recommendations to assure the quality, safety and efficacy ... · 10 the named authors [or editors...

1

WHO/TCV/DRAFT/3 April 2020 2

ENGLISH ONLY 3

4

5

Recommendations to assure the quality, safety and efficacy of 6

typhoid conjugate vaccines 7

8

Replacement of WHO Technical Report Series, No. 987, Annex 3 9

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12

NOTE: 13

This document has been prepared for the purpose of inviting comments and suggestions on the 14

proposals contained therein, which will then be considered by the Expert Committee on 15

Biological Standardization (ECBS). Publication of this early draft is to provide information 16

about the proposed WHO Recommendations to assure the quality, safety and efficacy of typhoid 17

conjugate vaccines (Replacement of WHO Technical Report Series, No. 987, Annex 3) to a broad 18

audience and to improve transparency of the consultation process. 19

20

The text in its present form does not necessarily represent an agreed formulation of the Expert 21

Committee. Written comments proposing modifications to this text MUST be received by 22

6 May 2020 in the Comment Form available separately and should be addressed to the World 23

Health Organization, 1211 Geneva 27, Switzerland, attention: Department of Health Products 24

Policy and Standards. Comments may also be submitted electronically to the Responsible 25

Officer: Dr Richard Isbrucker at email: [email protected] 26

27

The outcome of the deliberations of the Expert Committee will be published in the WHO 28

Technical Report Series. The final agreed formulation of the document will be edited to be in 29

conformity with the "WHO style guide, second edition" (KMS/WHP/13.1). 30

31 © World Health Organization 2020 32

All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, World Health 33 Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail: 34 [email protected]). Requests for permission to reproduce or translate WHO publications – whether for sale or for 35 noncommercial distribution – should be addressed to WHO Press, at the above address (fax: +41 22 791 4806; e-36 mail: [email protected]). 37 The designations employed and the presentation of the material in this publication do not imply the expression of any 38 opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, 39 city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps 40 represent approximate border lines for which there may not yet be full agreement. 41 42

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WHO/TCV/DRAFT/3 April 2020

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The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or 1 recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. 2 Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. 3 4 All reasonable precautions have been taken by the World Health Organization to verify the information contained in 5 this publication. However, the published material is being distributed without warranty of any kind, either expressed or 6 implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the 7 World Health Organization be liable for damages arising from its use. 8

9 The named authors [or editors as appropriate] alone are responsible for the views expressed in this publication. 10

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Contents 1

2

3

Introduction 4

5

General considerations 6

7

Part A. Guidelines on manufacture and control 8

9

A.1 Definitions 10

A.2 Guidelines on general manufacturing 11

A.3 Control of starting material 12

A.4 Control of vaccine production 13

A.5 Filling and containers 14

A.6 Control of the final product 15

A.7 Records 16

A.8 Samples 17

A.9 Labelling 18

A.10 Distribution and shipping 19

A.11 Stability, storage and expiry date 20

21

Part B. Nonclinical evaluation of new typhoid conjugate vaccines 22

23

B.1 General principles 24

B.2 Priduct characterization and process development 25

B.3 Nonclinical immunogenicity studies 26

B.4 Nonclinical toxicity and safety 27

28

Part C. Clinical evaluation of new typhoid conjugate vaccines 29

30

C.1 General considerations for clinical trials 31

C.2 Outline of the clinical development program 32

C.3 Assessment of the immune response 33

C.4 Immunogenicity 34

C.5 Efficacy 35

C.6 Safety 36

37

Part D. Guidelines for NRAs 38

39

D.1 General guidelines 40

D.2 Official release and certification 41

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Authors and acknowledgements 1

2

References 3

4

Appendix 1: Model protocol for the manufacturing and control of typhoid conjugate 5

vaccines 6

7

Appendix 2: Model NRA Lot-release Certificate for typhoid conjugate vaccines 8

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Introduction 1

Guidelines on the quality, safety and efficacy of typhoid conjugate vaccines (TCVs) were 2

developed through a series of international consultations in 2012 and 2013 and adopted by the 3

WHO Expert Committee on Biological Standardization (ECBS) at its 64th meeting in October 4

2013. Since that time there have been several major developments with respect to typhoid 5

conjugate vaccines. These include: 6

• The establishment of WHO International Standards for Vi antigens and Vi antibodies 7

(human) 8

• The licensing of TCVs in some countries. 9

• The publication of a WHO SAGE position paper in 2018 recommending the use of 10

typhoid conjugate vaccines from 6 months to 45 years of age and that the introduction of 11

TCVs in routine immunization programmes be prioritized in countries with the highest 12

burden of typhoid disease or a high burden of antimicrobial resistant Salmonella Typhi. 13

• Gavi approval of funding support for the introduction of TCVs in Gavi eligible countries 14

starting in 2019 15

• The WHO Prequalification in 2017 of Typbar-TCV, a typhoid conjugate vaccine from 16

India. 17

18

The impact of these developments on the production and quality control of TCVs and on 19

their non-clinical and clinical evaluation is reflected in the present revision. As TCVs have been 20

licensed since the development of the original guidelines in 2013, the current document contains 21

recommendations for their evaluation rather than guiding principles. As a consequence of the 22

increasing demand for TCVs, together with the above mentioned Gavi decision on funding, new 23

vaccine developers and manufacturers are entering the field and should benefit from updated 24

WHO guidance. Further clinical evaluation of TCVs, the detailed investigation of immune 25

responses to these vaccines and the search for a true immunological correlate or surrogate of 26

protection are on-going and Part C of this document may need further updating as new data 27

become available. 28

General considerations 29

Typhoid fever is an acute generalized infection of the mononuclear phagocyte system 30

(previously known as the reticuloendothelial system), intestinal lymphoid tissue and gall bladder 31

caused by Salmonella enterica serovar Typhi (S. Typhi). Paratyphoid fever is a clinically 32

indistinguishable illness caused by S. enterica serovar Paratyphi A or B (or, more rarely, C) (1-33

3). Typhoid and paratyphoid fevers are referred to collectively as enteric fever. In most endemic 34

areas, typhoid accounts for approximately 75–80% of cases of enteric fever. However, in some 35

regions, particularly in some parts of Asia, S. Paratyphi A accounts for a relatively larger 36

proportion of all enteric fevers (4-6). 37


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Pathogen 1

2

S. Typhi is a member of the family Enterobacteriaceae. It is a Gram-negative, non-lactose 3

fermenting bacillus that produces trace amounts of hydrogen sulfide. Its antigens include an 4

immunodominant lipopolysaccharide (LPS) O9, flagellar H phase 1 antigen “d” and capsular 5

polysaccharide Vi. 6

Vi acts as a virulence factor by preventing anti-O antibody from binding to the O antigen, 7

and inhibits the C3 component of the complement system from fixing to the surface of S. Typhi 8

(7). The Vi antigen is not unique to S. Typhi – it is also expressed by S. Paratyphi C, Citrobacter 9

freundii s.l. and some clades of S. enterica serovar Dublin. The genes responsible for the 10

biosynthesis of Vi polysaccharide are located in a locus (viaB) within Salmonella pathogenicity 11

island 7 (SPI-7) in the S. Typhi chromosome. Several other loci participate in the complex 12

regulation of Vi expression. Almost all S. Typhi isolates from blood cultures express Vi. 13

Nevertheless, Vi-negative strains have been identified occasionally, both in sporadic cases as 14

well as during outbreaks (8). Some of these strains are regulatory mutants that can revert to a Vi-15

positive state (9). However, some Vi-negative isolates from blood have been shown to harbour 16

deletion mutations in critical genes (e.g. tviB) within the viaB locus that render the strains unable 17

to synthesize Vi. This raises the theoretical concern that large-scale usage of Vi-containing 18

vaccines (either polysaccharide or conjugate) could lead to selective pressure that creates a 19

biological advantage for the emergence of Vi-negative strains (10). 20

Pathogenesis 21

22

Typhoid infection begins with ingestion of S. Typhi in contaminated food or water. In the small 23

intestine, the bacteria penetrate the mucosal layer, and ultimately reach the lamina propria. 24

Translocation from the intestinal lumen mainly occurs by S. Typhi targeting M cells overlying 25

gut-associated lymphoid tissue. Within this lymphoid tissue and in the lamina propria, S. Typhi 26

invokes an influx of macrophages and dendritic cells that ingest the bacteria but fail to destroy 27

them. Thus, some bacteria remain within macrophages in the lymphoid tissue of the small 28

intestine and flow into the mesenteric lymph nodes where there is an inflammatory response 29

mediated by the release of various cytokines. Bacteria enter the bloodstream via lymphatic 30

drainage, thereby seeding organs of the mononuclear phagocyte system (such as the spleen, liver 31

and bone marrow) and gall bladder by means of a silent primary bacteraemia. After a typical 32

incubation period of 8–14 days the clinical illness begins, usually with the onset of fever, 33

abdominal discomfort and headache. An accompanying low-level secondary bacteraemia occurs. 34

Before the availability of fluoroquinolone antibiotics, clinical relapses were observed in 35

5–30% of patients treated with antibacterial agents such as chloramphenicol and 36

sulfamethoxazole/trimethoprim. These post-treatment relapses occurred when typhoid bacilli re-37

emerged from their protected intracellular niches within the macrophages of the mononuclear 38

phagocyte system, where the antibacterial agents could not penetrate. 39


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Several lines of evidence indicate that in a small proportion of patients infected with S. 1

Typhi who may have premorbid abnormalities of the gall bladder mucosa, such as occurs 2

consequent to gallstones, gall bladder infection becomes chronic (i.e. excretion lasts for longer 3

than 12 months) (11). Such chronic carriers, who are themselves not clinically affected by the 4

presence of typhoid bacilli in their system, may excrete the pathogen in their faeces for decades 5

(12). They are thought to serve as a long-term epidemiological reservoir in the community, and 6

foster transmission of typhoid wherever there is inadequate sanitation, untreated water supplies 7

or improper food handling. 8

Epidemiology 9

10

Typhoid fever is restricted to human hosts and in the late nineteenth and early twentieth century 11

was endemic in virtually all countries in Europe and the Americas. Subsequently, the 12

widespread use of chlorination, sand filtration, and other means of water treatment drastically 13

reduced the incidence of typhoid fever despite the high prevalence of chronic carriers (11). 14

Typhoid remains endemic in most developing countries and is an important public health 15

problem mainly because large segments of the population lack access to safe water and basic 16

sanitation services (13). In addition, there are limited programs for detecting carriers and 17

restricting them from handling food. 18

Disease burden 19

20

Variable estimates of the annual epidemiologic burden (incidence and total number of cases) of 21

typhoid fever have been published in the scientific literature by extrapolation of data from 22

various sources. The true incidence of typhoid fever in most regions of developing countries is 23

not known. A study published in 2004 estimated that 22 million cases occur each year, causing 24

216,000 deaths, predominantly in school-age children and young adults; the annual incidence 25

was estimated to be 10-100 per 100,000 population (14). A systematic review of population-26

based studies from 1984 through 2005 reported an annual incidence ranging from 13 to 976 per 27

100,000 persons each year based on diagnosis by blood culture (15). 28

More recent analysis shows that typhoid fever remains a major cause of enteric disease of 29

children in low and middle-income countries with global estimates of disease burden ranging 30

between 11 and 21 million typhoid fever cases and approximately 145,000 to 161,000 deaths 31

annually (16). The majority of cases occur in Asia and Sub-Saharan Africa but many of the 32

island nations of Oceania also experience moderate to high typhoid fever incidence and large 33

outbreaks (17). 34

Several factors affect the calculation of the burden of typhoid disease, one of the most 35

critical being how one confirms that a patient with acute febrile illness has typhoid fever. 36

Unfortunately, there is no rapid, affordable, accurate point-of-care or laboratory diagnostic test to 37

confirm a case of acute typhoid fever. Bone marrow culture is widely recognized as the gold 38

standard but is impractical for widespread use. Blood culture is the most practical accurate 39


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confirmatory test but blood culture alone identifies only 40 – 80% of the cases that are detectable 1

using bone marrow culture (18-20). Culture of bile containing duodenal fluid and of skin snips of 2

rose spots can be positive when blood cultures are negative (15). Prior patient treatment with 3

antibacterial agents and the volume of blood cultured also affect the yield of cultures. Relying on 4

clinical diagnosis alone is not advisable because several other febrile syndromes caused by other 5

microorganisms, such as malaria, dengue, brucellosis and leptospirosis, can be confused with 6

typhoid in both adults and children. 7

The incidence of typhoid, its age-specific distribution and the severity of clinical disease 8

gleaned from passive surveillance implemented at health facilities often appears quite different 9

from data acquired through active surveillance, during which households are visited 10

systematically once or twice weekly to detect fever among their members and mild or early 11

clinical illness is detected. In 2008, a study by Ochiai and colleagues reported the incidence of 12

typhoid detected through passive surveillance (and modified passive surveillance in two 13

countries where additional health clinics were introduced into the community) in five Asian 14

countries (21). The incidence of typhoid fever ranged from 15.3 per 100,000 person-years among 15

people aged 5–60 years in China, to 451.7 per 100,000 person-years among children aged 2–15 16

years in Pakistan (21). More recently, the incidence in Nepal ranged from 297 to 449 per 17

100,000 with greater incidences occurring during the summer months (22). Incidence data from 18

the placebo control groups in vaccine trials also provide information on the incidence of typhoid 19

fever in multiple geographical areas and venues. However, because vaccine efficacy trials are 20

typically carried out in areas with high endemicity, caution must be taken when extrapolating 21

these incidence rates to other populations. New data on age-specific occurrence in certain 22

geographic regions, as in some sites in South Asia, confirm that typhoid fever of sufficient 23

severity to seek medical care is common in the 1 to 4 year old age group with a large proportion 24

of disease occurring in children between 6 months and 2 years of age (13). There has also been 25

an increasing record of major outbreaks often associated with antimicrobial resistant S. Typhi 26

(13). Well characterized outbreaks of confirmed typhoid fever in Sub–Saharan Africa have been 27

reported (23-27), the increased occurrence of outbreaks due to multi drug resistant typhoidal 28

Salmonella serovars being of particular concern. Extensively drug-resistant variants of S. Typhi 29

have also recently emerged in India, Bangladesh and Pakistan that severely limit treatment 30

options and are leading to a situation where the disease is increasingly difficult to treat (28, 29). 31

The S. Typhi H58 clade, with IncHI1 plasmids carrying multidrug resistance genes and target 32

site mutations causing fluoroquinolone resistance is responsible for much of the recent and 33

dramatic spread of resistant strains in countries like Pakistan in 2018 (29, 30). This clade is 34

believed to have emerged on the Indian subcontinent about 30 years ago and then spread to 35

south-east Asia and more recently to Sub-Saharan Africa (29). The development of extensively 36

antibiotic resistant S. Typhi, resistant to first and second line antibiotics, and its implications for 37

disease control was reviewed in the background paper for SAGE in 2017 (16). The global pattern 38

of drug resistant S. Typhi is dynamic and changing in each location and over time. For example, 39

in Ho Chi Minh City, Vietnam, in 1998 strains with decreased susceptibility to fluoroquinolones 40

increased from less than 5% to 80% within a few months (31). The large-scale outbreak of 41

extensively drug-resistant typhoid in Pakistan demonstrates the importance of understanding the 42


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local resistance patterns to enable the selection of appropriate antibiotics for the management of 1

typhoid fever cases (29). 2

Prior to the availability of antibacterial agents, typhoid resulted in a case-fatality rate of 3

approximately 10–20% (32). Current estimates covering the post antibiotic era range from 1 to 4

4% of those who receive adequate therapy (33). Most of the mortality occurs in developing 5

countries, predominantly in Asia. A review by Crump and colleagues reported community-based 6

mortality ranging from 0–1.8% across five studies in developing countries; hospital-based 7

mortality ranged from 0–13.9% (across all ages in 12 studies); and in children younger than 15 8

years mortality ranged from 0–14.8% (across 13 studies) (15). Hospitalization rates of 2 to 40% 9

have been reported indicating that the disease can be severe in a considerable proportion of 10

patients (21). The evolution and spread of multiple antibiotic resistant S. Typhi described above 11

further complicates the situation and leads to an increased proportion of patients experiencing 12

clinical treatment failure and complications, increasing hospital admission and prolonged 13

hospital stay (16). 14

Few studies have estimated the prevalence of chronic carriers of typhoid and paratyphoid 15

in developing countries. A survey in Santiago, Chile, conducted when typhoid fever was highly 16

endemic there in the 1970s, estimated a crude prevalence of 694 typhoid carriers per 100 000 17

population (34). In Kathmandu, Nepal, among 404 patients (316 females and 88 males) with gall 18

bladder disease undergoing cholecystectomy, S. Typhi was isolated from 3.0% of bile cultures 19

and S. Paratyphi A from 2.2% (35). Since the overall prevalence of cholelithiasis in the 20

population of Kathmandu was not known, the overall prevalence of chronic carriage in that 21

population could not be calculated. The role of chronic carriers in the transmission of typhoid 22

fever is still unclear (13) but thought to vary between settings of high, medium and low disease 23

incidence (14, 36). However, chronic carriers may represent a long-term reservoir of infection 24

and contribute to the persistence of typhoid fever through on-going shedding of S. Typhi and S. 25

Paratyphi into the environment, possibly contaminating water supplies. 26

Clinical features 27

28

S. Typhi infection results in a wide spectrum of clinical features, most often characterized 29

by persisting high-grade fever, abdominal discomfort, malaise and headache. Important clinical 30

signs in hospitalized patients include hepatomegaly (41%), toxicity (33%), splenomegaly (20%), 31

obtundation (2%) and ileus (1%) (37). Before antibacterial agents became available, gross 32

bleeding from the gastrointestinal tract and perforations occurred in 1–3% of untreated patients, 33

and hospital-based reports suggest that more than 50% of patients may have serious 34

complications. In 2005, Huang and colleagues analysed in which systems various complications 35

were likely to occur: the central nervous system (3–55%), the hepatobiliary system (1–26%), the 36

cardiovascular system (1–5%), the pulmonary system (1–6%), bones and joints (less than 1%), 37

and haematological system (rarely) (38). Intestinal perforations leading to peritonitis and death 38

continue to be reported, albeit rarely, in some settings. Interestingly, the emergence of the 39

multiple drug resistant strains has been associated not only with failure to respond to antibiotic 40

treatment but also with changes in the severity and clinical profile of enteric fever (2, 39). 41


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Immune responses to natural infection 1

2

Natural typhoid infection is usually associated with the detection of serum antibodies and 3

mucosal secretory immunoglobulin A (IgA) intestinal antibody against various S. Typhi 4

antigens; cell-mediated immune responses are also measurable (40-44). In areas where typhoid is 5

endemic, there is an age-related increase in the prevalence and geometric mean titre of anti-Vi 6

antibodies (45). Anti-flagella (H antigen) serum IgG antibodies following natural infection are 7

long-lived and have been studied for sero-epidemiological surveys (46). 8

While serological responses to LPS and flagella antigens tend to be fairly strong and are 9

commonly found in patients with culture-confirmed acute typhoid fever, only about 20% of such 10

patients exhibit significant levels of anti-Vi antibody (47, 48). In contrast, high concentrations of 11

anti-Vi serum IgG antibody are detected in 80–90% of chronic carriers (47, 48). 12

Cell-mediated immunity also appears to play a part in protection – it has been observed 13

that peripheral blood mononuclear leukocytes of otherwise healthy adults residing in typhoid-14

endemic areas, who have no history of typhoid, proliferate on exposure to S. Typhi antigens (49). 15

Disease Control 16

17

Similar to other enteric and diarrhoeal diseases, typhoid fever exists predominantly in 18

populations with inadequate access to safe water and basic sanitation. Effective typhoid control 19

requires a comprehensive approach that combines immediate measures, such as accurate and 20

rapid diagnostic confirmation of infection and timely administration of appropriate antibiotic 21

treatment, as well as sustainable longer-term solutions such as providing access to safe water and 22

basic sanitation services, health education, appropriate hygiene among food handlers and typhoid 23

vaccination. There is evidence that immunization against typhoid can substantially reduce 24

typhoid fever burden when targeted towards high risk age groups and geographic areas and when 25

combined with improved sanitation (50). The most recent WHO guidelines for use of typhoid 26

vaccines were published in 2018 (13). 27

Typhoid vaccines: 28

Inactivated whole cell vaccine 29

30

Inactivated S. Typhi bacteria (heat-inactivated and phenol-preserved) were first utilized to 31

prepare parenteral vaccines more than 100 years ago. In the 1960s, WHO sponsored field trials 32

that evaluated the efficacy of inactivated parenteral whole-cell vaccines in several countries (51, 33

52), and documented a moderate level of efficacy lasting up to 7 years (53). Data from studies of 34

human immune responses and immunogenicity studies in rabbits suggested that anti-H 35

antibodies might represent an immune correlate of protection (54); later extrapolation from the 36

results of mouse protection studies suggested that responses to Vi antigen may also correlate 37

with protection (55, 56). However, these vaccines were associated with considerable rates of 38


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systemic adverse reactions (57) and as they never became widely-accepted public health tools 1

are no longer produced or marketed. 2

Live, attenuated Ty21a oral vaccine 3

4

In the early 1970s, an attenuated strain of S. Typhi was developed through chemical-induced 5

mutagenesis of pathogenic S. Typhi strain Ty2 (55). The resultant mutant strain lost the activity 6

of the epimerase enzyme encoded by the galE gene, and no longer expressed the Vi antigen. The 7

vaccine was found to be stable, safe and efficacious in adults as well as children (58-62). The 8

level of protective immunity achieved varied according to the formulation of the vaccine, the 9

number of doses administered and the interval between doses. 10

For example, three doses of a provisional formulation of vaccine or placebo administered 11

to about 32 000 children (aged 6-7 years) in Alexandria, Egypt, gave a point estimate of efficacy 12

of 95% (95% confidence interval (CI), 77-99%) during 3 years of follow-up (63). Three doses of 13

enteric-coated capsules administered to Chilean schoolchildren aged 6–19 years using two 14

different dose intervals (either alternate days or 21 days between doses) gave a point estimate of 15

efficacy of 67% (95% CI, 47–79%) during 3 years of follow-up. For the group receiving doses 16

on alternate days, the point estimate of protection over 7 years was 62% (95% CI, 48–73%) (58, 17

64). The estimate of protection was 49% (95% CI, 24–66%) with the 21-day interval between 18

doses. Another trial used four doses administered within 7 days to Chilean schoolchildren and 19

demonstrated even greater protection (65). Human challenge studies showed that 5-8 doses of 20

Ty21a oral vaccine resulted in high rates of anti-LPS antibody seroconversion and 87 % 21

protective efficacy (66). However, more recent human challenge studies showed that a three dose 22

Ty21a immunization schedule resulted in a protective efficacy of only 35% after challenge when 23

using endpoints of fever and/or bacteremia as a diagnosis of typhoid (67). When efficacy was 24

recalculated using a similar definition for typhoid diagnosis (fever with subsequent 25

microbiological confirmation) as was used in the original vaccine/challenge study, Ty21a 26

efficacy reached 80% (67), which is similar to that reported in the older challenge studies. 27

As of 2013, almost all countries where Ty21a is licensed utilize a three-dose course of 28

enteric-coated capsules taken on alternate days, except the United States and Canada, which 29

recommend a four-dose course. 30

Two other field trials in Chile (62) and Indonesia (61) compared the enteric-coated 31

capsules with three doses of the liquid formulation. The liquid formulation conferred greater 32

efficacy than the capsules in both trials. In Chile, where doses were given on alternate days, 33

results with the liquid formulation were superior to Indonesia where the doses were administered 34

1 week apart (the point estimate of efficacy in Chile was 77%; in Indonesia it was 53%). In 35

Chile, 78% protection was documented up to 5 years after vaccination with the liquid 36

formulation (64). There is also indirect evidence that large-scale vaccination with Ty21a may 37

provide some degree of protection against typhoid in people who have not been vaccinated 38

through the mechanism of herd protection. This vaccine, first licensed in Europe in 1983 and in 39

the USA in 1989, is approved for use in individuals older than 6 years. Because the vaccine is 40


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highly acid labile, stomach acidity must be neutralized or by passed when Ty21a is fed orally. 1

The previously marketed liquid formulation is no longer produced, and only enteric coated 2

capsules are currently available (13). 3

Vi polysaccharide vaccine 4

5

Technological advances in the late 1960s made it possible to purify Vi polysaccharide without 6

damaging its antigenic properties and to prepare vaccines that are almost totally free of 7

contaminating LPS (68); these vaccines are associated with low rates of febrile reactions (1–2%). 8

The Vi PS vaccine was first licensed in the USA in 1994 and since then several products have 9

been licensed for use in individuals aged 2 years and older. One product, Typhim ViTM, has been 10

prequalified by the WHO.1 11

The immunological basis of protection by purified Vi polysaccharide parenteral vaccines 12

is the generation of serum anti-Vi IgG antibodies in 85–90% of vaccine recipients older than 2 13

years. 14

Clinical trials with the vaccine showed a rise in anti-Vi antibody titres in adults and 15

children (69-71). However, subsequent inoculations with Vi did not boost the antibody response. 16

Although a single dose has been associated with the persistence of antibodies for up to 3 years in 17

some recipients, many adult recipients in non-endemic areas showed a marked drop in antibody 18

levels after 2 years (72, 73). An epidemic of typhoid fever among French soldiers deployed in 19

Côte d’Ivoire showed that the risk of typhoid fever was significantly higher in persons 20

vaccinated more than 3 years previously (74). 21

Field trials in children and adults in Nepal given a single (25-µg) dose showed 72% 22

vaccine efficacy during 17 months of follow up (69); and a field trial in schoolchildren in South 23

Africa also using a single (25-µg) dose showed 60% protection during 21 months of follow-up 24

(70). In South Africa, protection declined to 55% at 3 years (75). Another field trial in China in 25

people aged 3–50 years given a single 30-µg dose showed 69% efficacy during 19 months of 26

follow-up (76). Thus, whilst a single dose of an unconjugated Vi vaccine provides moderate 27

protection, available data suggest that protective efficacy does not last beyond 3 years, so 28

revaccination is necessary within that time. 29

Most data suggest that children who are younger than 5 years respond poorly to Vi 30

polysaccharide vaccines (77). However, one cluster-randomized trial in Kolkata, India (78), 31

showed that protective efficacy among young children (aged 2–4 years) was 80%, which was 32

higher than that observed in children aged 5–14 years (56%) and in older persons (46%). In 33

contrast, a cluster-randomized field trial of similar design and using the same Vi vaccine in 34

Karachi, Pakistan, reported an adjusted total protective effectiveness of -38% (95% CI, -192% to 35

35%) for children aged 2–5 years compared with 57% (95% CI, 6% to 81%) for children aged 5–36

16 years (77). 37

1 https://extranet.who.int/gavi/PQ_Web/


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Thus, a single dose of Vi vaccine can provide moderate protection for a limited duration, 1

but the vaccines have the usual limitations associated with polysaccharide vaccines, including 2

poor immunogenicity in infants and young children, short-lived immunity and lack of 3

anamnestic immune responses to subsequent doses (72, 78, 79). 4

Vi polysaccharide–protein conjugate vaccine 5

6

Experience with several polysaccharide–protein conjugate vaccines (such as Hib, meningococcal 7

and pneumococcal vaccines) has shown that conjugation to a carrier protein overcomes many of 8

the limitations associated with unconjugated bacterial polysaccharides. On the basis of this 9

experience and to try to address the limitations of the typhoid vaccines described above, several 10

Vi polysaccharide–protein conjugate vaccines have been developed or are under development. 11

These include Vi conjugated to tetanus or diphtheria toxoid, to CRM197, as well as the prototype 12

Vi conjugate vaccine with Vi conjugated to nontoxic recombinant Pseudomonas aeruginosa 13

toxin (Vi-rEPA) (80). Also, a TCV that uses Vi prepared from C. freundii s.l. and CRM197 as 14

the carrier protein has been shown to elicit a higher level of anti-Vi IgG compared to a licensed 15

Vi polysaccharide vaccine control in European adults who had never been exposed to typhoid 16

fever (81). Vi preparations from C. freundii s.l. have been shown to be immunologically 17

indistinguishable from and structurally similar to those from S. Typhi (81, 82), although size 18

differences have been observed for Vi polysaccharide from S. Typhi and C. freundii s.l. using 19

size-exclusion high-performance liquid chromatography (HPLC). 20

Four TCVs have been licensed in India since 2008, three consisting of Vi polysaccharide 21

conjugated to tetanus toxoid, and one to CRM197. Other TCVs are in late-stage development in 22

some Asian countries. Typbar-TCV (a Vi-TT vaccine) was licensed in India in 2013 for use in 23

children over 6 months and in adults up to 45 years of age on the basis of immunogenicity and 24

safety demonstrated in a Phase III study in an endemic setting (13, 83); results showed that anti-25

Vi antibody titres were significantly higher among recipients of TCV compared with the 26

unconjugated Vi polysaccharide vaccine. Furthermore, the high geometric mean titres of IgG 27

anti-Vi antibodies elicited by a single dose of Typbar TCV persisted for up to 5 years in 28

approximately 84% of children. The vaccine was prequalified by WHO in December 2017. A 29

protective efficacy of 87.1 % (95 CI 47.2, 96.9) against persistent fever followed by positive 30

blood culture for S. Typhi was subsequently demonstrated in human challenge studies (84). 31

Interim data on the efficacy of Typbar TCV in an endemic population in Nepal have also 32

recently been published (22). These data, from a phase III participant-observer blinded 33

randomized study in children 9 months to <16 years of age, confirm that a single dose of TCV is 34

safe, immunogenic and effective in this field setting with an efficacy of 81.6 % (95%) (22). This 35

conclusion is supported by new data from Pakistan (VE of ~89%) and Navi Mumbai (VE of 36

82%) (personal communication). In view of the improved immunological properties of TCVs 37

compared to the other available typhoid vaccines, its suitability for use in young children and 38

expected duration of protection, the WHO SAGE recommended this as the preferred vaccine for 39

use in all age groups and that the introduction of TCV be prioritized in countries with the highest 40

burden of typhoid fever or with a high burden of antimicrobial resistant S. Typhi (13). 41


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No internationally agreed correlates or surrogates of protection have yet been agreed for 1

Vi conjugate vaccines, although suggested correlates have been proposed. Based on the assay 2

used to measure anti-Vi IgG serum antibodies generated in response to the prototype US 3

National Institutes of Health Vi-rEPA conjugate vaccine in Vietnam, a threshold value of 4.3 4

ug/ml anti-Vi antibody measured by ELISA was suggested to be associated with a high level of 5

sustained protection lasting 4 years after vaccination (85, 86). A placebo-controlled, randomized, 6

double-blind study in Vietnamese children aged 2–5 years in the highly endemic area had given 7

an estimated efficacy of 89% for the Vi-rEPA vaccine over 46 months of follow-up (87, 88). 8

Although the findings of Voysey and Pollard (89) confirmed that higher anti-Vi IgG levels are 9

associated with greater protection against typhoid infection, they failed to identify a threshold 10

level where the probability of infection becomes negligible within the range of antibody levels 11

induced by vaccination. 12

It is acknowledged that there are difficulties in comparing any immunogenicity data 13

generated with new TCVs and current ELISA protocols to the data generated in the original Vi-14

rEPA trial in Vietnam (16). However, the inclusion of a working reference serum calibrated 15

against the WHO International Standard serum anti-Vi antibodies (90, 91) can improve the 16

interpretation of data from clinical trials. The use of the WHO International Standard enables the 17

achievement of consistency in determining serum titers and provides a basis for the comparison 18

of data generated by different assays and/or different laboratories. 19

It has been suggested that variability in biophysical properties of antibodies induced by 20

Vi polysaccharide and Vi-TT conjugated vaccines, such as antibody subclass distribution and 21

avidity, may also impact protective outcomes. A recent study has identified Vi IgA as a 22

biomarker of protective immunity against typhoid fever and quantifies the concentration of Vi 23

IgA in vaccine recipients using the WHO International Standard (92). However, no correlate of 24

protection was identified, and further work is needed to determine whether IgA represents a true 25

correlate of protection, or a surrogate marker of another underlying immune response. 26

Challenge Studies 27

28

The development of vaccines against typhoid fever has been complicated by the human host-29

restriction of S. Typhi, the lack of clear correlates of protection, the scale required to run field 30

trials of efficacy and uncertainty about estimation of vaccine impact. Historically, only the 31

chimpanzee model of the 1960s demonstrated a pathogenesis and clinical illness that somewhat 32

recapitulated typhoid fever in humans (93-96). However, the chimpanzee model is no longer 33

permissible and recent animal models, including ones based on “humanized” small animals, have 34

not been able to mimic the disease process of human typhoid, despite many attempts (97-102). 35

Instead, a human challenge model has been used to overcome some of these difficulties and to 36

provide direct estimation of efficacy in vaccine recipients who are deliberately challenged with 37

the pathogen in a controlled setting (103, 104). The first setting during the 1950s to early 1970s 38

involved volunteers in a penal institution (66, 105-107) whereas more recently the model has 39

involved community volunteers (103, 104). 40


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Part A. Guidelines on manufacture and control 1

2

A.1 Definitions 3

4

A.1.1 International name and proper name 5

6

The international name of the vaccine should be typhoid conjugate vaccine. The proper name 7

should be the equivalent of the international name in the language of the country of origin. The 8

use of the international name should be limited to the vaccines that satisfy the specifications 9

formulated below. 10

A.1.2 Descriptive definition 11

12

A typhoid conjugate vaccine is a preparation of S. Typhi or C. freundii s.l. Vi polysaccharide 13

covalently linked to a carrier protein. It may be formulated with a suitable adjuvant. It should be 14

presented as a sterile, aqueous suspension or as freeze-dried material. The preparation should 15

satisfy all the specifications given below. 16

A.1.3 International reference materials 17

18

Two international standards for Vi polysaccharide (16/126 and 12/244) have been developed to 19

measure the polysaccharide content of typhoid vaccines based on Vi polysaccharide. The Vi 20

polysaccharide content was assessed by qNMR: standard 16/126 for Vi polysaccharide of S. 21

Typhi has a content of 2.03 ± 0.10 mg Vi polysaccharide per ampoule, and standard 12/244 for 22

Vi polysaccharide of C. freundii s.l. has a content of 1.94 ± 0.12 mg Vi polysaccharide per 23

ampoule (108, 109). Both standards are suitable for physicochemical and immunoassays used for 24

batch release of Vi polysaccharide vaccines. For example, the Vi polysaccharide standards can 25

be used as coating antigens for in-house ELISAs (109-111). Note that a standard of homologous 26

Vi polysaccharide should be used when analyzing the content of Vi polysaccharide vaccines 27

(108, 109). For example, if the conjugate has been made with Citrobacter Vi polysaccharide, a 28

Citrobacter Vi polysaccharide standard is more appropriate to use for determining its content. 29

Use of these standards decreased the variability of in-house assays (108, 109). 30

International standard 16/138: serum anti-Vi polysaccharide antibodies (human), is available and 31

consists of pooled post-immunisation sera, following vaccination with plain Vi polysaccharide or 32

conjugated Vi polysaccharide according to the immunisation schedule of Jin et al. (84). 33

International standard 16/138 has been evaluated in commercial ELISAs and in-house ELISAs 34

and has been assigned a concentration of 100 International Units (IU)/mL (110, 111). This 35

primary reference standard should be used as a calibrant for in-house and working standards that 36

are used to evaluate the immunogenicity of licensed vaccines and vaccine candidates in clinical 37

studies (110, 111). A second collaborative study used international standards 16/138 and 12/244 38


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to evaluate a standardized ELISA based on a co-coating of Vi PS and poly-L-lysine and showed 1

this generic assay to be an alternative for the commercial Vi PS ELISA (91). 2

These International Standards are available from the National Institute for Biological Standards 3

and Control, Potters Bar, the United Kingdom.2 For the latest list of appropriate WHO 4

international standards and reference materials, the WHO Catalogue of International Reference 5

Preparations should be consulted.3 6

A.1.4 Terminology 7

8

The definitions given below apply to the terms used in these Recommendations. They may have 9

different meanings in other contexts. 10

Carrier protein: the protein to which the Vi polysaccharide is covalently linked for the 11

purpose of eliciting a T cell-dependent immune response to the Vi polysaccharide. 12

Final bulk: the homogeneous preparation from one or more lots of purified bulk 13

conjugate in a single container from which the final containers are filled, either directly or 14

through one or more intermediate containers. 15

Final lot: a number of sealed, final containers that are equivalent with respect to the risk 16

of contamination that may have occurred during filling and freeze-drying (if performed). 17

Therefore, a final lot should have been filled from a single container and freeze-dried in one 18

continuous working session. 19

Master-seed lot: bacterial suspensions for the production of Vi polysaccharide or the 20

carrier protein should be derived from a strain that has been processed as a single lot and is of 21

uniform composition. The master-seed lot is used to prepare the working-seed lots. Master-seed 22

lots should be maintained in the freeze-dried form or be frozen below –45 °C. 23

Activated carrier protein: a carrier protein that has been chemically or physically 24

modified and prepared for conjugation to the polysaccharide. 25

Activated polysaccharide: purified polysaccharide that has been modified by a chemical 26

reaction or a physical process in preparation for conjugation to the activated carrier protein. 27

Purified bulk conjugate: a purified bulk conjugate is prepared by the covalent bonding 28

of activated Vi polysaccharide to the carrier protein, followed by the removal of residual 29

reagents and reaction by-products. This is the parent material from which the final bulk is 30

prepared. 31

Purified polysaccharide: the material obtained after final purification of polysaccharide. 32

The lot of purified polysaccharide may be derived from a single harvest or a pool of single 33

harvests that have been processed together. 34

2 www.nibsc.org/products 3 www.who.int/bloodproducts/catalogue

http://www.nibsc.org/products


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Single harvest: the material obtained from one batch of culture that has been inoculated 1

with the working-seed lot (or with the inoculum derived from it), harvested and processed 2

together during one production run. 3

Working-seed lot: a quantity of live strains for the production of Vi polysaccharide or 4

the carrier protein that are of uniform composition and that have been derived from the master 5

seed lot by growing the organisms and maintaining them in freeze-dried aliquots or frozen at or 6

below -45 °C. The working-seed lot is used to inoculate the production medium. 7

A.2 Guidelines on general manufacturing 8

9

The general manufacturing recommendations contained in Good Manufacturing Practices: main 10

principles for pharmaceutical products (112) and Good Manufacturing Practices for biological 11

products (113) should be applied at establishments manufacturing Vi polysaccharide conjugate 12

vaccines. 13

The production method should be shown to consistently yield Vi polysaccharide 14

conjugate vaccines of satisfactory quality as outlined in these Recommendations. All assay 15

procedures used for quality control of the conjugate vaccine and vaccine intermediates should be 16

validated. Post-licensure changes to the manufacturing process and quality control methods 17

should also be validated and may require the National Regulatory Authority’s (NRA) approval 18

prior to implementation (114, 115). 19

Production strains for Vi polysaccharide and the carrier proteins may represent a hazard 20

to human health and should be handled under appropriate containment conditions based on risk 21

assessment and applicable national and local regulations (116). Standard operating procedures 22

should be developed to deal with emergencies arising from accidental spills, leaks or other 23

accidents. Personnel employed by the production and control facilities should be adequately 24

trained. Appropriate protective measures, including vaccination, should be implemented if 25

available. 26

A.3 Control of starting material 27

28

A.3.1 Certification of bacterial strain 29

30

A.3.1.1 Bacterial strain for preparing Vi polysaccharide 31

32

The bacterial strain used for preparing Vi polysaccharide should be from single, well 33

characterized stock that can be identified by a record of its history, including the source from 34

which it was obtained, number of passages and the tests used to determine the characteristics of 35

the strain. Information regarding materials of animal origin used during passages of the bacterial 36

strain should be provided, such as compliance with the current WHO Guidelines on 37


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Transmissible Spongiform Encephalopathies in relation to Biological and Pharmaceutical 1

Products (117) or a statement of risk assessment. 2

The strain should be capable of stably producing Vi polysaccharide. S. Typhi and C. 3

freundii s.l. have been shown to be suitable sources for Vi polysaccharide. 1H nuclear magnetic 4

resonance (NMR) spectroscopy and immunochemical tests are suitable methods for confirming 5

the identity of the polysaccharide. 6

A.3.1.2 Bacterial strain for preparing the carrier protein 7

8

The bacterial strains used for preparing the carrier protein should be identified by their history, 9

including the source from which they were obtained, number of passages and the tests used to 10

determine the characteristics of the strains. Information regarding materials of animal origin used 11

during passages of the bacterial strain should be provided, such as compliance with the WHO 12

Guidelines on Transmissible Spongiform Encephalopathies in relation to Biological and 13

Pharmaceutical Products (117) or a statement of risk assessment. 14

A.3.2 Bacterial-seed lot system 15

16

The production of both Vi polysaccharide and the carrier protein should be based on a seed-lot 17

system consisting of a master seed and a working seed. Cultures derived from the working seed 18

should have the same characteristics as the cultures of the strain from which the master-seed lot 19

was derived (see sections A.3.1.1 and A.3.1.2). 20

Each new seed lot prepared should be characterized using appropriate control tests to 21

ensure comparable quality attributes to those of the previous seed lot. The control tests may 22

include culture purity, strain identity and Vi polysaccharide structure. New seed lots should also 23

be shown to have comparable Vi polysaccharide production in routine manufacturing prior to 24

their use. 25

A.3.3 Bacterial culture media 26

27

Manufacturers are encouraged to avoid the use of materials of animal origin. However, if the 28

culture medium does contain materials of animal origin, they should comply with current WHO 29

Guidelines on Transmissible Spongiform Encephalopathies in relation to Biological and 30

Pharmaceutical Products (117) and should be approved by the NRA. 31

The culture medium used to prepare bacterial-seed lots and commercial vaccine lots 32

should also be free from substances likely to cause toxic or allergic reactions in humans. 33

Additionally, the liquid culture medium used to produce polysaccharide intermediate should be 34

free from ingredients that will form a precipitate upon addition of chemical compounds, such as 35

hexadecyltrimethylammonium bromide (CTAB), for the purification of the Vi polysaccharide. 36

A.4 Control of vaccine production 37


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1

A.4.1 Control of polysaccharide antigen production 2

3

The Vi polysaccharides that are used in licensed vaccines are defined chemical substances if they 4

are prepared to similar specifications, for example as described in Requirements for Vi 5

polysaccharide typhoid vaccine (118) and the requirements in the following sections. As a result, 6

it is expected that they will be suitable for the preparation of TCVs. 7

A.4.1.1 Single harvests for preparing Vi polysaccharide antigen 8

9

The consistency of the production process should be demonstrated by monitoring the growth of 10

the organisms and the yield of Vi polysaccharide. 11

A.4.1.1.1 Consistency of microbial growth for antigen production 12

13

The consistency of the growth of production strains should be demonstrated by monitoring the 14

growth rate, pH, pO2 and the final yield of Vi polysaccharide, although monitoring should not be 15

limited to these parameters. 16

A.4.1.1.2 Bacterial purity 17

18

When required, samples of the culture should be taken before inactivation and examined for 19

microbial contamination. The purity of the culture should be verified using suitable methods, 20

such as inoculation on appropriate culture media. If contamination is found, the culture and any 21

product derived from it should be discarded. 22

A.4.1.2 Bacterial inactivation and antigen purification 23

24

Generally, S. Typhi is inactivated by a suitable inactivating agent such as formaldehyde or by an 25

alternative method such as heating. The inactivation process should be validated. 26

After inactivation, the biomass of S. Typhi or C. freundii s.l. is removed using an 27

appropriate method such as centrifugation or tangential flow filtration. The Vi polysaccharide 28

may be then purified from the supernatant by precipitation with CTAB or other suitable methods 29

approved by the NRA. All reagents should be pharmaceutical grade and sterile. Bioburden 30

should be monitored during purification. To ensure stability, purified Vi polysaccharide in 31

powder form should be stored at 2–8 °C, and purified Vi polysaccharide in solution should be 32

stored below -20 °C. The duration during which the polysaccharide will remain stable should be 33

validated. 34

A.4.1.3 Control of purified Vi polysaccharide antigen 35

36


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Each lot of purified Vi polysaccharide should be tested for identity and purity, as well as the 1

additional parameters described below. All tests should be validated by and agreed with the 2

NRA. 3

A.4.1.3.1 Identity 4

5

Vi polysaccharide is a linear homopolymer composed of (l → 4)-2-acetamido- 2-deoxy-α-D-6

galacturonic acid that is O-acetylated at carbon-3 (119). 7

A test should be performed on the purified polysaccharide to verify its identity, such as 8

NMR spectroscopy (120) or a suitable immunoassay, as is appropriate and convenient. 9

A.4.1.3.2 Molecular size or mass distribution 10

11

The molecular size or mass distribution of each lot of purified polysaccharide should be 12

estimated to assess the consistency of each batch. The distribution constant (KD) should be 13

determined by measuring the molecular size distribution of the polysaccharide at the main peak 14

of the elution curve obtained by a suitable chromatographic method. The KD value or the mass 15

distribution limits, or both, should be established and shown to be consistent from lot to lot for a 16

given product. For gel filtration or high performance size-exclusion chromatography (HPSEC), 17

to ensure consistency and a defined proportion of high molecular size polysaccharide, typically 18

at least 50% of the Vi polysaccharide should elute at a KD value less than a predefined value, 19

depending on the chromatographic method used. 20

An acceptable level of consistency should be agreed with the NRA. Alternatively, 21

calculation of the peak width at the 50% level can be used to analyse the distribution of 22

molecular weight (MW). 23

Due to its high viscosity on molecular sizing columns, the Vi polysaccharide does not 24

behave as do other polysaccharides; therefore, column matrices and eluents should be carefully 25

chosen to ensure a representative recovery (121). 26

Suitable detectors for this method include a refractive index detector (122), alone or in 27

combination with static light scattering detector (e.g. Multi-angle Laser Light Scattering, 28

MALLS) (97) and/or with a viscometer. The methodology used should be validated to 29

demonstrate sufficient resolution in the appropriate molecular weight range. Manufacturers are 30

encouraged to produce Vi polysaccharide that has a consistent distribution of molecular size. 31

A.4.1.3.3 Polysaccharide content 32

33

The concentration of the Vi polysaccharide in its O-acetylated, acid form in eluted fractions can 34

be measured using Hestrin’s method (123), or the acridine orange method (119, 124). More 35

specific methods, such as NMR (120) or high-performance anion exchange chromatography with 36

pulsed amperometric detection (HPAEC–PAD) (124) are also acceptable, and a suitable 37

immunoassay, for example rocket immunoelectrophoresis or ELISA, may be considered. A 38


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suitable reference preparation of Vi polysaccharide calibrated against the WHO 1st International 1

Standard for Citrobacter freundii (e.g. NIBSC 12/244) or S. Typhi (e.g. NIBSC 16/126) Vi 2

polysaccharide should be used where appropriate (see A.1.3). These methods should be validated 3

and agreed with the NRA. 4

A.4.1.3.4 O-acetyl content 5

6

The O-acetyl content of the purified Vi polysaccharide is important for the immunogenicity of 7

Vi (119, 121, 125); it should be at least 2.0 mmol/g polysaccharide (52% O-acetylation), unless 8

justified. Hestrin’s method (123), or NMR (120, 126) or other suitable methods may be used to 9

quantitatively determine O-acetylation. The methods used, and the acceptance criteria should be 10

agreed with the NRA. 11

A.4.1.3.5 Moisture content 12

13

If the purified polysaccharide is to be stored as a dried form, the moisture content should be 14

determined using suitable, validated methods, and the results should be within agreed limits; the 15

methods used, and the acceptable limits should be agreed with the NRA. 16

A.4.1.3.6 Protein impurity 17

18

The protein content should be determined using a suitable validated method, such as that of 19

Lowry et al., and using bovine serum albumin (BSA) as a reference (127). Sufficient 20

polysaccharide should be assayed to accurately detect protein contamination. Each lot of 21

purified polysaccharide should typically contain no more than 1% (weight/weight) of protein. 22

A.4.1.3.7 Nucleic acid impurity 23

24

Each lot of purified polysaccharide should contain no more than 2% of nucleic acid by weight as 25

determined by ultraviolet spectroscopy, on the assumption that the absorbance of a 10-g/L 26

nucleic acid solution contained in a cell of 1 cm path length at 260 nm is 200 (128). Other 27

validated methods may be used. Sufficient polysaccharide should be assayed to detect accurately 28

2% nucleic acid contamination. 29

A.4.1.3.8 Phenol content 30

31

If phenol has been used to prepare the Vi polysaccharide antigen, each lot should be tested for 32

phenol content using a validated method that has been approved by the NRA. The phenol content 33

should be expressed in µg per mg of purified Vi antigen and shown to be consistent and within 34

the limits approved by the NRA. 35

A.4.1.3.9 Endotoxin 36


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To ensure an acceptable level of pyrogenic activity in the final product, the endotoxin content of 1

each lot of purified Vi polysaccharide should be determined and shown to be within limits 2

agreed with the NRA. Suitable in vitro methods include the Limulus amoebocute lysate (LAL) 3

test or a monocyte activation test (MAT). 4

A.4.1.3.10 Residues of process-related contaminants 5

6

The residues of process-related contaminants in the purified polysaccharide (e.g. CTAB, 7

formaldehyde and antifoaming agents) should be determined, and shown to be within limits 8

agreed with the NRA. The routine testing of each lot before release for residual process-related 9

contaminants may be omitted once consistency has been demonstrated on a number of lots; this 10

number should be agreed with the NRA. 11

A.4.1.4 Activated polysaccharide 12

13

Purified Vi polysaccharide is usually activated to enable conjugation; it may also be partially 14

depolymerized either before or during the activation process. 15

A.4.1.4.1 Chemical activation 16

17

Several methods are satisfactory for the chemical activation modification of Vi polysaccharides 18

prior to conjugation. The method that is chosen should be approved by the NRA. As part of the 19

in-process control procedures, the processed Vi polysaccharide that will be used in the 20

conjugation reaction should be assessed to determine the number of functional groups 21

introduced. 22

A.4.1.4.2 Molecular size or mass distribution 23

24

If any size-reduction or activation steps are performed, the average size or mass distribution (i.e. 25

the degree of polymerization) of the processed Vi polysaccharide should be measured using a 26

suitable method. The size or mass distribution should be controlled using appropriate limits since 27

the size may affect the reproducibility of the conjugation process. 28

A.4.2 Control of carrier-protein production 29

30

A.4.2.1 Consistency of microbial growth for the carrier protein 31

32

The consistency of the growth of the microorganisms used should be demonstrated using 33

methods such as pH and the final yield of the appropriate protein or proteins; other methods may 34

also be used. 35

A.4.2.2 Characterization and purity of the carrier protein 36

37


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Proteins that have been used as carriers in licensed conjugate vaccines include TT, DT and 1

CRM197, but other carriers could be used. Proteins should be assayed for purity and 2

concentration and tested to ensure they are nontoxic and appropriately immunogenic. All tests 3

and proteins should be approved by the NRA. 4

For established carrier proteins, certain levels of purity may already be specified and 5

perhaps required. Preparations of TT and DT should satisfy the relevant recommendations 6

published by WHO (129, 130). CRM197 can be obtained from cultures of Corynebacterium 7

diphtheriae C7 /β197 (131) or expressed recombinantly by genetically modified micro-8

organisms (132). CRM197 with a purity not less than 90% as determined by high performance 9

liquid chromatography (HPLC) should be prepared by column chromatographic methods. The 10

content of residual host DNA should be determined, and results should be within the limits that 11

have been approved for the particular product by the NRA. When CRM197 is produced in the 12

same facility as DT, methods should be used to distinguish the CRM197 protein from the active 13

toxin. 14

A test should be performed on the purified carrier protein to verify its identity. Mass 15

spectrometry or a suitable immunoassay or physico-chemical assay could be performed as 16

appropriate and convenient. 17

Additionally, the carrier protein should be characterized using a combination of the 18

following physicochemical methods as appropriate: (a) sodium dodecyl sulfate–polyacrylamide 19

gel electrophoresis (SDS–PAGE); (b) isoelectric focusing; (c) HPLC; (d) amino acid analysis; 20

(e) amino acid sequencing; (f) circular dichroism; (g) fluorescence spectroscopy; (h) peptide 21

mapping; (i) or mass spectrometry (133). Outcomes should be consistent with the reference 22

material. 23

A.4.2.3 Degree of activation of the carrier protein 24

25

Adipic acid dihydrazide (ADH) or other appropriate linkers, such as N-Succinimidyl 3-26

(2pyridyldithio)-propionate, can be used to modify the carrier protein. The level of protein 27

modification should be monitored, quantified and be consistent. The use of an in-process control 28

may be required. The reproducibility of the method used for modification should be validated. 29

The level of modification of the carrier protein by ADH can be assessed by determining 30

the amount of hydrazide; this is done by using colorimetric reactions with 2,4,6-31

trinitrobenzenesulfonic acid and with ADH as a standard (134-136). Other suitable methods 32

include fluorescent tagging followed by HPLC, or quadrupole time-of-flight mass spectrometry. 33

A.4.3 Conjugation and purification of the conjugate 34

35

A number of methods of conjugation are in use; all involve multistep processes (124, 134-137). 36

Prior to demonstrating the immunogenicity of the Vi polysaccharide conjugate vaccine in 37

clinical trials, both the methods of conjugation and the control procedures should be established 38

to ensure the reproducibility, stability and safety of the conjugate. 39


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The derivatization and conjugation processes should be monitored and analyzed for 1

unique reaction products. Residual unreacted functional groups or their derivatives are 2

potentially capable of reacting in vivo and may be present following the conjugation process. 3

The manufacturing process should be validated, and the limits for unreacted activated functional 4

groups (those that are known to be clinically relevant) at the conclusion of the conjugation 5

process should be agreed with the NRA. 6

After the conjugate has been purified, the tests described below should be performed to 7

assess the consistency of the manufacturing process. The tests are critical for ensuring 8

consistency from lot to lot. 9

A.4.4 Control of the purified bulk conjugate 10

11

Tests for releasing purified bulk conjugate should be validated. 12

A.4.4.1 Identity 13

14

A suitable immunoassay or other methods should be performed on the purified bulk conjugate to 15

verify the identity of the Vi polysaccharide. Depending on the buffer used, NMR spectroscopy 16

may be used to confirm the identity and integrity of the polysaccharide in the purified bulk 17

conjugate (126, 138-140). The identity of the carrier protein should also be verified using a 18

suitable method. 19

A.4.4.2 Endotoxin 20

21

The endotoxin content of the purified bulk conjugate should be determined using a Limulus 22

amoebocyte lysate (LAL) test unless otherwise justified and shown to be within limits agreed 23

with the NRA. 24

A.4.4.3 O-acetyl content 25

26

The O-acetyl content of the purified bulk conjugate should be determined by NMR or by other 27

appropriate methods. The specification for the O-acetyl content of the purified bulk conjugate 28

should be agreed with the NRA. The specification for O-acetyl content of the conjugate bulk 29

should not be higher than that set for the purified Vi polysaccharide. 30

A.4.4.4 Residual reagents 31

32

The purification procedures for the conjugate should remove any residual reagents that were 33

used for conjugation and capping. The removal of reagents, their derivatives and reaction 34

byproducts, such as ADH, phenol and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide 35

(known as EDC, EDAC or EDCI), should be confirmed using suitable tests or by validation of 36

the purification process. 37


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The specifications of the process and the quantifiable methods to be used should be 1

agreed upon in consultation with the NRA. 2

The process validation should also demonstrate that no significant covalent modification 3

of the Vi polysaccharide itself has occurred, and the percentage of the modified Vi 4

monosaccharides should not exceed what was shown to be safe and immunogenic in clinical 5

studies. For example, many common conjugation procedures use EDC, and a frequent side 6

reaction can result in Vi carboxylates being covalently modified to form an N-acylurea. Such 7

modification may alter the structure of the Vi, and this modification is known to be 8

immunogenic, leading to antibodies that cross-react with other EDC-modified polysaccharides, 9

such as those in Hib, pneumococcal and meningococcal conjugate vaccines; thus, this 10

modification may interfere with these vaccines. The N-acylurea content can be readily measured 11

using NMR. 12

A.4.4.5 Polysaccharide content 13

14

The content of Vi polysaccharide should be determined using an appropriate validated assay, 15

such as HPAEC–PAD (85, 108, 124), or immunological methods (e.g. rate nephelometry, rocket 16

electrophoresis). See section A 1.3 for the recommendation for suitable reference materials. 17

A.4.4.6 Conjugated and unbound (free) polysaccharide 18

19

A limit for the presence of unbound (free) Vi polysaccharide relative to total Vi polysaccharide 20

should be set for the purified bulk conjugate; this limit should be agreed with the NRA. Methods 21

that have been used to assay unbound polysaccharide include RP-HPLC (SEC) (141), Capto 22

Adhere anion-exchange resin binding (142), and deoxycholate precipitation (143), followed by 23

HPAEC-PAD or another method as described in section A 4.4.5. Other suitable methods may be 24

developed and validated. 25

A.4.4.7 Protein and unbound (free) content 26

27

The protein content of the purified bulk conjugate should be determined using an appropriate 28

validated assay. Each batch should be tested for conjugated and unbound protein. The 29

unconjugated protein content of the purified bulk conjugate should comply with the limit for the 30

product that has been agreed with the NRA. Appropriate methods for determining unbound 31

protein include HPLC or capillary electrophoresis. 32

A.4.4.8 Conjugation markers 33

34

The success of the conjugation process can be assessed by characterizing the conjugate using 35

suitable methods. For example, an increase in the MW of the protein component of the conjugate 36

compared with the carrier protein can be determined using the Coomassie blue stain with SDS–37

PAGE; an increase in the MW of the conjugate compared with both the Vi polysaccharide and 38


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the protein components should be demonstrated by the gel filtration profile, HP-SEC, capillary 1

electrophoresis and other suitable methods. The conjugate should retain antigenicity for both Vi 2

and the carrier protein as demonstrated by dot blot or western blot. 3

Where the chemistry of the conjugation reaction results in the creation of a unique 4

linkage marker, such as a unique amino acid, it should be quantified for each conjugate batch to 5

assess the extent of the covalent reaction between the Vi polysaccharide and the carrier protein. 6

This assessment of the unique linkage marker may be omitted once the consistency of the 7

conjugation is established with the agreement of the NRA. 8

A.4.4.9 Absence of reactive functional groups 9

10

The validation batch should be shown to be free of reactive functional groups or their derivatives 11

that are suspected to be clinically relevant on the polysaccharide and the carrier protein. 12

Where possible, the presence of reactive functional groups – for example, those derived 13

by ADH treatment – should be assessed for each batch. Alternatively, the product of the capping 14

reaction may be monitored, or the capping reaction can be validated to show that reactive 15

functional groups have been removed. 16

A.4.4.10 Ratio of polysaccharide to carrier protein 17

18

The ratio of polysaccharide to carrier protein in the purified bulk conjugate should be calculated. 19

For this ratio to be a suitable marker of conjugation, the content of each of the conjugate 20

components prior to their use should be known. For each purified bulk conjugate, the ratio 21

should be within the range approved by the NRA for that particular conjugate and should be 22

consistent with the ratio in vaccine that has been shown to be effective in clinical trials. 23

A.4.4.11 Molecular size or mass distribution 24

25

It is important to evaluate the molecular size or mass of the polysaccharide–protein conjugate to 26

establish the consistency of production, product homogeneity and stability during storage. 27

The relative molecular size of the polysaccharide–protein conjugate should be determined 28

for each purified bulk conjugate using a gel matrix appropriate to the size of the conjugate (121). 29

The method should be validated and should have the specificity to distinguish the 30

polysaccharide–protein conjugate from other components that may be present (e.g. unbound 31

protein or polysaccharide). The specification of molecular size or mass distribution should be 32

vaccine-specific and consistent with that of lots shown to be immunogenic in clinical trials. 33

Typically, the size of the polysaccharide–protein conjugate may be examined by methods 34

such as gel filtration using high-performance size- exclusion chromatography (HPSEC) on an 35

appropriate column. Since the ratio of polysaccharide to protein is an average value, 36

characterization of this ratio over the molecular size or mass distribution (e.g. by using dual 37


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monitoring of the column eluent) can provide further proof of the consistency of manufacturing 1

(133, 144). 2

A.4.4.12 Bacterial and mycotic bioburden 3

4

The purified bulk conjugate should be tested for bacterial and mycotic bioburden according to 5

the methods described in Part A, section 5.2, of the revised General requirements for the sterility 6

of biological substances (145), or using methods approved by the NRA. If a preservative has 7

been added to the product, appropriate measures should be taken to prevent it from interfering 8

with the test. 9

A.4.4.13 Specific toxicity of the carrier protein 10

11

When appropriate, the bulk conjugate should be tested to confirm the absence of specific toxicity 12

by the carrier protein. Alternatively, the absence of specific toxicity of the carrier protein may be 13

performed at the purified carrier protein stage if agreed with the NRA. 14

A.4.4.14 pH 15

16

If the purified bulk conjugate is a liquid preparation, the pH of each batch should be tested, and 17

the results should be within the range of values, determined to be safe in clinical trials, and 18

compatible with stability studies. For a lyophilized preparation, the pH should be measured after 19

reconstitution with the appropriate diluent. 20

A.4.4.15 Appearance 21

22

The appearance of the bulk purified conjugate should be examined. It should be clear to 23

moderately turbid, and colourless to pale yellow. 24

A.4.5 Preparation and control of the final bulk 25

26

A.4.5.1 Preparation 27

28

The final bulk is prepared by mixing a preservative or stabilizer, or both (if used), with a suitable 29

quantity of the bulk conjugate to meet the specifications of vaccine lots that have been shown to 30

be safe and efficacious in clinical trials. If an adjuvant is used, it should be mixed with the final 31

bulk at this stage. The use of a preservative in either single-dose or multi-dose vaccine vials is 32

optional. If the multi-dose vaccine does not contain preservative, its use should be time-restricted 33

after opening of the vial. If a preservative were to be added, its effect on antigenicity and 34

immunogenicity must be assessed to ensure that the preservative does not affect immune 35

response. 36

A.4.5.2 Test for bacterial and mycotic sterility 37


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1

Each final bulk should be tested for bacterial and mycotic sterility according to the requirements 2

of Part A, sections 5.1 and 5.2, of the revised General requirements for the sterility of biological 3

substances (145), or using methods approved by the NRA. If a preservative has been added to 4

the final bulk, appropriate measures should be taken to prevent it from interfering with the test. 5

A.5 Filling and containers 6

7

The recommendations concerning filling and containers given in Good Manufacturing Practices 8

for biological products (113) should be applied. 9

A.6 Control of the final product 10

11

A.6.1 Inspection of the final containers 12

13

All filled final containers should be inspected as part of the routine manufacturing process. 14

Those containers showing abnormalities – such as vial defects, improper sealing, clumping or 15

the presence of endogenous or exogenous particles – should be discarded. The test should be 16

performed against a black, and a white, background, according to pharmacopoeial specifications. 17

A.6.2 Control tests on the final lot 18

19

The following tests should be performed on several final container vials or syringes from each 20

final lot of vaccine (that is, in the final container), and the tests used should be validated and 21

approved by the NRA. The permissible limits for the different parameters listed under this 22

section, unless otherwise specified, should be approved by the NRA. 23

A.6.2.1 Appearance 24

25

The appearance of the final vial and final container (once reconstituted) should be verified and 26

should meet the established criteria with respect to its form and colour. 27

A.6.2.1 Identity 28

29

Identity tests on the Vi polysaccharide and the carrier protein should be performed on each final 30

lot. An immunological test or a physicochemical assay may be used for the Vi polysaccharide 31

and the carrier protein. 32

A.6.2.2 Bacterial and mycotic sterility 33

34


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The contents of the final containers should be tested for bacterial and mycotic sterility according 1

to the requirements of Part A, sections 5.1 and 5.2, of the revised General requirements for the 2

sterility of biological substances (145), or using a method approved by the NRA. If a 3

preservative has been added, appropriate measures should be taken to prevent it from interfering 4

with the sterility test. 5

A.6.2.3 Polysaccharide content 6

7

The amount of Vi polysaccharide conjugate in the final containers should be determined and 8

shown to be within the specifications agreed with the NRA. 9

The formulations of conjugate vaccines produced by different manufacturers may differ. 10

A quantitative assay for the Vi polysaccharide should be carried out. The specification is likely 11

to be product-specific. Examples of tests that may be used include: (a) colorimetric methods; (b) 12

chromatographic methods (including HPLC or HPAEC–PAD); or (c) immunological methods 13

(including rate nephelometry and rocket immunoelectrophoresis). 14

A.6.2.4 Unbound (free) polysaccharide 15

16

A limit for the presence of free Vi polysaccharide should be set for each type of conjugate 17

vaccine. Assessing the level of unconjugated polysaccharide in the final lot may be technically 18

demanding (142); as an alternative, the molecular size of the conjugate could be determined for 19

the final lot to confirm the integrity of the conjugate. An acceptable value should be consistent 20

with the value seen in batches used for clinical trials that showed adequate immunogenicity; the 21

value should be approved by the NRA. 22

A.6.2.5 O-acetyl content 23

24

The O-acetyl content of the Vi polysaccharide conjugate in the final container should be 25

determined for each final lot by NMR (120) or by other appropriate methods, such as Hestrin’s 26

method (123). Routine release testing of each lot for O-acetyl content in the final product may be 27

omitted if the NRA agrees and if the O-acetyl content is measured at the level of conjugate bulk 28

and process validation data obtained during the product’s development confirmed that 29

formulation and filling do not alter the integrity of the functional groups. A limit for the O-acetyl 30

content of the Vi polysaccharide conjugate should be approved by the NRA (125). 31

A.6.2.6 Molecular size or mass distribution 32

33

The molecular size or mass distribution of the polysaccharide conjugate should be determined 34

for each final lot using a gel matrix appropriate to the size of the conjugate. The analysis of 35

molecular size or mass distribution for each final lot may be omitted provided that the NRA 36

agrees, and the test has been performed on the conjugate bulk (see section A.4.4.11). 37

A.6.2.7 Endotoxin 38


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1

The endotoxin should be tested using a validated Limulus amoebocyte lysate test or a suitable in 2

vitro assay. The endotoxin content should be consistent with levels found to be acceptable in 3

vaccine lots used in clinical trials and within the limits agreed with the NRA. 4

A.6.2.8 Adjuvant content and degree of adsorption 5

6

If an adjuvant has been added to the vaccine, its content should be determined using a method 7

approved by the NRA. The amount of the adjuvant should also be agreed with the NRA. If 8

aluminium compounds are used as adjuvants, the amount of aluminium should not exceed 1.25 9

mg per single human dose. 10

The consistency of adsorption of the antigen to the adjuvant is important; the degree of 11

adsorption should be tested in each final lot and should be within the range of values measured in 12

vaccine lots shown to be clinically effective. The methods used, and the specifications should be 13

approved by the NRA. 14

A.6.2.9 Preservative content 15

16

If a preservative has been added to the vaccine, its content should be determined using a method 17


The amount of preservative in each dose of the vaccine should be shown not to have any 19

deleterious effect on the antigen or to impair the safety of the product in humans. The 20

effectiveness of the preservative should be demonstrated, and the concentration used should be 21


A.6.2.10 General safety (innocuity) 23

24

The general safety test (also referred to as the abnormal toxicity test or innocuity test) is no 25

longer recommended. The Expert Committee on Biological Standardization (ECBS) has 26

recommended the discontinuation of the innocuity test listed in all WHO Recommendations, 27

Guidelines and manuals for biological products (146). 28

A.6.2.11 pH 29

30

If the vaccine is a liquid preparation, the pH of each final lot should be tested, and the results 31

should be within the range of values found for lots shown to be safe and effective in clinical 32

trials. For a lyophilized preparation, the pH should also be measured after reconstitution with the 33

appropriate diluent. 34

A.6.2.12 Moisture content 35

36


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If the conjugate is dried, the acceptable level of residual moisture should be established, and the 1

limit should be agreed with the NRA. 2

A.6.2.13 Osmolality 3

4

The osmolality of the final lots should be determined and shown to be within the limits agreed 5

with the NRA. 6

A.6.3 Control of diluents 7

8

The recommendations in Good Manufacturing Practices: main principles for pharmaceutical 9

products (112) should apply to the manufacture and quality control of the diluents used to 10

reconstitute conjugate typhoid vaccines. An expiry date should be established for the diluents 11

based upon stability data. For lot release of the diluent, tests should be done to assess the 12

appearance, identity, volume, sterility and content of key components. 13

A.7 Records 14

15

The recommendations in Good Manufacturing Practices for biological products (113) should be 16

followed as appropriate for the level of development of the candidate vaccine. 17

A.8 Samples 18

19

A sufficient number of lot samples of the product should be retained for future studies and needs. 20

Vaccine lots that are to be used for clinical trials may serve as reference materials in the future, 21

and a sufficient number of vials should be reserved and stored appropriately for that purpose. 22

A.9 Labelling 23

24

The recommendations in Good Manufacturing Practices for biological products (113) that are 25

appropriate for a candidate vaccine should be applied, and the following additional information 26

should also be included. 27

The label on the cartons enclosing one or more final containers, or the leaflet 28

accompanying each container, should include: 29

• a statement that the candidate vaccine fulfils Part A of these Guidelines; 30

• the information that if the vaccine is a lyophilized form it should be used immediately 31

after reconstitution; if data have been provided to the licensing authority to indicate that 32

the reconstituted vaccine may be stored for a limited time then the length of time should 33

be specified; 34

• where needed, information on the volume and nature of the diluent to be added to 35

reconstitute the lyophilized vaccine; this information should specify that the diluent 36

approved by the NRA should be supplied by the manufacturer. 37


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1

A.10 Distribution and shipping 2

3

The recommendations appropriate for candidate vaccines given in Good Manufacturing 4

Practices for biological products (113) should be followed. 5

Shipments should be maintained within specified temperature ranges, and packages 6

should contain cold-chain monitors (147). 7

A.11 Stability, storage and expiry date 8

9

The relevant recommendations outlined in Good Manufacturing Practices for biological 10

products (113) should be followed. The statements concerning storage temperature and expiry 11

date that appear on primary or secondary packaging should be based on experimental evidence 12

and should be approved by the NRA. 13

A.11.1 Stability testing 14

15

Adequate stability studies form an essential part of vaccine development. These studies should 16

follow the general principles outlined in WHO Guidelines on stability evaluation of vaccines 17

(148) and WHO Guidelines on the stability evaluation of vaccines for use under extended 18

controlled temperature conditions (149). The shelf life of the final product and the hold time of 19

each process intermediate (such as the purified polysaccharide, the carrier protein and the 20

purified bulk conjugate) should be established based on the results of real-time, real-condition 21

stability studies, and approved by the NRA. 22

The stability of the vaccine in its final container and at the recommended storage 23

temperature should be demonstrated to the satisfaction of the NRA on at least three lots of the 24

final product manufactured from different bulk conjugates. In addition, a real-time real-condition 25

stability study should be conducted on at least one final container lot produced each year. 26

A protocol should be established and followed for each stability study which specifies the 27

stability-indicating parameters to be monitored as well as the applicable specifications. Some 28

stability-indicating parameters may change over the shelf life as discussed in section below. 29

Different specifications should be established for release and end of shelf-life as described in 30

WHO Guidelines on the stability evaluation of vaccines for use under extended controlled 31

temperature conditions (149). The specification at the end of shelf-life should be linked to lots 32

demonstrated to be safe and effective/immunogenic in clinical trials, while the release 33

specification is often based on manufacturing capability. 34

The polysaccharide component of conjugate vaccines may be subject to gradual 35

hydrolysis at a rate that may vary depending upon the type of conjugate, the formulation or 36

adjuvant, the excipient, and conditions of storage. The hydrolysis may result in a reduced 37


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molecular size of the Vi polysaccharide component, a reduction in O-acetyl content, a reduction 1

in the amount of polysaccharide bound to the carrier protein, a change in pH, or in a reduced 2

molecular size of the conjugate, or some combination of these. 3

The O-acetyl content should be monitored quantitatively for stability testing and release 4

testing. The quantity of free protein should be monitored for stability testing and release testing. 5

The molecular size or mass distribution should be monitored for stability testing and release 6

testing. 7

If applicable, the residual moisture should be monitored for stability testing and release 8

testing. 9

Where applicable, the level of adsorption of the conjugate to the adjuvant should be 10

shown to be within the limits agreed with the NRA, unless data show that the immunogenicity of 11

the final product does not depend on the adsorption of the antigen to the adjuvant. 12

Accelerated stability studies may provide additional supporting evidence of the stability 13

of the product or as product characteristics, or both, but are not recommended for establishing 14

the shelf-life of the vaccine under a defined storage condition. 15

When any changes are made in the production process that may affect the stability of the 16

product, the vaccine produced by the new method should be shown to be stable. 17

If manufacturers consider incorporating a vaccine vial monitor (VVM) into the label, 18

they should provide appropriate data to justify a correlation between the stability kinetics of the 19

vaccine and the selected VVM (150). 20

A.11.2 Storage conditions 21

22

The recommended long-term storage conditions should be based on the findings of the stability 23

studies and ensure that all stability-indicating parameters (e.g. free saccharide) of the conjugate 24

vaccine meet the specifications at the end of the shelf-life. The labelled and packaged vaccine 25

products should be stored at the recommended long-term storage conditions. 26

If approved by the NRA, use of a vaccine under ECTC requires specific monitoring as 27

described in Guidelines on the stability evaluation of vaccines for use under extended controlled 28

temperature conditions (149). 29

A.11.3 Expiry date 30

31

The expiry date should be based on the shelf-life supported by stability studies and approved by 32

the NRA. The start of the dating period, e.g. the date of formulation of final bulk or the date of 33

filling, should be agreed with the NRA. The expiry dates for vaccines and diluents may be 34

different from one another. 35

A.11.4 Expiry of reconstituted vaccine (if applicable) 36

37


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For single-dose containers, the reconstituted vaccine should be used immediately. For multidose 1

containers, the use of reconstituted container should follow WHO Multi-dose vial policy 2

(Internet at: http://www.who.int/immunization/documents/en/), and shall be according to the 3

Summary of Product Characteristics and supplied instructions. 4

http://www.who.int/immunization/documents/en/


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Part B. Nonclinical evaluation of new typhoid conjugate vaccines 1

B.1 General principles 2

3

Detailed WHO guidelines on the design, conduct, analysis and evaluation of nonclinical studies 4

of vaccines are available separately (151), and they should be read in connection with Part B of 5

these Recommendations. Specific issues to be considered in relation to candidate Vi conjugate 6

vaccines are considered in section B.3. Plans for nonclinical studies to be conducted during the 7

development of the vaccine should be discussed with the NRA early in the review process. 8

B.2 Product characterization and process development 9

10

It is critical that vaccine production processes are appropriately standardized and controlled to 11

ensure consistency in manufacturing and the collection of nonclinical data that may suggest 12

safety and efficacy in humans. 13

Candidate formulations of Vi conjugate vaccines should be characterized to define the 14

critical structural and chemical attributes that indicate the polysaccharide, the conjugating protein 15

and the conjugate product are sufficiently pure and stable, and their properties are consistent. The 16

extent of product characterization may vary depending on the stage of development. Vaccine lots 17

used in nonclinical studies should be adequately representative of those intended for use in 18

clinical investigations – that is, the safety data should support the initiation of clinical studies in 19

humans. Ideally, the lots should be the same as those used in the clinical studies. If this is not 20

feasible, then the lots should be comparable with respect to physicochemical data, stability and 21

formulation. 22

B.3 Nonclinical immunogenicity studies 23

24

Immunogenicity studies in animal models may be poorly predictive of the efficacy of 25

glycoconjugate vaccines in humans and should only be conducted when they provide proof-of-26

concept information in support of a clinical development plan. Immunogenicity data derived 27

from appropriate animal models may be useful in establishing the immunological characteristics 28

of the Vi polysaccharide conjugate product, and may guide the selection of doses, schedules and 29

routes of administration that will be evaluated in clinical trials. However, any animal test plan 30

should incorporate 3Rs (Replace, Reduce, Refine) best practices. When animal models are used 31

for preclinical testing of vaccine immunogenicity they should elicit an anti-Vi IgG response that 32

is significantly greater than that of the control group (for example, non-conjugated Vi vaccine), 33

and a booster response should occur after the second dose (134). Immunogenicity studies of Vi 34

polysaccharide conjugates have been conducted in mice (97, 124, 152-154); in humans, 35

correlation has been made between the level of anti-Vi IgG and protection against clinical 36

disease (69, 86, 155). Therefore, the primary end-point for nonclinical studies of the 37

immunogenicity of Vi conjugate vaccines should be the level of anti-Vi elicited. 38


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Nonclinical studies of immunogenicity may include an evaluation of seroconversion rates 1

or geometric mean antibody titres, or both. When possible, nonclinical studies should be 2

designed to assess relevant immune responses, including the functional immune response (e.g. 3

by evaluating serum bactericidal antibodies, opsonophagocytic activity and serum-dependent 4

opsonophagocytic killing) (see section C.2.2). These studies may also address the interference 5

that can occur among antigens when multiantigen vaccines are used (see section C.2.3). In such 6

cases, the response to each antigen should be evaluated. 7

Although there have been advances in animal models, no ideal animal model exists that 8

establishes direct serological or immunological correlates of clinical protection. In the absence of 9

such a model, it is important to ensure that the production batches have the same protective 10

efficacy as those used and shown to be protective in clinical trials. Therefore, emphasis is 11

increasingly placed on ensuring consistency in manufacture through the use of modern physical, 12

chemical and immunological quality-control methods. Once validated, these non-animal methods 13

are considered more appropriate for use in lot release processes (Part A). 14

B.4 Nonclinical toxicity and safety 15

16

WHO Guidelines on nonclinical evaluation of vaccines (151) should be followed when assessing 17

toxicity and safety. Toxicity studies for Vi polysaccharide conjugate typhoid vaccines may be 18

performed in an appropriate animal model. These studies should entail careful analysis of all 19

major organs, as well as of tissues proximal to and distal from the site of administration, to detect 20

unanticipated direct toxic effects. These effects should be assessed for a wide range of doses, 21

including those exceeding the intended clinically relevant dose. If novel proteins are used to 22

manufacture conjugate vaccines, toxicity studies should be performed on these proteins first. 23

Nonclinical safety studies should be conducted in accordance with the GLPs that have been 24

described elsewhere (156, 157). For ethical reasons, it is desirable to apply the 3Rs concept of 25

“Replace Reduce Refine” to minimize the use of animals in research where scientifically 26

appropriate. 27


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Part C. Clinical evaluation of new typhoid conjugate vaccines 1

2

C.1 General considerations for clinical studies 3

4

The general principles described in the WHO Guidelines on clinical evaluation of vaccines: 5

regulatory expectations (158) apply to Vi polysaccharide conjugate vaccines and should be 6

followed. Some issues specific to the clinical development program for Vi conjugate vaccines 7

are discussed below and should be read in conjunction with the general guidance mentioned 8

above. WHO Guidelines for good clinical practice (GCP) for trials on pharmaceutical products 9

(159) are also available and should be consulted. 10

Vi conjugate vaccines have already been licensed in some countries for use in children 11

over 6 months and adults up to 45 years of age and one has been prequalified by WHO (13). The 12

licensure of effective Vi conjugate vaccines in some countries and their availability through the 13

WHO prequalification program has implications for the pathway to approval and design of 14

clinical studies in children above the age of 6 months and adults up to 45 years old. Information 15

supporting the safety, immunogenicity, efficacy and effectiveness of Vi conjugate vaccines in 16

typhoid endemic regions, as well as insights into putative immune correlates of protection, are 17

continually emerging (22, 83, 160-163). The principles for clinical evaluation outlined below are 18

based on the current situation and should be read in light of the circumstances in any one NRA’s 19

jurisdiction. 20

C.2 Outline of the clinical development program 21

22

It is recommended that the major part of the pre-licensure clinical development program is 23

conducted in subjects who are representative of the intended target population. 24

C.2.1 Dose and Schedule 25

26

The early clinical development program should provide a preliminary assessment of safety and 27

should be suitable for identifying an appropriate dose of conjugated Vi antigen and dose 28

regimen(s) for the target age group(s). Such studies are necessary for each candidate Vi 29

conjugate vaccine that is developed, since it is not possible to extrapolate the dose and schedule 30

identified for one conjugate vaccine to another. This consideration applies even if the same 31

carrier protein is used for two different Vi conjugate vaccines, since experience with other 32

conjugated polysaccharide vaccines has indicated that differences in conjugation chemistry can 33

affect immune responses to the polysaccharide(s). 34


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In the absence of a pre-licensure efficacy study, pathways to approval of a candidate Vi 1

conjugate vaccine in any one NRA’s jurisdiction may depend on the following: 2

• If there is a licensed Vi conjugate vaccine for which protective efficacy has been 3

documented (these data may come from pre- and/or post-licensure efficacy studies and/or 4

from post-approval studies of effectiveness), and subject to any pertinent national 5

legislation, the efficacy of a candidate Vi conjugate vaccine may be inferred based on 6

adequately designed comparative immunogenicity studies to bridge to the efficacy data 7

for the licensed vaccine. 8

9

• If there are data that point to a specific anti-Vi antibody concentration that strongly 10

correlates with efficacy, approval of a candidate Vi conjugate vaccine may be based on 11

immunogenicity studies in which vaccine efficacy is inferred by estimating the 12

proportion of vaccinated subjects predicted to be protected on the basis of immune 13

responses that exceed the concentration identified. In this situation, it may still be 14

appropriate for a NRA to request that the sponsor compares the immune response to the 15

candidate vaccine with the immune response to a licensed Vi conjugate vaccine for which 16

protective efficacy has been demonstrated. 17

18

• If there is no widely-accepted antibody concentration that strongly correlates with 19

efficacy and no licensed Vi conjugate vaccine for which protective efficacy has been 20

documented, it may be appropriate to infer efficacy of a candidate conjugate vaccine by 21

comparing the immune response with a licensed unconjugated Vi vaccine in subjects 22

aged from 2 years. See further in section C4. 23

24

C.3 Assessment of the immune response 25

26

C.3.1 Immune parameters of interest 27

28

The primary parameter for assessing the humoral immune response to a vaccine is usually based 29

on a measure of functional antibody. However, there are no well-established or standardized 30

assays for assessing functional antibody responses to Vi-containing vaccines, and it is not known 31

how the results of such assays correlate with vaccine efficacy. 32

The assessment of the immune response to licensed unconjugated (78, 164, 165) and 33

conjugated (22, 84, 85, 166) Vi vaccines has predominantly used ELISA methods to measure 34

total anti-Vi IgG in serum. For unconjugated Vi polysaccharide vaccines, approval has often 35

been based on comparing proportions that achieve anti-Vi IgG at least 1 µg/mL and/or on the 36

proportions that achieve at least a 4-fold increase in anti-Vi IgG from pre- to post-vaccination 37

(22). A regional or in-house working reference serum preparation calibrated against the WHO 38

International Standard serum anti-Vi antibodies (see Part A.1.6) should be used in the 39

interpretation of immunogenicity data from clinical trials. The use of the WHO International 40


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Standard improves consistency in the determination of serum titers and provides a basis for the 1

comparison of data generated by different assays and/or different laboratories. 2

At present, there is no established or widely agreed immune correlate of protection for 3

typhoid vaccines, although correlations between total serum antibody (75), total anti-Vi IgG (77, 4

87, 88, 167, 168), or anti-Vi IgA (92) in serum and protection against typhoid have been 5

described. A putative immune correlate of protection based on anti-Vi IgG has been proposed 6

based on long-term follow-up of Vietnamese children who received a candidate Vi conjugate 7

vaccine in a large efficacy trial but the value reported is specific to the assay that was applied 8

during that study and it is not yet clear what the corresponding values may be when using 9

alternative assays. 10

C.3.2. Considerations regarding the carrier protein 11

12

Proteins such as CRM197, DT, TT and rEPA have been used in the production of various Vi 13

conjugate vaccines. Based on experience with other types of conjugate vaccines that use 14

CRM197, DT or TT as the carrier protein, there is some potential that the immune response to the 15

Vi conjugated antigen may be reduced or enhanced in subjects who have pre-existing high levels 16

of tetanus or diphtheria antitoxin before vaccination. This phenomenon should be explored 17

during the development of Vi conjugate vaccines; this may be accomplished by analysing post-18

vaccination responses and comparing these with pre-vaccination antibody concentrations to the 19

protein carrier. The potential clinical significance of any effect requires careful consideration. 20

Depending on the target age range, it may be important to assess the effects of co-21

administering Vi conjugate vaccines with other routine vaccinations. Guidance on such studies, 22

including instances in which co-administered vaccines contain the carrier protein, may be found 23

in the WHO Guidelines on clinical evaluation of vaccines: regulatory expectations (158). 24

C.3.3 Immune memory 25

26

Vi conjugate vaccines are expected to elicit T-cell-dependent immune responses, which can be 27

assessed by administration of a post-priming Vi conjugate dose after an interval of at least 6–12 28

months has elapsed. Details of the clinical assessment of priming may be found in the WHO 29

Guidelines on clinical evaluation of vaccines: regulatory expectations (158). Whether or not 30

booster doses will be needed to maintain protection after successful priming with Vi conjugate 31

vaccines is not yet known. Until such time as this question is answered, there should be plans in 32

place to document antibody persistence and to evaluate vaccine effectiveness. 33

C.4 Immunogenicity 34

35

This section should be read in conjunction with the guidance on comparative immunogenicity 36

trials provided in the WHO Guidelines on clinical evaluation of vaccines: regulatory 37

expectations (158). The selection of the most appropriate licensed vaccine for use as a 38

comparator in clinical studies must be agreed between the sponsor and the NRA. 39


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C.4.1 Studies that compare conjugated Vi vaccines 1

2

If the aim of the study is to immunobridge efficacy documented with a licensed Vi 3

conjugate vaccine to a candidate vaccine, the study should be designed to demonstrate the non-4

inferiority of the immune response to the candidate vaccine when compared with a licensed Vi 5

conjugate vaccine. The primary immune parameter for purposes of immunobridging and the 6

acceptance criteria for concluding that the candidate vaccine will have at least similar efficacy to 7

the licensed vaccine should be pre-defined and agreed between NRAs and sponsors. 8

If efficacy data have supported derivation of an immune correlate of protection, the 9

proportion of subjects that achieves at least this concentration after vaccination with the 10

candidate vaccine should be the primary immune parameter. In this setting, a direct comparison 11

with a licensed Vi conjugate vaccine would not be essential although some NRAs may request 12

that a comparison is made with a licensed Vi conjugate vaccine for which vaccine efficacy has 13

been documented. Such a direct comparison also provides a randomised assessment of safety. 14

If the sponsor wishes to, or is requested to conduct a comparative study against a licensed 15

Vi conjugate for which efficacy is not documented, demonstrating non-inferiority for the 16

candidate vs. licensed vaccine does not inform on the potential efficacy of the candidate vaccine. 17

Therefore, either the immune responses to the candidate should be interpreted against an immune 18

correlate of protection or threshold value or, if neither exists, consideration should be given to 19

alternative study designs as described below. 20

C.4.2 Studies that compare conjugated Vi vaccines with unconjugated Vi vaccines 21

22

Studies that compare candidate Vi conjugate vaccines with licensed unconjugated Vi vaccines 23

should only be conducted in subjects who are aged at least 2 years. It is recommended that such 24

studies are conducted only if a licensed Vi conjugate vaccine comparator is not available, but it 25

is perceived important to generate comparative safety and immunogenicity data vs. a licensed 26

vaccine (see section C.2). If such studies are to be the basis for approval, data should be 27

generated for the age range for which a claim for use of the candidate vaccine will be sought. 28

Studies should stratify subjects by appropriate age subgroups, or separate studies should be 29

conducted in different age groups. 30

See section C.3. regarding possible approaches to the primary comparison of immune 31

responses. 32

The immune responses should be measured in samples collected at day 28 after the initial 33

vaccination series have been completed (i.e. after a single dose or after the last assigned dose of 34

the primary series), or in samples collected at an alternative time point if this is justified by data 35

on antibody kinetics. 36

C.4.3 Studies that compare vaccinated groups with unvaccinated groups 37

38


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There are two situations in which such studies may be considered: 1

i) As explained in section C.2, if there is an established immune correlate of protection, 2

a direct comparison of immune responses with a licensed vaccine is not necessary. 3

However, such a comparison may still be useful for interpreting the safety data and 4

for putting the immune responses to the candidate vaccine into some context. 5

6

ii) In the absence of an immune correlate of protection or the possibility of 7

immunobridging the candidate Vi conjugate vaccine to the protective efficacy of a 8

licensed Vi conjugate vaccine, a study that compares a candidate Vi conjugate 9

vaccine with an unvaccinated group could be considered for subjects who are younger 10

than 2 years. A comparison between a candidate Vi conjugate vaccine and a licensed 11

Vi polysaccharide vaccine would not be appropriate due to lack of reliable protective 12

immune responses to the latter in children aged < 2 years. In this setting, it is 13

recommended that studies employ randomized allocation to the candidate Vi 14

conjugate vaccine (i.e. the vaccinated group) or to a licensed non-typhoid vaccine 15

from which study subjects may derive some benefit (i.e. the control group). 16

17

The anti-Vi immune responses in the group receiving the candidate Vi conjugate vaccine 18

should be superior to those in the control (unvaccinated) group. To put the immune responses 19

observed into context, anti-Vi titres may be compared with one or both of: 20

• the immune response to an unconjugated Vi vaccine in subjects > 2 years 21

• the immune response to the candidate Vi conjugate vaccine in subjects aged > 2 years 22

23

C.5 Efficacy 24

25

This section should be read in conjunction with the guidance on efficacy trials and effectiveness 26

studies provided in the WHO Guidelines on clinical evaluation of vaccines: regulatory 27

expectations (158). 28

Protective efficacy studies against typhoid can be conducted only in endemic areas with 29

relatively high rates of disease. If a protective efficacy study is conducted, it should compare 30

rates of febrile illnesses associated with a positive blood culture for S. Typhi between a group 31

that receives the candidate Vi conjugate vaccine and an appropriate control group. A double-32

blind design is recommended but this would require that the control group is randomized to a 33

licensed Vi conjugate vaccine, if available, or unconjugated Vi vaccine if appropriate for the age 34

group selected. Otherwise, a non-typhoid vaccine that is indistinguishable in appearance from the 35

candidate conjugate vaccine and is administered in the same way (i.e. route and schedule) may 36

be considered. 37

Successful typhoid challenge studies conducted in healthy adults using an appropriate 38

and validated model (i.e. one in which some protective efficacy of unconjugated Vi vaccines is 39

detectable) could provide considerable supporting evidence of the efficacy of a Vi conjugate 40


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vaccine. Human challenge studies may provide information on the relationship between the 1

immune response and various efficacy parameters. If, in consultation with the NRA, sponsors 2

decide to conduct typhoid challenge studies in humans, they should be undertaken only by 3

physicians with appropriate expertise, and in a carefully controlled setting, to ensure the safety of 4

the volunteers (104). Healthy adults that are expected or known to be naïve to typhoid and 5

typhoid vaccines should be screened to detect any underlying pre-existing conditions that could 6

impact on safety. In particular, subjects who might be at risk of complications of typhoid should 7

be excluded, including any subject with gall bladder disease. The challenge strain should be well 8

characterized and there should be complete information on its susceptibility to antibacterial 9

agents. 10

An issue to consider after initial licensure is the possibility that widespread use of a Vi 11

conjugate vaccine and high immunization coverage in a population where typhoid fever is 12

endemic may lead to the emergence of otherwise rare Vi-negative variants of S. Typhi (169-13

172); these variants exist and can cause typhoid fever, albeit they have lower attack rates (105, 14

106). 15

C.6 Safety 16

17

Current evidence suggests there are no major specific safety issues for Vi conjugate vaccines 18

(173) and that reports of adverse events are similar to those of other polysaccharide conjugate 19

vaccines. It is recommended that the assessment of safety in pre-licensure studies should follow 20

the usual approaches to ensure comprehensive monitoring and data collection (158). 21

22


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Part D. Guidelines for NRAs 1

2

D.1 General guidelines 3

4

The general guidelines for NRAs and national control laboratories (NCLs) given in the WHO 5

Guidelines for national authorities on quality assurance for biological products (114) and the 6

WHO Guidelines for independent lot release of vaccines by regulatory authorities (174) should 7

be followed. These Guidelines specify that no vaccine substance or product should be released 8

until the manufacturer has demonstrated consistency in manufacturing and quality. 9

The detailed procedures for production and quality control, and any significant changes 10

in these that may affect the quality, safety or efficacy of a Vi polysaccharide conjugate typhoid 11

vaccine, should be discussed with and approved by the NRA. For control purposes, the relevant 12

international reference preparations currently in force should be obtained for the purpose of 13

calibrating national, regional, and working standards as appropriate. The NRA may obtain the 14

product-specific or working reference from the manufacturer for use in lot release testing. 15

Consistency of production has been recognized as an essential component in ensuring the 16

quality of vaccines. The NRA should carefully monitor production records and the results of 17

quality-control tests for clinical lots, as well as results for a series of consecutive lots of the final 18

bulk and final product. 19

D.2 Official release and certification 20

21

A vaccine lot should be released only if it fulfils the national requirements and Part A of these 22

Recommendations (174). 23

A model protocol for the manufacturing and control of TCVs is shown in Appendix 1; 24

this protocol should be signed by the responsible official of the manufacturing establishment and 25

should be prepared and submitted to the NRA in support of a request to release the vaccine for 26

use. This protocol may also be referred to as the Product Specification File. 27

A Lot Release Certificate signed by the appropriate official of the NRA should be 28

provided to the manufacturing establishment and should certify that the lot of vaccine meets all 29

national requirements and/or Part A of these Recommendations. The certificate should also state 30

the lot number, the number under which the lot was released, and the number appearing on the 31

labels of the containers as well as the basis of the release decision (by summary protocol review 32

or independent laboratory testing). In addition, the date of the last satisfactory determination of 33

critical quality parameters (such as the ratio of free Vi polysaccharide to bound Vi 34

polysaccharide) as well as the expiry date assigned on the basis of the shelf-life of the vaccine 35

should be stated. A model NRA Lot-release Certificate is given in Appendix 2. A copy of the 36

model protocol should be attached to the lot-release certificate – the purpose of which is to 37

facilitate the exchange of TCVs between countries. 38


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Authors and acknowledgements 1

2

The scientific basis for the revision of the Guidelines on the quality, safety and efficacy of 3

typhoid conjugate vaccines published in WHO Technical Report Series, No. 987, was discussed 4

at a WHO drafting group meeting, which met in Potters Bar, United Kingdom, 25-26 November, 5

2019 and was attended by the following: Dr A.D. Bentsi-Enchill, World Health Organization, 6

Geneva, Switzerland; Dr A. Pollard, University of Oxford, Department of Paediatrics, Oxford, 7

United Kingdom; Dr B. Bolgiano, National Institute for Biological Standards and Control, 8

Potters Bar, United Kingdom; Dr I. Fevers, Consultant, Suffolk, United Kingdom; Dr F. Gao, 9

National Institute for Biological Standards and Control, Potters Bar, United Kingdom; Dr E. 10

Griffiths, Consultant, Kingston upon Thames, Surrey, United Kingdom; Dr R. Isbrucker, World 11

Health Organization, Geneva, Switzerland; Dr I. Knezevic, World Health Organization, Geneva, 12

Switzerland; Dr M. Levine, University of Maryland School of Medicine, Baltimore, Maryland, 13

United States of America; Dr J.L. Mathew, Postgraduate Institute of Medical Education and 14

Research, Chandigarh, India; Dr L. Parsons, Center for Biologics Evaluation and Research, Food 15

& Drug Administration, United States of America; Dr S. Rijpkema, National Institute for 16

Biological Standards and Control, Potters Bar, United Kingdom; Dr T. Wu, Health Canada, 17

Ottawa, Canada; Dr S-H. Yoo, World Health Organization, Geneva, Switzerland. 18

This document was prepared by the above drafting group, which also included Dr M. Powell 19

(Health Products Regulatory Authority, Dublin, Ireland). Comments received from vaccine 20

manufacturers (Dr L.B. Martin, GSK Vaccines Institute for Global Health, Siena, Italy, and Dr P. 21

Talaga, Dr E. Zablackis, and Dr S. Gaudin of Sanofi Pasteur, Lyon, France) were also addressed. 22

23


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Appendix 1 1 2

Model protocol for the manufacturing and control of typhoid conjugate 3

vaccines 4 5 The following protocol is intended for guidance and indicates the minimum information that should be 6 provided by the manufacturer to an NRA. Information and tests may be added or omitted as required by an 7 NRA, and should include information if the intermediates, drug substances, drug product, or final vaccine 8 is manufactured by more than one commercial entity. 9 10

It is thus possible that a protocol for a specific product may differ in detail from the model 11 provided. The essential point is that all relevant details demonstrating compliance with the licence and 12 with the relevant WHO recommendations for a particular product should be given in the protocol 13 submitted. 14 15

The section concerning the final product must be accompanied by a sample of the label and a 16 copy of the leaflet that accompanies the vaccine container. If the protocol is being submitted in 17 support of a request to permit importation, it must also be accompanied by a lot-release certificate from 18 the NRA of the country where the vaccine was produced, stating that the product meets national 19 requirements as well as the recommendations in Part A of this document. 20

21

Summary information on the final lots: 22 23 International name of product: 24 Commercial name: 25 Product license (marketing authorization) number: 26 Country: 27 Name and address of manufacturer: 28 Final packing lot number: 29 Type of containers: 30 Number of containers in this packing lot: 31 Final container lot number: 32 Number of filled containers in the final lot: 33 Date of manufacture: 34 Nature of final product: 35 Preservative used and nominal concentration: 36 Volume of each recommended single human dose: 37 Number of doses per final container: 38 Summary of composition: 39

(Include a summary of the qualitative and quantitative composition of the vaccine per single human 40

dose; include the conjugate, any adjuvant used and other excipients) 41

Shelf-life approved (months): 42 Expiry date: 43 Storage conditions: 44

The following sections are intended for reporting the results of the tests performed during the 45

production of the vaccine, so that the complete document will provide evidence of consistency in 46


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production; thus, if any test had to be repeated, this information must be indicated. Any 1

abnormal results should be recorded on a separate sheet. 2

Detailed information on manufacture and control 3 SUMMARY OF STARTING MATERIALS 4

It is possible that a number of bulk lots may be used to produce a single final lot. A summary of the bulk 5 polysaccharide, activated saccharide, bulk carrier protein and bulk conjugate lots that contributed to the final 6 lot should be provided. 7 8 CONTROL OF TYPHOID Vi POLYSACCHARIDE 9

Bacterial strain 10

Identity of Salmonella Typhi Ty2 or 11

Citrobacter freundii: 12 Origin and short history: 13

Authority that approved the strain: 14 Date approved: 15

16 Bacterial culture media for seed-lot preparation and Vi production 17 Free from ingredients that form precipitate when 18

CTAB is added: 19 Free from toxic or allergenic substances: 20 Any components of animal origin (list): 21 Certified as TSE-free: 22

23

Master-seed lot 24 Lot number: 25 Date master-seed lot established: 26 27

Working-seed lot 28 Lot number: 29 Date working-seed lot established: 30 Type of control tests used on working-seed lot: 31 Date seed lot reconstituted: 32 33

Control of single harvests 34

For each single harvest, indicate the medium used; the dates of inoculation; the temperature of 35

incubation; the dates of harvests and harvest volumes; the results of tests for bacterial growth rate, 36

pH, purity and identity; the method and date of inactivation; the method of purification; and the yield 37

of purified polysaccharide. 38

39


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Control of purified typhoid Vi polysaccharide 1

Lot number: 2 Date of manufacture: 3 Volume: 4 5

Identity 6

Date tested: 7 Method used: 8 Specification: 9 Result: 10

11

Molecular size or mass distribution 12


17

Polysaccharide content 18


23

O-acetyl content 24


29

Moisture content 30


35

Protein impurity 36


41

Nucleic acid impurity 42


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5

Phenol content 6


11

Endotoxin content 12


17

Residues of process-related contaminants 18


23 Control of modified polysaccharide 24

Lot number: 25 Method of chemical modification: 26

27

Extent of activation for conjugation 28


33



39 CONTROL OF CARRIER PROTEIN 40

Microorganisms used 41

Identity of strain used to produce carrier protein: 42


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Origin and short history: 1 Authority that approved the strain: 2 Date approved: 3 4 Bacterial culture media for seed-lot preparation and carrier-protein production 5

Free from ingredients that form precipitate when CTAB 6 is added: 7

Free from toxic or allergenic substances: 8 Any components of animal origin (list): 9 Certified as TSE-free: 10

11 Master-seed lot 12

Lot number: 13 Date master-seed lot established: 14 15 Working-seed lot 16

Lot number: 17 Date established: 18 Type of control tests used on working-seed lot: 19 Date seed lot reconstituted: 20 21 Control of carrier-protein production 22

List the lot numbers of harvests: indicate the medium used; the dates of inoculation; the temperature 23

of incubation; the dates of harvests and harvest volumes; the results of tests for bacterial growth rate, 24

pH, purity and identity; the method and date of inactivation; the method of purification; and the 25

yield of purified carrier protein. Provide evidence that the carrier protein is nontoxic. 26

27

Purified carrier protein 28

Lot number: 29 Date produced: 30 31

Identity 32


37

Protein impurity 38



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1

Nucleic acid impurity 2


7 Modified carrier protein 8

Lot number: 9 Date produced: 10 Method of modification: 11 12

Extent of activation 13


18 CONTROL OF PURIFIED BULK CONJUGATE 19

Production details of bulk conjugate 20

List the lot numbers of the saccharide and carrier protein used to manufacture the conjugate vaccines, 21

the production procedure used, the date of manufacture and the yield 22

23

Tests on purified bulk conjugate 24

Identity 25

Date tested: 26 Method used: 27 Specification: 28 Result: 29 30

Endotoxin content 31


36

O-acetyl content 37

Date tested: 38 Method used: 39 Specification: 40


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Result: 1 2

Residual reagents 3


8

Vi polysaccharide content 9


14

Conjugated and unbound (free) polysaccharide 15


20

Protein content 21


26

Conjugation markers 27


32

Absence of reactive functional groups (capping markers) 33


38

Ratio of polysaccharide to protein 39



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1



7

Bacterial and mycotic bioburden 8

Method used: 9 Media: 10 Volume tested: 11 Date of inoculation: 12 Date of end of test: 13 Specification: 14 Result: 15

16

Specific toxicity of carrier protein (where appropriate) 17

Method used: 18 Strain and type of animals: 19 Number of animals: 20 Route of injection: 21 Volume of injection: 22 Quantity of protein injected: 23 Date of start of test: 24 Date of end of test: 25 Specification: 26 Result: 27

28

pH 29


34

Appearance 35


40

Depending on the conjugation chemistry used to produce the vaccine, tests should also be included 41

to demonstrate that amounts of residual reagents and reaction by-products are below a specified 42

level. 43


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1

CONTROL OF FINAL BULK 2

Lot number: 3 Date prepared: 4 5

Preservative (if used) 6

Name and nature: 7 Lot number: 8 Final concentration in the final bulk: 9

10

Stabilizer (if used) 11


15

Adjuvant (if used) 16


20 Tests on final bulk 21

Bacterial and mycotic sterility 22

Method used: 23

Media: 24

Volume tested: 25 Date of inoculation: 26

Date of end of test: 27

Specification: 28

Result: 29 30 FILLING AND CONTAINERS 31

Lot number: 32 Date of sterile filtration: 33 Date of filling: 34 Volume of final bulk: 35 Volume per container: 36 Number of containers filled (gross): 37 Date of lyophilization (if applicable): 38 Number of containers rejected during inspection: 39 Number of containers sampled: 40 Total number of containers (net): 41 Maximum duration approved for storage: 42 Storage temperature and duration: 43


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1 CONTROL TESTS ON FINAL LOT 2

Inspection of final containers 3

Date tested: 4 Method used: 5 Specification: 6 Results: 7 Appearance before reconstitution:4 8 Appearance after reconstitution:4 9 Diluent used: 10 Lot number of diluent used: 11 12

Tests on final lot 13

Identity 14


19

Sterility 20

Method used: 21 Media: 22 Number of containers tested: 23 Date of inoculation: 24 Date of end of test: 25 Specification: 26 Result: 27

28

Polysaccharide content 29


34

Unbound (free) polysaccharide 35


40

4 This applies to lyophilized vaccines.


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O-acetyl content 1


6



12

Endotoxin or pyrogen content 13


18

Adjuvant content and degree of adsorption (if applicable) 19

Date tested: 20 Nature and concentration of adjuvant per single 21

human dose: 22 Method used: 23 Specification: 24 Result: 25

26

Preservative content (if applicable) 27


32

General safety 33


38

pH 39

Date tested: 40 Method used: 41 Specification: 42


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Result: 1

2

Moisture content5 3


8

Osmolality 9


14 Control of diluent (if applicable) 15

Name and composition of diluent: 16 Lot number: 17 Date of filling: 18 Type of diluent container: 19 Appearance: 20 Filling volume per container: 21 Maximum duration approved for storage: 22 Storage temperature and duration: 23 Other specifications: 24 25 CONTROL OF ADJUVANT6

26

Summary of production details for the adjuvant 27

When an adjuvant suspension is provided to reconstitute a lyophilized vaccine, a summary of the 28

production and control processes should be provided. The information provided and the tests performed 29

depend on the adjuvant used. 30

31

Summary information for the adjuvant 32 Name and address of manufacturer: 33 Nature of the adjuvant: 34 Lot number: 35 Date of manufacture: 36 Expiry date: 37 38 Tests on the adjuvant 39

5 This applies only to lyophilized vaccines 6 This section is required only when an adjuvant is provided separately to reconstitute a lyophilized vaccine.


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Adjuvant content 1


6

Appearance 7


12

Purity or impurity 13


18

pH 19


24

Pyrogenicity7 25


30

Sterility 31

Method used: 32 Media: 33 Number of containers used: 34 Date of inoculation: 35 Date of end of test: 36 Specification: 37 Result: 38

39

CERTIFICATION BY THE MANUFACTURER 40

7 A pyrogen test of the adjuvant is not needed if a pyrogen test was performed on the adjuvanted reconstituted vaccine


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Name of head of quality control (typed) 1

Certification by the person from the control laboratory of the manufacturing company taking 2

overall responsibility for the production and quality control of the vaccine 3

I certify that lot no. of typhoid conjugate vaccine, whose 4 number appears on the label of the final containers, meets all national requirements and/or satisfies Part 5 A8 of WHO Recommendations on the quality, safety and efficacy of typhoid conjugate vaccines (20).9 6

Signature 7 Name (typed) 8 Date 9 10

Certification by the NRA 11

If the vaccine is to be exported, attach a Lot-release Certificate from the NRA (as shown in Appendix 2), a 12 label from a final container and an instruction leaflet for users. 13

8 With the exception of provisions on distribution and shipping, which the NRA may not be in a position to assess. 9 WHO Technical Report Series, No. 987, Annex 3


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Appendix 2 1

2

Model NRA Lot-release Certificate for typhoid conjugate vaccines 3 4 Certificate no. 5 6 The following lot(s) of typhoid conjugate vaccine produced by 10 in7 ,11 8 whose numbers appear on the labels of the final containers, meet all national requirements12 and Part A13 9 of the WHO Recommendations on the quality, safety and efficacy of typhoid conjugate vaccines (2020)14 10 and complies with WHO Good manufacturing practices: main principles for pharmaceutical products;15 11 Good manufacturing practices for biological products;16 and Guidelines for independent lot release of 12 vaccines by regulatory authorities.17 13 14 The release decision is based on 18

15 16 Final lot number: 17 Number of human doses released in this final lot: 18 Expiry date: 19 20 The Director of the NRA (or other appropriate authority) 21 22 Name: (typed) 23 Signature: 24 Date: 25 26

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10 Name of manufacturer 11 Country of origin. 12 If any national requirements have not been met, specify which one(s) and indicate why the release of the lot(s) has nevertheless been authorized by the NRA. 13 With the exception of provisions on distribution and shipping, which the NRA may not be in a position to assess. 14 WHO Technical Report Series, No. 987, Annex 3. 15 WHO Technical Report Series, No. 961, Annex 3. 16 WHO Technical Report Series, No. 822, Annex 1. 17 WHO Technical Report Series, No. 978, Annex 2. 18 Evaluation of the summary protocol, independent laboratory testing, or procedures specified in a defined document etc., as appropriate

recommendations to assure the quality, safety and efficacy ... · 10 the named authors [or editors...

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