recommendations to assure the quality, safety and efficacy ......1 2 draft may 27, 2010 3 english...

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

Draft

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

Draft

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

Draft

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

Draft

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

Draft

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

Draft

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

Draft

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

Draft

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

Draft

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

Draft

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

Draft

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

Draft

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

Draft

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

Draft

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

Draft

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

Draft

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

Draft

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

Draft

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

Draft

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

Draft

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



657

A.12 Labelling 658


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

Draft

A.13 Distribution and shipping 671



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

Draft

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

Draft

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

Draft

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

Draft

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

Draft

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

Draft

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

Draft

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

Draft

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

Draft

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

Draft

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

Draft

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

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

Draft

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


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


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

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


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

Draft

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

Draft

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

Draft

• 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

Draft

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

Draft

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

Draft

• 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

Draft

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

Draft

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:

Draft

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: __________________________

Draft

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:

Draft

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:

Draft

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


Mycoplasmas

Method: Media: Volume inoculated: Date of start of test : Date of end of test : Result:

Mycobacterium spp. (if applicable) Method:

Draft

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)


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:

Draft

Bacteria and fungi


Potential hazards e.g. residual chemical(s) (if relevant)


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


Albumin content (if mammalian cells and animal serum are used for production)

Draft


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


Identity


Adjuvant or mineral vehicle concentration (if applicable)


Degree of adsorption (if applicable) Method: Specification: Date of test: Result:

Draft

pH


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


Identity

Draft


Bacteria and fungi

Method Media and temperature of incubation: Volume inoculated: Date of start of test : Date of end of test : Result:

pH


Osmolality Method: Specification: Date of test: Result:

Preservatives (if applicable)


Pyrogenic substances


Adjuvant content

Method: Specification:

Draft

Date of test: Result:

Protein content (or calculated value)


Degree of adsorption (if applicable)


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)

Draft

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)


1403

Draft

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

Draft

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

recommendations to assure the quality, safety and efficacy ......1 2 draft may 27, 2010 3 english...

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