potency tests should reflect the activity of the vaccine and should be able to distinguish vaccines of 1208
low potency which may be of reduced immunogenicity in humans. At the time when the WHO 1209
requirements for recombinant hepatitis B vaccines were published it was considered that assays that 1210
determine the HBsAg content of adjuvanted vaccines would be difficult to standardize. Therefore, it 1211
was proposed that immunogenicity in mice should form the basis for determining vaccine potency 1212
by comparing the antibody response induced by the test and the vaccine reference preparation and 1213
that the specification for potency should be approved by the NRA. 1214
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1215
Several manufacturers have since developed and validated in vitro potency tests which are suitable 1216
for use with their individual vaccines. As a result of the implementation of the in vitro test, the 1217
mouse potency test is no longer being performed by these manufacturers on every final lot. Since the 1218
vaccines in question were well established and had been used in millions of individuals, an 1219
amendment to the Requirements was published in 1997 (6) to permit the use of a validated in vitro 1220
test to determine vaccine potency as one parameter to monitor consistency of production with 1221
specifications approved by the NRA. 1222
1223
The in vivo assay should be used to establish consistency of production of a new hepatitis B vaccine 1224
and in vaccine stability studies. In addition, the in vivo potency test should be used to characterize 1225
the vaccine after significant changes in the manufacturing process. 1226
1227
In vivo potency tests 1228
A suitable quantitative potency test in mice is as follows. Groups of 10-20 mice, five weeks of age 1229
or weight range (17-22 g),, are immunized intraperitoneally with a series of at least three dilutions of 1230
the reference and test vaccine using a suitable diluent. Some manufacturers use a diluent which 1231
contains the same concentration of alum as the vaccine. The strain of mice used for this test must 1232
give a suitable dose-response curve with the reference and test vaccine. The concentrations of 1233
vaccine tested should be selected to permit the calculation of 50% seroconversion to antibodies 1234
against HBsAg. The ED50 for both test and reference vaccine should lie within the doses 1235
administered. Terminal bleeds are taken after 28 or 42 days or when an adequate antibody response 1236
has developed. Individual sera are assayed for antibodies to HBsAg 1237
1238
Points which should be considered in establishing such an assay are: 1239
• Strain and sex of mice used must give a suitable dose response to the reference and test 1240
vaccine. 1241
• Number of mice per dilution required to meet the validity criteria of the test. 1242
• Nature and composition of the diluent used to prepare the dilutions of the test vaccine (e.g. 1243
containing the adjuvant at the same concentration as used in the vaccine). 1244
• Number of dilutions and appropriate selection of doses to be tested. 1245
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• The concentrations of vaccine tested should be selected to permit the calculation of the 1246
dilution giving 50% seroconversion (i.e. ED50). 1247
• Assay used to determine the concentration of antibodies to HBsAg in sera. 1248
• Calculation of the cut-off value (threshold). The value is either calculated from the responses 1249
of control group of mice immunized with diluent (e.g. mean OD of negative control + 2 1250
standard deviations) or using a threshold expressed in mIU/ml as an arbitrary value eg 10 1251
mIU/ml which is the level indicative of seroprotection in humans.,. It is important that the 1252
choice of cut-off generates an optimal dose response with the specific dilution range. 1253
• Statistical approach used to analyse the results (e.g. probit). 1254
• Interpretation of results - ED50/relative potency 1255
• The establishment of an in-house mouse anti-HBsAg reference serum or panel of high, 1256
medium and low titre sera could be used to monitor kit performance and assist in the 1257
evaluation of new kits or comparison of results from different laboratories (e.g. manufacturer 1258
and NCL) 1259
• The 95% confidence limits of the potency estimates for each test vaccine should fall in the 1260
range 33 - 300%. 1261
1262
In vitro potency tests 1263
Several manufacturers have validated in vitro potency tests based on the determination of HBsAg in 1264
dilutions of the vaccine using a commercial detection kit or another method which quantifies the 1265
HBsAg antigen content present in the vaccine. Two manufacturers have described a method based 1266
on an inhibition approach in which dilutions of the vaccine are incubated with a fixed amount of 1267
polyclonal anti-HBsAg antibodies where detection of the unbound antibody is directly related to the 1268
amount of HBsAg in the vaccine (25, 26, 27). Whichever type of assay is used, the validation studies 1269
should show that the assay is suitable to verify consistency of production. 1270
1271
In vitro potency tests should be able to distinguish vaccine of low potency, which may include 1272
vaccines of low immunogenicity in humans (e.g. lots tested during dose finding clinical studies) and 1273
vaccine samples with artificially reduced potency obtained following incubation for 7 days or 15 1274
days at 60°C or by incubation overnight at 37°C with 100 ppm of hydrogen peroxide (26, 27). 1275
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Although vaccines which have been frozen are known to be of low immunogenicity in man due to 1276
an effect on the adjuvant, such vaccines may not necessarily give low potencies in in vitro assays. 1277
1278
When a manufacturer introduces a change in the test method, this must be fully validated according 1279
to the ICH guidelines and the test run in parallel with the previous assay so that any potential 1280
variation are detected and managed appropriately. 1281
1282
Important parameters to determine during validation include the assessment of commercial lots and 1283
reference vaccine concurrently at optimal dilutions to produce dose-response curves suitable for 1284
quantitative analysis by an appropriate statistical method. The statistical validity of the assay should 1285
be assessed, the potency of the test relative to the reference vaccine should be estimated and the 1286
assay's precision (i.e. confidence interval) should be calculated. The 95% confidence limits of the 1287
potency estimates for each test vaccine should fall in the range 80 - 125%. Acceptance criteria for 1288
the in vitro assay should be established based on the assay of a suitable number of consecutive final 1289
lots. 1290
1291
The vaccine formulation may influence the parameters used in the assay, therefore, it is 1292
recommended that optimization of the assay is carried out when a new formulation is tested (e.g. 1293
when the level of preservative is modified). Each manufacturer should set a specification for the in 1294
vitro test which ensures that vaccines that pass this test would also pass the mouse immunogenicity 1295
test. 1296
1297
Several factors must be considered when validating an assay, namely 1298
• The type of commercial kit used, where the type of HBsAg used may differ from the 1299
vaccine HBsAg (e.g. manufacturing process, expression system etc). 1300
• The adjuvant used in the vaccine and the possible need for a pre-treatment step (e.g. with 1301
detergent). 1302
• The reference preparation used (e.g. monovalent HBsAg, combination vaccine etc). 1303
• Nature of the diluent used to prepare the dilutions of the test and reference vaccine. 1304
• Statistical approach use to analyse the results. 1305
• Establishment of test specification based on data from assays on a series of typical 1306
production batches of vaccines which pass the mouse immunogenicity test. 1307
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1308
The validation of an in vitro potency test should be based on ICH principles (ICH Topic Q 2 1309
CPMP/ICH/381/95) and include: 1310
• Specificity 1311
• Accuracy 1312
• Precision (including repeatability, intermediate precision and reproducibility) 1313
• Linearity 1314
• Range (limits of quantification) 1315
• Robustness should be documented during assay development 1316
1317
In vitro assay for antigen quantification of aqueous bulk (purified non-adjuvanted antigen). 1318
The same in vitro assay used to determine vaccine potency is generally used to determine HBsAg 1319
content of the aqueous bulk. Therefore, it is important to minimize the impact of changes in 1320
commercial kits and to use appropriate reference preparations. This reference material could be a 1321
representative bulk of known HBsAg protein content or a highly purified preparation of HBsAg of 1322
known protein content stored in single use aliquots. It is important to note that the reference 1323
materials based on adjuvanted product is not suitable for use in assays of non-adjuvanted 1324
intermediate bulks of HBsAg. Although formulation of final vaccine bulk is generally based on 1325
protein content, some manufacturers have chosen to use HBsAg content for this in-process step and 1326
the standardization and monitoring of this assay is therefore critical. 1327
1328
Establishment of product-specific reference 1329
The vaccine potency (in vivo and in vitro) should be assessed against a product-specific reference 1330
preparation. The first (primary) reference preparation should be established using a vaccine lot 1331
found to be effective and safe in clinical trials or alternatively a vaccine lot that is traceable to 1332
vaccine lot of proven effectiveness and safety. Points to consider when establishing a product-1333
specific reference preparation include: 1334
• Source 1335
• Quantity (availability) 1336
• Full characterisation 1337
• Evaluation in the mouse potency test 1338
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• Evaluation in an in vitro potency test 1339
• Stability studies (accelerated degradation and real time stability) 1340
• Establishment of control charts to monitor reference performance. 1341
1342
Replacement of product-specific reference 1343
Standards should be routinely monitored and they should be replaced before they begin to show loss 1344
of activity. The shelf-life of standards may be longer than the shelf life of vaccine for routine use if 1345
data to demonstrate stability of an individual vaccine for this period are available. The shelf-life 1346
should be established under the defined storage conditions and maintenance of sterility. A 1347
replacement working standard should be a typical batch of vaccine preferably of similar potency to 1348
the previous standard. 1349
1350
Points to consider in the establishment and replacement of a reference vaccine: 1351
• Documentation of the procedure for replacing standards. 1352
• Information of product/reference stability and establishment of shelf-life. 1353
• Procedure to monitor loss of potency (e.g. trending of relevant values such as changes in 1354
dose response curves, changes of values compared to an internal reference preparation such 1355
as a non-adjuvanted stable HBsAg etc). 1356
• Definition of acceptable limits of trended values (e.g. mean initial potency minus 3 standard 1357
deviations). 1358
• Review of batch record of new reference vaccine to ensure it complies with the 1359
specifications in the marketing authorisation. 1360
• Calibration of new reference vaccine against current reference using both in vivo and in vitro 1361
tests. 1362
1363
Issues relating to potency tests on the hepatitis B component of combination vaccines 1364
1365
Optimization of in vitro assays should be undertaken when used with combination vaccines 1366
containing HBsAg as there is evidence that some vaccine components may interfere in such tests 1367
(ref). If an in vitro assay is not suitable for a particular combination, an in vivo assay should be used. 1368
This should be performed at the level of the final bulk. 1369
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1370
If a monovalent hepatitis B vaccine is used in in vivo potency assays of combination vaccines, 1371
consideration must be given to the adjuvant effect of whole cell pertussis and whether the release 1372
specification applied to monovalent vaccines is applicable. 1373
1374
1375
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Appendix 2 1376
1377
Summary protocol for manufacturing and control of hepatitis B vaccines. 1378
1379 The following protocol is intended for guidance, and indicates the information that should be 1380
provided as a minimum by the manufacturer to the NRA. 1381
Information and tests may be added or deleted if necessary , to be in line with the marketing 1382
authorization approved by the NRA. 1383
1384
It is thus possible that a protocol for a specific product may differ in detail from the model provided. 1385
The essential point is that all relevant details demonstrating compliance with the license and with the 1386
relevant WHO recommendations of a particular product should be given in the protocol submitted. 1387
1388
The section concerning the final product must be accompanied by a sample of the label and a copy 1389
of the leaflet that accompanies the vaccine container. If the protocol is being submitted in support of 1390
a request to permit importation, it should also be accompanied by a lot release certificate from the 1391
NRA of the country in which the vaccine was produced/released stating that the product meets 1392
national requirements as well as Part A recommendations of this document published by WHO. 1393
1394
1395
1. Summary information on the finished product (final vaccine lot)
International name:
Trade name:
Batch number(s):
Finished product (final vaccine lot)
Final vaccine bulk:
Type of container:
Total number of containers in this batch:
Number of doses per container:
Composition (antigen concentration) / volume
of single human dose:
Date of last potency test by the manufacturer
Date of expiry:
Storage temperature:
Product license (marketing authorization)
number:
Marketing authorization issued by --------------------------------------------
Name and address of manufacturer:
Name and address of product license holder if
different:
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2. Production information
Batch number(s) of aqueous bulk(s):
Site(s) of manufacture of aqueous bulk(s):
Date of manufacture of aqueous bulk:
Site of manufacture of finished product (final
vaccine lot)
Date of manufacture of finished product (final
vaccine lot)::
1396
A genealogy of the lot numbers of all vaccine components used in the formulation of the final 1397
product will be informative. 1398
1399
The following sections are intended for the reporting of the results of the tests performed during the 1400
production of the vaccine 1401
1402
3. Starting materials
The information requested below is to be presented on each submission. Full details on
Master and working seed-lots and cell banks upon first submission only and whenever a
change has been introduced.
Cell banks Source of HBsAg (expression system) Master cell bank (MCB) lot number & preparation
date: Population doubling level (PDL) of MCB Date of approval of protocols indicating
compliance with the requirements of the relevant
monographs and with the marketing authorisation:
Manufacturer’s working cell bank (MWCB) lot
number & preparation date: Population doubling level (PDL) of MWCB
Date of approval of protocols indicating
compliance with the requirements of the relevant
monographs and with the marketing authorisation:
Production cell lot number: Storage condition: __________________________
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Identification of cell substrate Method: Specification: Date of test : Result:
Nature and concentration of antibiotics or selecting
agent (s) used in production cell culture
maintenance medium:
Identification and source of starting materials used
in preparing production cells including excipients
and preservatives (particularly any materials of
human or animal origin e.g. albumin; serum):
4. Fermentation
Mammalian cells
Provide information on cells corresponding to each single harvest.
Ratio or proportion of control to production cell
cultures: Volume of control cells: Period of observation of cultures: Percentage rejected for non-specific reasons: Result: Haemadsorbing viruses
Type(s) of RBC: Storage time and temperature of RBC: Incubation time and temperature of RBC: % cultures tested: Date of start of test : Date of end of test : Result:
Tests on supernatant fluids for other adventitious
agents (if relevant) Date of sampling from production cell
cultures: Type of simian cells:
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Quantity of sample inoculated: Incubation temperature: Date of start of test : Date of end of test : % of viable culture at the end Result:
Type of human cells:
Quantity of sample inoculated: Incubation temperature: Date of start test: Date of end of test: % of viable culture at the end: Result:
Type(s) of other diploid cells:
Quantity of sample inoculated: Incubation temperature: Date of start of test: Date of end of test : % of viable culture at the end: Result:
Mammalian and yeast cells
Bacteria and fungi
Method Media and temperature of incubation: Volume inoculated: Date of inoculation : Date of end of observation : Result:
Mycoplasmas (for mammalian cells) Method: Media: Volume inoculated: Date of start of test : Date of end of test : Result:
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5. Single harvests (or pools) Batch number(s): Date of inoculation: Date of harvesting: Volume(s) of fermentation paste, storage
temperature, storage time and approved storage
period: Culture purity or sterility for bacteria and fungi
Method: Media and temperature of incubation: Volume inoculated: Date of start of test : Date of end of test : Result:
Plasmid retention
Method: Specification: Date of test: Result:
In addition, the following tests if mammalian cells are used
Adventitious agents
Method: Specification: Date of test: Result:
Mycoplasmas
Method: Media: Volume inoculated: Date of start of test : Date of end of test : Result:
Mycobacterium spp. (if applicable) Method:
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Media and temperature of incubation: Volume inoculated: Date of start of test : Date of end of test : Result:
Reverse transcriptase assay Method: Specification: Date of test: Result:
6. Control of aqueous bulk (purified antigen) Batch number(s) of purified bulk: Date(s) of purification(s): Volume(s), storage temperature, storage time and
approved storage period: Purity (add PAGE photographs)
Method: Specification: Date of test: Result:
Protein content Method: Specification: Date of test: Result:
HBsAg Antigen content / Identity Method: Specification: Date of test: Result:
Ratio of HBsAg antigen : protein content Specification: Result:
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Bacteria and fungi
Method: Media and temperature of incubation: Volume inoculated: Date of start of test : Date of end of test : Result:
Potential hazards e.g. residual chemical(s) (if relevant)
Method: Specification: Date of test: Result:
Lipid Method: Specification: Date of test: Result: Carbohydrate Method: Specification: Date of test: Result:
Residual DNA (if applicable) Method: Specification: Date of test: Result:
Bacterial endotoxins
Method: Specification: Date of test: Result:
Albumin content (if mammalian cells and animal serum are used for production)
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Method: Specification: Date of test: Result:
7. Final vaccine bulk
Batch number(s) of aqueous bulk:
Formulation date: Batch number(s) of all components used during
adjuvant formulation: Volume, storage temperature, storage time and
approved storage period: Bacteria and fungi
Method: Media and temperature of incubation: Volume inoculated: Date of start of test : Date of end of test : Result:
Identity
Method: Specification: Date of test: Result:
Adjuvant or mineral vehicle concentration (if applicable)
Method: Specification: Date of test: Result:
Degree of adsorption (if applicable) Method: Specification: Date of test: Result:
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pH
Method: Specification: Date of test: Result:
Preservative Method: Specification: Date of test: Result:
Potency test: in vivo assay (if applicable)
Species, strain, sex and weight specifications: Dates of vaccination, bleeding: Date of assay: Batch number of reference vaccine and
assigned potency: Vaccine doses (dilutions) and number of
animals responding at each dose: ED50 of reference and test vaccine: Potency of test vaccine vs. reference vaccine
with 95% fiducial limits of mean:
Validity criteria:
8. Final vaccine lot
Batch number: Date of filling: Type of container: Filling volume: Number of containers after inspection: Appearance
Method: Specification: Date of test: Result:
Identity
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Method: Specification: Date of test: Result:
Bacteria and fungi
Method Media and temperature of incubation: Volume inoculated: Date of start of test : Date of end of test : Result:
pH
Method: Specification: Date of test: Result:
Osmolality Method: Specification: Date of test: Result:
Preservatives (if applicable)
Method: Specification: Date of test: Result:
Pyrogenic substances
Method: Specification: Date of test: Result:
Adjuvant content
Method: Specification:
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Date of test: Result:
Protein content (or calculated value)
Method: Specification: Date of test: Result:
Degree of adsorption (if applicable)
Method: Specification: Date of test: Result:
Potency:
In vitro assay Method: Batch number of reference vaccine and
assigned potency: Specification: Date of assay: Result:
If an in vivo assay is used (may be performed at
final bulk stage) Species, strain, sex and weight specifications: No. of mice tested Dates of vaccination, bleeding: Date of assay: Batch number of reference vaccine and
assigned potency: Vaccine doses (dilutions) and number of
animals responding at each dose: ED50 of reference and test vaccine Potency of test vaccine vs. reference vaccine
with 95% fiducial limits of mean: Validity criteria: Date of start of period of validity:
General safety (unless deletion authorised)
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Test in mice
No. of mice tested : Volume and route of injection: Date of injection: Date of end of observation: Specification: Result:
Test in guinea-pigs No. of guinea-pigs tested: Volume and route of injection; Date of injection: Date of end of observation: Specification : Result: Freezing point (if applicable)
Method: Specification: Date of test: Result:
1403
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Appendix 3 1404
Model certificate for the release of recombinant hepatitis B vaccine by a national 1405
regulatory authority 1406
1407
This certificate is to be provided by the national regulatory authority of the country where the 1408
vaccines have been manufactured, upon request by the manufacturer 1409
1410
Certificate No________________ 1411
1412
LOT RELEASE CERTIFICATE 1413
1414
The following lot(s) of recombinant hepatitis B vaccine produced by 1415
______________________1 in _______________
2, whose numbers appear on the labels of the final 1416
containers, meet all national requirements3 and Part A
4 of the WHO recommendations to assure the 1417
quality, safety and efficacy of recombinant hepatitis B vaccines (_____)5, and comply with Good 1418
Manufacturing Practices for Pharmaceutical Products: Main Principles6 and Good Manufacturing 1419
Practices for Biological Products7. 1420
As a minimum, this certificate is based on examination of the summary protocol of 1421
manufacturing and control. 1422
1423
Final Lot No. No. of released human doses
in this final vaccine lot Expiry date
________________
________________
________________
1424
The Director of the National Regulatory Authority (or Authority as appropriate): 1425
1426
Name (Typed)
Signature
Date
1427
1 Name of manufacturer 1428
2 Country of origin 1429
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3 If any national requirements are not met, specify which one(s) and indicate why release of the lot(s) 1430
has nevertheless been authorized by the national regulatory authority 1431
4 With the exception of provisions on distribution and shipping, which the national regulatory 1432
authority may not be in a position to assess. 1433
5 WHO Technical Report Series, No. ___, YYYY, Annex __. 1434
6 WHO Technical Report Series, No. 908, 2003, Annex 4. 1435
7 WHO Technical Report Series, No. 822, 1992, Annex 1. 1436
1437
1438
1439 1440