Usp vaccines-biological medicines

Track II, Session II: Biological
Medicines–Vaccines
Wednesday, April 17, 2013 (11:30 a.m. to 1:30 p.m.)
IPC–USP Science & Standards Symposium
Partnering Globally for 21st Century Medicines

Moderator: Mahesh Bhalgat, Ph.D.
USP Medicines Compendium Expert Committee

Comparing the Opportunities and
Challenges in Thermostable Vaccines
with Conventional Vaccines
Rajesh Jain, Ph.D.
Joint Managing Director
Panacea Biotec Limited, New Delhi
Panacea Biotec Ltd. All Rights Reserved, Privileged & Confidential

April 17, 2013

Contents
• Company Overview
• Vaccines needed across all age groups

• Concept of Vaccines
• Cold Chain
• Thermostable Vaccines

Panacea Biotec Ltd., All Rights Reserved. Priviledged & Confidential

Company Overview
Business Lines

Ranking

Infrastructure

• Pharmaceuticals
• Vaccines & Biotherapeutics (MABs, Peptides, r-Proteins)

• Largest Vaccine Producer in India
• 3rd Largest Biotechnology Company in India (ABLE Survey 2011)
• Ranked 39th amongst Pharmaceutical Companies in India (ORG-IMS, 2011)
• Four Research & Development Centers
• Established Sales & Distribution Network in India, 50 branded products
• Direct presence in Germany for specialty hospital products segment
• Presence in 55 ROW and Emerging Markets
• cGMP Manufacturing Facilities

Manpower

• 3,800 Human Resource
• 291 in R&D
• 1,200 in Sales and Marketing

Financials

• Turnover : INR 1,130 Cr. (~USD 250 Mn.), CAGR of 21%
• Listed in BSE and NSE

Vaccine Business – Key Highlights
Robust Pipeline

Established Presence

• Innovative vaccines
• Universal Flu (Recombinant viral vectored)

• Established presence of over 25 years in vaccines

• Dengue (Chimeric Recombinant Tetravalent )

• Reliable partner to WHO, UNICEF: largest supplier of

• DTwP/DTaP- IPV combination (Fully Liquid
combination Vaccines)

vaccines to UNICEF from India
• First Indian Company to launch innovative branded

• Recombinant Anthrax Vaccine
• Seasonal Flu (Quadrivalent seasonal Vaccine)

combination vaccine – Easyfive (Hep. B+DTP+Hib) and

• Pneumococcal Conjugate (13 valent conjugate)

other combination vaccines – Easyfour, Ecovac

• Japanese Encephalitis (Inactivated cell culture
based viral vaccine)

• One of the 3 Companies chosen by Govt. of India to
develop Flu vaccine (Seasonal & Pandemic Flu)
• Joint venture with Chiron Corp. (Novartis) for marketing
of Branded combination vaccines in India

• Meningococcal (Tetravalent conjugate vaccine)
• Adolescent Vaccines
• Tdap (AdTdap)
• Tdap- IPV (AdTdap -pol)

Innovation Infrastructure

Laksh, Mohali
Drug Discovery: Small Molecules
• Target identification to development of pre-clinical
candidate
• Focus areas : Metabolic disorders, Anti-infectives, CNS

GRAND, Navi Mumbai
NDDS Product Development
• Platform NDDS technologies : Nanoparticles,
Liposomes, Micro-particles, Depot Injections, SPORT,
Oral films etc.
• High barrier to entry generics

OneStream, New Delhi
Drug Discovery: Novel Biologicals
• Target identification to development of pre-clinical candidate
• Focus areas for Novel Peptides: Metabolic Disorders
• Biosimilars & Vaccines

Sampann, Lalru
Generic Product Development
• High barrier to entry generics
• NDDS technologies: Depot Injections, Oral modified release,
SMEDDS in Softgels, MD tablets, Critical dose drugs
• Bio-therapeutics & Vaccine Formulation Development

Pipeline Overview

Pipeline

Generics

Generics
US:
• 2 ANDAs filed
• 45 ANDAs in
development

NDDS

NCE

NDDS

Europe:
• 1 MAA
granted
• 20 products in
development

• 7 best in class
NDDS products
in development
(Scientific
advice from US
/ European
agencies has
been taken for
5 products)

NCE & Peptides
• 4 Best in Class
NCE
• 1 Atypical NCE
• 2 Novel Peptides

Biosimilars,
Peptides,
Vaccines

Biosimilars,
Peptides,
Vaccines
• 4 Biosimilars in
development
• 4 Peptides in
development
• 10 Innovative
vaccines in
development

Commitment to make Affordable Vaccines…

The Pledge
June 2011,
London

“We at Panacea Biotec wish to express our continued, unstinted & unequivocal
commitment & support to further the cause of GAVI Alliance.
In this initiative we pledge to bring down cost of pentavalent vaccine (EasyFive) by
minimum 15% in the coming years as a tiny contribution to a mammoth cause!”

Vaccine - Manufacturing Capabilities
Vaccine Formulation Facility

Location

Delhi

Formulation Facilities

2 lines for Oral vaccines

2 lines. 1 for Vials , 1 for PFS
switch able to lyophilized in Vials

Baddi

One more filling line under
construction

Capacities
(million doses/ annum
)

1600

PFS-16
Single dose -39
10 Dose -350

Formulation Capacity of 2 billion doses p.a (Includes Vials & PFS)

Built Up Area

> 50,000 sq ft

> 129,167 sq ft

Vaccine - Manufacturing Capabilities
Indigenous & fully integrated bulk antigen manufacturing facilities
Location

Bulk Antigen Facilities

Built Up Area

Recombinant Vaccines
Bacterial Vaccines

> 18,000 sq. ft

Tetanus Vaccine

> 20,000 sq. ft

Cell Culture Vaccines

Lalru

> 40,000 sq. ft

> 30,000 sq. ft

Licensed Bulk Antigens –
• Diphtheria Toxoid • Tetanus Toxoid • Whole cell / acellular Pertussis
• Haemophilus influenzae type b conjugate • Recombinant Hepatitis B
• Inactivated H1N1 split viron influenza vaccine bulk (by traditional egg based technology)
Facility has provision for production of Bulk Antigens which are under development
Vaccines : Japanese Encephalitis ,Sabin IPV, Dengue Vaccine, egg based seasonal flu vaccine, yellow
fever vaccine
Bio therapeutics : Viral proteins , non-viral recombinant bio molecules on cell culture in both conventional
& disposable formats .

(US-FDA / UK-MHRA compliant)

Vaccines needed across all age groups

•

Need of pre-birth vaccination
• Mothers transfer fewer protective antibodies to their infants because of less exposed to
infectious agents, breast feeding is less common & of shorter duration. Also, as current
schedules starts @ 11/2 – 2 months of age, there is period of vulnerability during 4–6
months of life with significant mortality & morbidity

In 21st century vaccines will be needed across all age groups
Source: Rappuoli R. Nature reviews. Immunology, Vol 11, Dec 2011, pp. 865-872.

Vaccines
• Concept of Vaccine`
– Training of immune system to face various existing disease agents
– Generate memory cells.

•

Prophylactic treatment against disease.

•

Most effective treatment against any disease so far.

Vaccines-success stories so far
• Made eradication of many life threatening diseases possible.
– Smallpox – Eradicated in1979
– Polio – Significant elimination
– Measles – Significant elimination

– Rubella – Significant elimination

• Still in progress
– Hepatitis-B: 260,000 in the 1980s to about 60,000 in 2004.
– Diphtheria: No case reported in USA after 2003.

Vaccines - Limitations
• Limited production capacities

• Failure to protect immuno-compromised patients.
• Instability issues
– Heat and freeze degradation.

– Unfolding of structure.
– Hydrolysis, oxidation, deamidation.
– Inherent instability of large molecules.

• Additional costs of cold chain logistics and storage.

Temperature sensitivity of vaccines
• Vaccines lose potency over time
and the rate of potency loss is
temperature-dependent.

• Both,

high

temperature

as

well

as

(freeze)

low
are

detrimental to vaccine quality
– Most aluminum salt adjuvant containing
vaccines are freeze-sensitive (Require
Freeze stabilization)
– Live attenuated vaccines are sensitive to
heat (Require Heat Stabilization)

Cold-chain have been established to ensure that the
potency of vaccines is maintained until the point of
use.

Cold chain
• A

cold

chain

is

a

time

temperature-controlled

and

supply

chain which provides a series of
facilities

for

maintaining

ideal

storage conditions from the point of
origin to immunization site.
• Nearly all vaccines require cold
chain

for

proper

transportation

while maintaining potency.

Cold Chains- Risks associated
• Drawback : Costly and complex distribution logistics.
– In the developed world, maintaining the cold chain is estimated to cost up to $200 million a
year and increases the cost of vaccination by 14–20% (World Health Organization)

• 75-100% of vaccines at some stage of transportation experience “temp
excursions” . Usually occurs in the tail end of the cold chain.
• Risk of vaccine wastage and
associated costs.
– 50% losses in emerging nations
(GAVI report).
– 10% losses in established markets
(Australian MoH report).

• Need more space for storage and
transport.

WHO recommended storage temperature

Reality of cold chains in
underdeveloped countries

What we are looking for ???
• Thermo-stable vaccine which can be stored at room temperature
(Reformulation)
• Redefining the thermal stability of existing vaccines for relaxing the cold
chain requirements
• Developing new strains with inherent thermostability.
Vaccine transported or stored at room temperature ??

Thermo stable vaccines
• Vaccines neither requiring refrigeration nor affected by freezing.
• Thermostable vaccines will help in
– Decreasing the cost of vaccine stockpiling.
– Improving the efficacy of vaccine.
– Energy cost saving (due to cold chain)
– Reducing wastage of vaccines.

• Will ensure vaccine stability in remote areas of the world with limited or no
access to electricity for cold chain.
Thermostable vaccines represent a better opportunity to increase the outreach of
global immunization program.

Ambient storage condition-WHO
Measured mean annual data

Long-term stability
testing conditions

Zone

Climate/Definition

Temperature
(Open air, °C)

Partial vapor
pressure (hPa)

I

Temperate climate

≤15

≤11

21°C/45%

II

Subtropical and Mediterranean

>15-22

>11 to 18

25°C/60%

III

Hot and dry

>22

≤15

30°C/35%

IVa

Hot and humid

>22

>15 to 27

30°C/65%

IVb

Hot and vary humid

>22

>27

30°C/70%

How to overcome these problems?
• Explore genetically modified strains that address specific stability issues.
• Apply novel formulation concepts and processing technologies
– Spray drying, foam drying, and lyophilization, etc.

• Employ computational analysis of protein structure to inform formulation
design.

Thermostable vaccines – ongoing research
• Use of silk protein biomaterial matrices for stabilization of MMR vaccine (Tufts University, USA).
• Use of lipid particle vaccines platform (VBI ).
• Use of osmolytes for Hep-B vaccines (PATH, USA).

• ThermoVax platform (RiVax™, Velothrax™).
• HydRIS technology platform (Oxford University and Nova Bio-Pharma )
• Use of Glycerin, PEG-300, Propylene glycol for freeze sensitive formulation.
• Coated microneedle Patches (influenza virus by University of Queensland).
• Oral tablets (E-coli by Johns Hopkins Bloomberg School of Public Health & PATH, Polio
vaccines).
• Flu vaccine by Powderject, USA

Sugar Glass Technology
• A joint effort by Durer Chemical corporation USA, CSIR Australia

• Utilize stabilizing abilities of sugars is used for stabilizing vaccines.
• Measles , DTaP vaccines successfully stabilized.
• Pre-clinical investigations have demonstrated the immunogenicity and
potency of the trehalose-dried vaccine candidate.

ThermoVax Technology
•

A proprietary vaccine formulation platform from Soligenix,
thermostable properties to aluminum adjuvanted vaccines.

•

Makes alum vaccines resistant to freeze/thaw and Heat excursions.

•

Delivers long-term stabilization of labile antigen-adjuvant combinations

•

Maintains native structure

US

bestowing

– Applicable to many types of commercial vaccines Polysaccharide conjugates,
VLPs, recombinant subunit proteins and peptides
– Complex vaccines utilizing “secondary adjuvants”

– Combination as well as multivalent vaccines
•

Scientific merit of ThermoVax™ technology validated through $9.4M grant award
Grant provides for stabilization of its proprietary ricin vaccine (RiVax™) and anthrax
vaccine (Velothrax™)

Preservation by Vaporization (PBV)
•

Under study for YF-VAX 17D by Universal Stabilization Technologies, US

•

Higher activity titer after drying and thermostability during subsequent storage
(increased shelf-life).

•

Allows subsequent particle size reduction (micronization).

•

Allows short-term stability at 60°C to 90°C that could be used for encapsulation of dry
powders for buccal and transdermal delivery avoiding a need of reconstitution with
water.

Ambient storage condition-WHO

POC
VeloThrax, rPA, Soligenix

Thermostable IM Measles

Microneedle patch for flu

HepB Formulations, PATH

Preclinical

Phase I/II

Phase III

Marketed

Challenges in thermostable vaccine development
•

Commercial
–
–

Patents rights are in hand of very few companies and individuals.

–

•

High cost and time associated with development and licensure of thermostable vaccines

Higher Product Cost would impact the immunization programs in developing world

Regulatory
–

Addition of novel stabilizer/adjuvant/excipient/process require huge data and cost involved.

–

Choice of excipients/stabilizer to achieve limited Target population (Infants) have extremely low
regulatory tolerance for adverse effects.

–

Stability indicating markers and correlates of protection should be identified. Better clarity on the
stability targets from the policy makers is desired.

–

Thermostable vaccines without cold chain require new regulations, policies, and logistic systems in
additional to new formulation technologies.

Thermostable Vaccine – industry Perspective
• Cost vs. benefit has to be evaluated by industry.
– Funding model for bearing the developmental cost should be identified

– Developmental cost vs. saving by cold chain should be evaluated
– Priority development of vaccine candidate e.g. Pentavalent, MMR,Polio.

• Wider and open view of regulatory agencies
– Relaxation of transportation guideline for already stable vaccines (e.g. D, T & Hep-B).
– Separate transport and storage conditions for individual vaccines.

Developmental initiatives
• Bill & Melinda Gates Foundation (Measles vaccine project to Dr. Paul
Duprex , TransForm Pharmaceuticals, Inc., Massachusetts, United States -

US
• PATH (Freeze and heat stable Liquid HepB Formulation).
• BARDA (Awarded US$2.5 million funding to advance the development of
thermostable influenza vaccines)

Summary
•

Integrate stabilization approaches in early vaccine development.

•

Many vaccines are more stable than we assume e.g. Human rotavirus vaccine, HepB & tetanus toxoid. New supply chain model (combination of cold chain and
controlled temperature chain), distribution policies and logistic system need to
developed for them

•

Funding model to take-care the developmental cost and the risk associated with

them needs to be developed.

Evolving regulatory framework
for vaccine PQ
 IPC-USP 12th Science & Standards
Symposium
 New Delhi India 16-17 April 2013
Carmen Rodriguez Hernandez
QSS-EMP-HIS-WHO

Purpose of WHO vaccines prequalification
programme

 A service provided to UN purchasing agencies.
 Provides independent opinion/advice on the quality, safety
and efficacy of vaccines for purchase
 Ensures that candidate vaccines are suitable for the target
population and meet the needs of the programme
 Ensures continuing compliance with specifications and
established standards of quality

36 |

Evolving Regulatory framework for Vaccines PQ
17 April 2013
Carmen Rodriguez Hernandez QSS/EMP/HIS/WHO

The following displays the count of finalised
submission/review processes which are on time (Internal time is
less than or equals 12 months) and those which are overdue
(Internal time exceeds 12 months), by year of submission

2012: first streamlined submission under US FDA: Timeframe taken for PQ was
198 days . Quality review done by WHO . Two additional submissions received 30
September, 2012. Evaluation ongoing
37 |

17 April 2013

PQ activity

2009

2010

2011

2012
Jan-Sept

Reassessments

11

12

10

12

Annual reviews and
variations

6

21

74*

53**

Testing (lots)

124

159

183

105

Complaints/ other
issues of concern

3

13

16

12

AEFI

7

7

7

5

Meetings with
manufacturers

62

80

71

119

Meeting with NRAs
and others

33

86

64

109

38
* Refers to changes ** Refers to Vaccines containing 448 total changes

Vaccine PQ activities to facilitate access
 Secure the existing supply of vaccines
 Explore alternative sources
 Work with NRAs from user countries to facilitate
registration of vaccines
 Sustain functionality and secure risk mitigation strategies
(SOP to be published shortly)

 Mechanisms to minimize wastage of vaccines, facilitate
outreach (VVMs, MDVP, CTC)
39 |

17 April 2013

Procedure for expedited review of imported
prequalified vaccines for use in national
immunization programmes
 Concept proposed by 6 SEARO countries in 2005.
 Meeting with WHO HQ and RO in 2005

 Discussed need for registration in all countries
 Proposed a "facilitated process" for registration (MA) of imported
prequalified vaccines

EXPEDITED REVIEW PROCEDURE FOR
LICENSING PQ VACCINES
Expert Committee on Biological Standardization
40 |

17 April 2013

Support to NRAs
Implementation of Procedure for expedited review of imported
prequalified vaccines for use in national immunization
programmes (WHO/IVB/07.08)
Firstly used
for registration
of MenAfriVac
(16 countries)

41 |

17 April 2013

Implementation workshops- AFRO

July 2011

July 2011

1
2
3
4
5
6
7
8

Uganda
Ghana
Nigeria
Rwanda
Tanzania
Guinea Bissau
Kenya
Ethiopia

1

Burkina Faso

2

Benin

9

3

5

Central African
Republic
Côte d'Ivoire

6

Gabon

Cameroon

7

Gambia

8

Sénégal

11

Eritrea

9

Togo

2

Ethiopia

3

Gambia

4

Kenya

5

Malawi

6
7

Namibia
Uganda

8

Sierra Leone

Tchad

10

Botswana

Mauritanie

10

1

Burundi

4

July 2012

42 |

17 April 2013

Implementation workshops- WPRO

November 2012
1.

CAMBODIA

2.

LAO PEOPLE'S DEMOCRATIC
REPUBLIC

3.

MONGOLIA

4.

PAPUA NEW GUINEA

5.

PHILIPPINES

6.

SOLOMON ISLANDS
VANUATU

7.

43 |

17 April 2013

Status of agreements with NRAs
NRA/Country

Status

AIFA/ Italy

Discussions ongoing

ANSM (France)

Discussions ongoing

ANVISA (Brazil)

Signed

BELGIUM

Discussions ongoing

BULGARIA

Signed

CECMED (Cuba)

Signed

HC (Canada)

Signed

CDSCO – DCGI (India)

Discussions ongoing

BADANPON (Indonesia)

Signed

KFDA (Korea)

Discussions ongoing

JAPAN

Discussions ongoing

SWISSMEDIC (Switzerland)

Discussions ongoing

THAI FDA

Signed

US FDA (US) framework for Vaccines PQ
Evolving Regulatory

Signed

47 |

17 April 2013

Regulatory networks: DCVRN
Network of regulators from developing countries: 2004
– NRA Meets criteria of functionality or Government endorsed workplan &
Expertise
– Local PQ vaccine manufacture / Clinical trials

 Members: Brazil, China, Cuba, India, Indonesia, Korea, South Africa,
Thailand and Iran

 Strengthen capacity of National Regulatory Authorities

• both for Members and other Developing Countries
• Initial focus on regulatory control of Clinical trials
• through exchange of experience and information

48 |

17 April 2013

Regulatory networks: AVAREF
Network of regulators from African region: 2006
– To provide information to countries targeted for clinical trials of
vaccines.
– To promote and strengthen communication and collaboration
between NRAs and Ethics Committees in countries where
vaccines are being developed and those targeted for clinical trials
in the African Region.
– To provide expertise to African NRAs in support of regulation and
evaluation of vaccines.
Members:one representative each of the National Regulatory Authorities
and of the National Ethics Committees of 19 countries in the African
region
49 |

17 April 2013

Regulatory networks: Discussions
DCVRN

AVAREF

Meetings

Bi-annual-Annual FtF
Annual meetings: 7
meetings + web meeting:
12 meetings held + 2
web meetings

Participants

regulator from mature NRAs (USFDA, HC, EMA)
Other regulators from the region as observer

Sessions

Scientific session
Closed session

50 |

17 April 2013

QC labs- NETWORK OF WHO VPQ
CONTRACTED LABORATORIES
 ANSM France

 NIBSC UK

 Cantacuzino Romania

 PEI Germany

 Department of
Medical Sciences
(DMSC) Thailand

 RIVM

 Health Canada

 South African National
Control Laboratory

 Korea Food and Drug
Administration
 NCE Hungary
51 |

17 April 2013

 SIPH Belgium

 Swissmedic

 TGA Australia

Qualification of WHO contracted
laboratories
 Phase 1:

 Phase 2: Visit to the lab for
evaluating

 Review of SOPs and
related documents

 Organisation of the Laboratory

 Validation protocol and
report
 Testing 3 batches in
parallel with a
contracted laboratory

 Quality System
 Personnel

 Premises and equipment
 Handling of samples
 Reagents and Reference material
 Test methods


52 |

17 April 2013

Test reports

VPQ TESTING PROGRAM

Challenges
• Increased demand for
evaluation of vaccines
Novel vaccines to be evaluated:
Need for new tests
• High demand for evaluation of
combination vaccines: Increased
complexity, results are not
consistent, need for standardization
and harmonization

53 |

17 April 2013

Solutions
• Identification of new
laboratories/additional
capacity
Transfer of methodologies to
WHO contracted labs

• Project on stardardization of Hib:
WHO-EDQM
• Pre-testing phase
• Suitability of the method:
Collaborative study
• ECBS

National cold room during the campaign
54 |

Contribution to development
of Controlled Temperature
Chain
Project Optimize: PATH/WHO
Mali, polio
campaign,

Photos: WHO/Olivier
Ronveaux

Nicaragua, rotavirus delivery,

Photo: Gates Foundation

Transport to health centre

Allow specific vaccines to be kept and administered at
ambient temperatures, up to 40oC
For one, limited period of time immediately preceding administration
For vaccines meeting a number of stability conditions

Current focus: vaccines administered during campaigns and special strategies:
eg Meningo conjugate A, Yellow Fever, Pneumo, Hepatitis B, Rota, Cholera

Manufacturers

Regulators

Studies to enable
on label use of
vaccines under
CTC and regulatory
submissions

Regulatory
pathways

55 |

Review data
for licensing
under CTC

WHO
CTC Guidelines(QSS)
Work w/regulators to
define Regulatory
Pathways and
prequalification (QSS)
Field studies to show
programmatic
challenges ,
opportunities and
impact of CTC (EPI)

Capacity building in countries

NRAs
• NRA Observers
in PQ
evaluation
procedures
• Joint reviews of
PSF with NRAs
of producing
countries/NRA
networks

56 |

Laboratories

• Collaboration between
targeted testing program
and NCL networks
globally
• Support to NCL for the
establishment of critical
testing methods relevant
to PQ vaccines
• Harmonization of test
methodologies

17 April 2013

PQ Programme to
leverage
networking
approach to
strengthen
regulatory capacity
including
regulation of
clinical trials
(DCVRN, AVAREF)

Alternative to Animal Testing in the
Quality Control of Vaccines and
Regulatory Acceptance
Mahesh Bhalgat, Ph.D.
Biological E Limited

Overview



Current approaches to vaccine manufacturing and
testing
– The use on in vivo testing methods



Regulatory acceptance of in vitro methods



Opportunities for advancement of alternate
methods



Demonstrating product quality, safety and efficacy
using the consistency approach



Conclusions

Steps In Vaccine Manufacturing
Production of the established
(inactivated) vaccines
Virulent micro-organism/toxin
Culture
Concentration
Detoxification/inactivation
Purification
Blending (adjuvant, antigens)

Inactivated
vaccine/toxoid final lot


Safety tests



Potency tests

Current Considerations In Lot Release Of Vaccines


S

 Starting

point is uniqueness of every lot
produced

 Focus

of lot release testing on final

product
 Use

of a international reference preparation
expressed in IU/ml

 Reliance

on animal models for safety

and potency

Limitations Of Current “In Vivo” Based Approaches

 Scientific Aspects

 Validity aspects : reproducibility tests, questionable relevance.
 Use of reference preparation (not like to like)
 Science: based on models developed > 50 yrs ago

 Ethical Aspects

 Practical

: Extensive animal use

aspects : Costs, time required

The Relevance Of Biological Assays

 Ligand/receptor assays (not cell based)
 SPR

Variability

Relevance

 Modern cell based bioassays (early read out)
 Receptor binding
 KIRA
 PACE
 Reporter gene assay

?

 Classical cell based bioassays (late read out)
 Proliferation assay
 Death of cells as read out

 In vivo bioassays
 Includes in vivo clearance event

63

Speed

Species Difference Can Be Unpredictable



For humans the circulating leukocyte profile is 50-70% neutrophils
but for rodents it is 50-100% lymphocytes. (Haley, 2003)
– Relevance of measuring leukocyte related changes (immunogenicity) in alternate
species?

***********************************************************************


Mouse spleens are major sites for lifelong hematopoietic activity
while humans have little hematopoietic activity in embryonic spleens
and virtually none in adult spleens. (Haley,2003)
– Relevance of using mouse spleen cells as target cells in immunotoxicity assays?

***********************************************************************


TCDD (a Dioxin derivative) causes a dose-dependent suppression
of the T-cell Dependent Antibody Response in adult female B6C3F1
mice, but enhances the TDAR in F344 and Long-Evans rats even at
high doses. (Smialowicz et al., 1994)
– Relevance to humans?

Animal Usage In Human And Veterinary Vaccines
•

Used in:
•
•
•

Vaccine development (research, validation of efficacy)
Production (sometimes in animals, primary cell cultures, eggs)
Batch control testing (safety and potency testing)

•

Routine batch control testing is responsible for 80% of animal use in
vaccine industry and regulation

•

Batch control testing of vaccines accounts for ~10% of all animal
use in biomedical research, using 10 million animals every year

•

Biologicals testing has the highest proportion and number of
experiments causing severe pain and distress to animals out of
various types of experiments (basic research, toxicity testing, etc.)

Sadhana Dhruvakumar, PETA, Feb 2005

The 3R’s Approach



Replacement:
– Substitution of insentinent material for conscious living
higher animals



Reduction:
– Reduction in the number of animals used to obtain
information of given amount of precision



Refinement:
– Decrease in the incidence of severity of inhumane
procedures applied to those animals which still have to
be used

Russell and Birch

Overview



Current approaches to vaccine manufacturing and
testing
– The dependency on in vivo methods



Regulatory acceptance of in vitro methods



Opportunities for advancement of alternate
methods



Demonstrating product quality, safety and efficacy
using the consistency approach



Conclusions

European Center For The Validation Of Alternative Methods
(ECVAM) – Selected Examples


Replace:
•

Validated commercially available ELISA kits for rabies potency testing
(1999)
• Validated human-blood-based pyrogenicity test (2005)
• Validated Vero cell test for specific toxicity testing of diphtheria toxoid


Refine:
•

Sponsored development of humane endpoints for rabies, pertussis, and
erysipelas challenge tests (1999)
• Validated ELISA and ToBI test for batch potency testing of human
tetanus vaccine (2000)
• Validated ELISA test for swine erysipelas vaccine


Reduce:

•

Validated ELISA and ToBI test for batch potency testing of human
tetanus vaccine (2000)
• Validated ELISA test for swine erysipelas vaccine

European Directorate for Quality Medicine(EDQM)/European
Pharmacopeia (Ph. Eur.) – Selected Examples


Replace:
•
•
•
•
•



Refine:

•
•


Accepts vaccination-serology tests for tetanus, diphtheria, and cholera vaccines
(in lieu of vaccination-challenge)
Recommends use of humane endpoints in vaccination-challenge procedures

Reduce:
•
•



Accepts antigen quantification test for rabies (1998)
Deleted Abnormal Toxicity Test (in favor of production consistency approach)
Deleted guinea pig test for diphtheria (residual toxin and irreversibility of
diphtheria toxoid)
Deleted in vivo test for polio (for some manufacturers*)
Deleted residual pertussis toxin test for acellular pertussis (for some
manufacturers*)

Accepts single dilution assays for diphtheria, tetanus, and acellular pertussis
vaccines (in lieu of multiple dilution assays)
Accepts vaccination-serology tests for tetanus, diphtheria, and cholera vaccines
(in lieu of vaccination-challenge)

*A licensing authority can waive tests in monographs if it is assured of production consistency

WHO Animal Use Alternatives Example: Polio Neurovirulence
Testing


Transgenic mouse test introduced as alternative to monkey NVT
(TRS 904, 2002)



Future direction: can transgenic mice be considered as fully
equivalent to monkey NVT?



Future challenges: (a) independent testing by National Regulatory
Authority in Tg mouse test, (b) maintenance of competence for
testing as polio nears / beyond eradication, (c) need for NVT to
control Sabin-IPV?; (d) Need for other molecular tests such as nonisotope method, MALDI-TOF, microarray hybridization, Massively
Parallel Sequencing –the ultimate solution for monitoring molecular
consistency of live viral vaccines?

International Workshop on Alternate Methods-J Shin, WHO, Sept 2010

WHO Reduction And Refinement Examples: DTP Vaccines


Reduction of animal testing during the lot release of DT vaccines



Addendum to Recommendations for potency of DT vaccines (TRS 927,
2003)



-Introduces possibility to use (a) serological assays or (b) challenge assay
with a single dilution, both involving reduced number of animals, as an
approach for lot release.



-Conditionality; consistency in production and quality control has been
confirmed on a continuous basis.



Recommendation for the use of validated humane end points in
recording results of potency testing



-Revised recommendations for whole cell pertussis vaccines (TRS 941,
annex 6, 2007)


WHO Reduction And Refinement Examples: T Containing
Vaccines

“The innocuity test on the final lot may be omitted
for routine lot release once consistency of
production has been demonstrated, subject to the
approval of the NRA.”

WHO Reduction and Refinement Examples: T Containing
Vaccines

“The innocuity test on the final lot may be omitted
for routine lot release once consistency of
production has been demonstrated, subject to the
approval of the NRA.”

WHO Reduction And Refinement Examples: Rabies
Vaccines



Recommendation for the use of validated humane end points in
recording results of potency testing



-Revised recommendations for rabies vaccines (TRS 941, annex 2, 2007)



-plus possibility to use single dilution assay for NIH test



Statement that there is no additional value in performing an
accelerated stability test for the purpose of lot release.



-Since this test is based on the NIH test for potency after exposure to the
elevated temperature, this statement led to discontinuation of this test on a
lot-to-lot basis in a number of countries.


WHO Reduction And Refinement Examples: Yellow Fever
Vaccines



-Amendment of requirements for yellow fever vaccine
potency assay (TRS 872, 1998): 2008



-recent establishment of WHO IS for yellow fever
potency



* allows possibility to qualify the cell culture assay in
place of the mouse potency assay with improved interlaboratory comparison


WHO Continues To Evaluate Additional Opportunities



Lot release guidelines



Recommending mutual recognition of animal tests in
exporting and importing countries



Mumps vaccine neurovirulence tests



Repository of mumps vaccine seed strains being
established to facilitate evaluation of alternatives to
monkey NVT and international collaborative study under
development


US FDA Perspective on Potency



610.10 Potency



“Tests for potency shall consist of either in vitro
or in vivo tests, or both, which have been
specifically designed for each product so as to
indicate its potency in a manner adequate to
satisfy the interpretation of potency given by the
definition in 600.3(s)….”

International Workshop on Alternate Methods-T. Finn, US-FDA, Sept 2010

US FDA Perspective On Vaccine Safety And Potency Testing



Tests performed on final bulk/container sample(s) to assure safety….
– e.g.: general safety test, histamine sensitization test, endotoxin…etc



….and potency
– e.g.: D-antigen ELISA test for polio types 1, 2 and 3, ELISA for pertussispotency,
diphtheria and tetanus potency… etc.



Lot-release testing



610.1: Test prior to release…



“No lot of any licensed product shall be released…prior to the
completion of tests for conformity with standards applicable to each
product….”


US FDA Perspective On Alternative Safety And Potency
Tests



CBER encourages alternatives to reduce, refine and
replace the use of animals in safety and potency testing



Relevance



Data to support use



Validation



•Goal: Safe, pure and potent vaccines


US FDA Process On Changing A Potency Or Safety Test



Supplement to the License



•601.12: Changes to an approved application
– Potency: Rationale and data to support proposed
alternative
– Safety: Rationale and data to support proposed
alternative or demonstration of lack of need


US FDA Conducts Research On 3Rs

Active research
– One of CBER’s Research Priorities: Evaluating,
developing and integrating novel scientific
technologies and preclinical models for use in product
regulation, including development and analysis of
novel approaches that reduce, refine, or replace (3R’s)
the use of animals high resolution novel technologies
for their potential to improve product characterization,
e.g.: molecular-based assays (microarray and high
throughput sequencing)
– alternative to lethal test for anthrax vaccine potency
– alternative to monkey neurovirulence test for polio
vaccine

Indian Authorities Continue to Embrace 3R’s - Examples



Hepatitis B vaccine batches can be released
without any in vivo testing for potency (1 in 5 or 1
in 10 lots to be tested)



Indian Pharmacopeia considering deletion of
abnormal toxicity test



HIB vaccine lot release testing does not require
animal immunogenicity testing



Indian Pharmacopeia considering establishing
guidelines to utilize in vitro D-antigen test instead
of in vivo test

USP Is Implementing 3R’s in USP-MC Monographs



USP-MC monograph for Hepatitis B does not
include any “required” in vivo testing.



Haemophilus influenzae B vaccine monograph
under development applies same approach.

Status Of Alternatives In Common Vaccines: Bacterial Vaccines
Type

Examples

Animal test

Alternatives (accepted by)

Toxoids

Tetanus

• Safety: absence of toxin, irreversibility of toxoid,
specific toxicity

• Deletion of specific toxicity test (EU)
• Combined absence and irreversibility of toxin tests (EU)
• In vitro endopeptidase test for toxin detection has been developed
but not validated

• Potency: multidilution vaccination challenge on
guinea pigs or mice

• Single dilution test (EU)
• Antibody estimation by ELISA or ToBI (EU, WHO)

• Safety: absence of toxin (5 guinea pigs for bulk
lot), irreversibility of toxoid, specific toxicity

• Deletion of specific toxicity test (EU)
• Combined absence and irreversibility of toxin tests (EU)
• In vitro Vero cell test for toxin detection (WHO)

Diphtheria

•Potency: multidilution vaccination challenge on
guinea pigs with ~ 20 control animals

• Single dilution test (EU, WHO)
• Antibody estimation by Vero cell test (WHO) – ELISA and ToBI have
also been developed

Acellular
pertussis
vaccines
(ACPVs)

Bacterins

• Safety: absence of toxin (5 mice), irreversibility
of toxoid (5 mice)

• In vitro CHO clustering test can be done on bulk but not final lot

• Potency: multidilution vaccination + serology on
6 groups of mice

• Single dilution test (EU)

Whole cell
pertussis
vaccines

• Safety: mouse weight gain test with 10 mice for
testing specific toxicity

• Modified to use 5 guinea pigs (EU)
• In vitro alternatives include LAL pyrogen test (WHO)

• Potency: Kendrick test - multidilution vaccination
and intracerebral challenge in 136 mice – large
numbers of animals, severe distress, poor
precision and reliability

• Humane non-lethal endpoints (EU)
• Aerosol challenge instead of intracerebral
• Antibody estimation by whole cell ELISA (validated in 2000)

Cholera

6 mice, guinea pigs, or rabbits

• This serology test is accepted by the EU

Haemophil
us type B
conjugate

16 mice

• This serology test is accepted by the EU
• Moving testing upstream: if final bulk testing is satisfactory,
can omit potency testing of final lot

Status Of Alternatives In Common Vaccines: Viral Vaccines

Examples

Animal test

Alternatives (accepted by)

Rabies

• Safety: extraneous agent testing

• Cell culture test (WHO) + EU for vet vaccines
• Humane endpoints (EU)
• Single dilution (EU)
• Vaccination + antibody estimation using 5
mice (EU)
• Antigen quantification (WHO)

•Potency: NIH test (multi-dilution
vaccination + intra-cerebral challenge
test in up to 170 mice per batch)

Hep A (inactivated) and
Hep B (recombinant)

• Vaccination + serological test in
mice or guinea pigs

• Antigen quantification (EU, WHO)

Inactivated Poliovirus
(IPV)

• Multi-dilution vaccination + serology
in at least 60 rats

• Molecular analyses, e.g., MAPREC for
poliovirus type 3 (WHO)
• Neurovirulence in transgenic mice for

Oral Poliomyelitis (OPV)

• Neurovirulence testing in over 80
monkeys by intra-spinal injection

• Molecular analyses, e.g., MAPREC for
• Neurovirulence in transgenic mice for

Already not tested in animals: Influenza (tested in eggs), Meningococcal and Pneumococcal (only need pyrogen
test which can be done in vitro), Oral typhoid, Varicella, Measles, Mumps, Rubella
Currently no alternatives available: BCG (2 safety tests on 6 guinea pigs each)

Not covered here: Yellow Fever, Smallpox, Japanese Encephalitis, Anthrax

Tetanus Toxoid Influence on
IFN-gamma levels

Tetanus Toxoid Influence on IL-4 levels

Biosensor Analysis of Diphtheria
Toxoids

Biomarkers for Pertussis Vaccine Toxicity

New Concepts Followed In Vaccine Manufacturing



cGMP



QBD



Real-time fermentation data



In-process monitoring



In-process testing



Environmental monitoring



Testing using newer techniques



QA oversight and release

What Is Process Consistency

‘…… a concept which includes GMP, process
validation and in process and final product tests
and is aimed at verifying if a manufacturing
process produces final lots which are consistent
with one that fulfils all the criteria of Quality,
Safety and Efficacy as defined in the marketing
authorization, with the ultimate goal of replacing
animal tests’
(De Mattia et al. 2011)

Consistency Testing In Vaccine Quality Control: Procedure



Test first few lots thoroughly; in non-animal models but
also in laboratory animals and in target species
(clinical/historical batch).



Based on this information, specify the profile of the
vaccine (fingerprint) based on clinical, manufacturing and
testing criteria. Set alert and acceptance criteria and
criteria for deviations from consistency.



Subsequent vaccine lots produced should have the
same profile as the clinical lot. The consistency in profile
is monitored by non-animal techniques.



If so, the vaccine lot can be released for use.

Consistency Based Testing Is A Paradigm Shift

Traditional concept of vaccine
lot release testing

New paradigm: Consistency
testing

 Each lot produced by a

 Each lot produced by a

manufacturer is considered to
be a unique product

manufacturer is one of a series
and is NOT unique

 Use of Reference preparation

 Use of clinical lot

 Emphasis in quality control of
each vaccine lot is on final product

 Makes use of :
- strict application of quality
systems (GMP, QA)
- quality by design
- extensive in-process testing
- new innovative analytical tools

 Quality control includes several
animal models and is animal
demanding

Testing of D and T Vaccines-Potential Parameters For
Consistency Based Testing

Production parameters
•
•
•
•
•
•
•
•
•
•
•

Optical density
pH
Flocculation titre
Endotoxin
Protein Nitrogen
Protein
Residual formalin
In vitro safety test (Vero)
Reversion (Vero)
Osmolarity
etc.

Product quality parameters
•
•
•
•
•

Kf (flocculation time)
purity
various physico-chemical tests
Moab binding (biacore)
DAFIA (Direct Alhydrogel Formulation
Immunoassay)
• etc.

Parameters can be used to set alert criteria and acceptance criteria

Consistency Approach Offers Benefits

 Scientific

Benefits:
More meaningful batch release as quality is linked to a
clinical lot and better understanding of your product



Ethical Benefits:
Apart from clinical lot (first few lots) NO animal use is
required for lot release testing

 Practical

Benefits:
Quality control will be less time consuming (a few days
instead of 2 months)

Consistency Approach Also Has Its Challenges
 Tests:

what set of tests is needed and will this be
the same for every vaccine. Product specific ??

 ‘Risk

assessment’: what products are ready for
implementing the consistency principle.
Consistency is NOT a one-for-all strategy!
blending: adjuvant and antigen –
antigen interaction.

 Vaccine

 Validation:

how to compare fundamentally
different approaches.

Conclusions
 Current

paradigm for lot release testing is based on
extensive testing of final antigen/final vaccine

 Reliance

on in vivo testing has poses challenges

 With

introduction of cGMP/QA/in-process testing in
vaccine production, consistency based release testing
can be considered

 All

major international bodies such as
EDQM/EU/WHO/USP/IP/US-FDA have all embraced the
transition from in vivo to in vitro methods

 Researchers

and manufacturers need to pursue
development and implementation of alternate methods

Diverse Techniques Can Be Used In-process And Final
Lot Testing

Physico-chemical

Application



circular dichroism

secondary & tertiary structure proteins



fluorescence spectrometry

protein conformation, protein modifications



colourimetric assays

free amino groups in proteins

Immuno-chemical


biosensor analysis

epitope quality, antigen-antibody kinetics



ELISA (with Mabs)

peptide mapping, ag quantification



electrophoresis

purity, protein modification, stability

In vitro functional


binding assays



Immune cells

antigen binding
antigen processing, B/T cell responses, cytokine

Novel Vaccine Adjuvants
Manish Gautam, Ph.D.
Serum Institute of India Limited

Outline of Presentation



Overview of vaccine adjuvants



Novel Vaccine adjuvants: Mechanistic
aspects.



Preclinical Evaluation of Novel Adjuvants



Novel adjuvant development at SIIL- A case
study of SIIL-3

Vaccine Adjuvant - Definitions and Guidelines
1)

Adjuvants are the substances that are intended to
enhance relevant immune response and subsequent
clinical efficacy of the vaccines (WHO guidelines on
nonclinical evaluation of vaccines, WHO Technical
Report Series, No. 927, 2005)

2)

A vaccine adjuvant is a component that potentiates the
immune responses to an antigen and/or modulates it
towards the desired immune responses. (EMEA
guideline on adjuvants in vaccines for human use. 2005)

3)

New Draft Guidance on Preclinical Evaluation of Vaccine
Adjuvants is currently being developed by WHO aiming
towards harmonization of requirements. (Available at
WHO website for comments and discussion).

Novel Vaccine Adjuvants
• Previously, Adjuvant development was largely based on
approaches focusing largely on humoral immune
responses.
• Advances in basic sciences, immunology and vaccinology
per se, have led to better understanding of host immune
response against infection. These advances also impacted
antigen discovery and adjuvant development.
• Vaccines such as HIV. Malaria, HPV, cancer have brought
cellular immunity and its induction in focus
• Novel adjuvants targeting cellular immunity are currently
being sought. Immunomodulation is becoming the central
principle of preclinical assessment of adjuvants
• Regulatory frameworks to support such adjuvant
development are currently in development.

Novel Vaccine Adjuvants: Changing Paradigms

Key to effective vaccines
Delivery system

Reduces
booster
frequency

Immunomodulation (innate and adaptive
immunity ;B and T cell Immunity Inducers
Reduces
vaccine
dose

Immune
directing

Vaccine
adjuvant
Boosts
immunogenicity
of sub-unit
vaccines

Rapid
seroprotection
Boosts
immunogenicity
in neonates
and elderly

Adjuvant Development

Europe

Alum

liposomes
(HAV, flu) AS04 (MPL)
(HPV, HBV)
MF59
AS03, MF59, AF03
(flu elderly)
(pan flu)

1900 1920 1940 1960 1980 2000 2020
Alum
USA

 The


AS04 (MPL)
(HPV)

slow process of adjuvant discovery.

Alum was the first adjuvant to be licensed in the 1920s. in the USA. The squalene-based
oil-in-water emulsion MF59 was first licensed in Europe for a flu vaccine (FLUAD) in 1997.



The LPS analog monophosphoryl lipid A (MPL) formulated with alum (AS04) was first
approved for an HBV vaccine (Fendrix) in Europe in 2005. The oil-in-water emulsion AS03
was approved for a pandemic flu vaccine (Prepandrix) in 2008.
Taken from M.Friede 2011, WHO

Vaccine Adjuvants and Immune System Targets
Innate Immunity

Adptive Immunity
Antigen-specific
B and T cell responses

“Inflammatory responses’
Hours

Days

Innate immune
receptors
Signal 2
= adjuvants
Vaccine vehicle (vector)
Signal-1
=Antigen

Antigen
Presenting
cells

Months-Years

Signal 3
Cytokines
Co-stimulation
T cells
MHC-peptide

Min

TCR

Signal 1 + 2 (+3)
= Immune
response
Signal 1 only
= Tolerance
/Ignorance

Novel Adjuvants are Engineered to Target APCs, the Key Players of
the Innate Immunity

Stimulated by discovery and better understanding of role of
following targets in host immune response
 Discovery of toll-like receptors
NLRP3 nucleosomes
•Th1/Th2 Immunity

Aluminium Salts: New Insights

Formation of inflammasome

From DeGregorio, 2009

Stimulates Th2 directed cytokines

Alum induces cell death and the release of
not only uric acids but also host cell DNA
at injection sites
Uric acids

70
60
50
40

6.0E+05

4500
4000

5.0E+05

3500
3000

Cell numbers

Concentration(ng/mL)

Cocentration(nmol/mL)

80

Dead cells

dsDNA

2500
2000

30

1500

20

1000

10

500

0h

4h

24h

3.0E+05

2.0E+05
1.0E+05

0

0

4.0E+05

0.0E+00
0h

4h

24h

OVA / Alum
OVA
Marichal T, Ohata K et al Nat Med. 2011 In press

0h

4h

24h

48h

Adjuvants are the Ligand for Innate Immune Receptors
Receptors

Ligands

Adjuvants

TLR2/1
or
TLR2/6

Lipo-proteins
Lipo-peptide
Peptide glycans

E coli heat-labile enterotoxins
MDP
MALP2

TLR3

dsRNA

Poly I:C

TLR4

LPS

MDP, MPL

TLR5

Flagellin

Flagellin

TLR7,8

ssRNA

Imiquimod, R848, ssRNA

TLR9

CpG DNA
Hemozoin

CpG ODNs

RIG-I, MDA5

dsRNA, ssRNA

Poly I:C, ssRNA

NALP3
(AIM2)

DNA,RNA
ATP+PAMPs
Uric acid cristal (MSU)

Alum , particulate adjuvants
(Silica)

LPS : lipopolysaccharide MDP : muramyl dipeptide MALP２ : macrophage-activating lipopeptide 2
Poly I:C : Polyinosinic–polycytidylic acid MPL : monophosphoryl lipid A

MoA
aluminium,
Oil-in-water
emulsions

From: DeGregorio 2009

New adjuvants: immune potentiators and
antigen delivery systems
Optimize delivery of
Ag (or other
adjuvants) to APC in
lymphoid tissues

Delivery
systems
Alum, MF59 etc

Immune
potentiators
MPL, CpG,
Saponins etc

Antigens
Combo 2+ immune modulators
• Synergy through different
MOA (immune cell type or
pathways)

Recombinant
proteins

Activates innate
immune cells by
mimicking “danger
signals” normally
provided by infection
to enhance immune
responses

Combo: Immune modulator
+ delivery vehicle
• Enhance responses
through improved
delivery of antigen
and/or immune
modulator
• E.g., ISCOMS

Long-lived B & T cell memory

Adjuvant in Development
Class
TLR3
TLR4

TLR5
TLR7
TLR8
TLR9

Saponins
O/W emulsion

W/O emulsion
Polysaccharides
Cationic liposomes
Virosomes
poly-electrolytes

component
Poly I:C
MPL
MPL
RC530
GLA
flagellin
Imiquimod
Resiquimod
CpG, IC41

QS21
QS21
squalene
tocopherol
squalene
mineral oil
Inulin
DDA
Polyoxidonium

phase 1
cancer
leish

phase II

phase III

licensed

herpes
pneumonia

malaria
cancer

HPV, HBV
Allergy

HIV
flu
influenza

influenza
TB
pneumonia
HIV
HIV

cancer
cancer
Allergy
cancer
cancer
Alzheimer
HBV, CMV

HBV

malaria
Seasonal flu
Pandemic flu

malaria
cancer
HBV, flu
TB
malaria

influenza
HAV, flu
influenza

Dendritic Cells (DCs).

• DCs of two lineages: lymphoid and myeloid
differentially influence maturation of TH1 and TH2.
• Immature DCs: phagocytic, express CCR5 & CCR6,
low levels of MHC Class II and B7.
• Mature DCs: lose phagocytic capacity, increase
presentation ability, enhanced expression of MHC
Class II and B7.
• Maturation influenced by PAMPs. Influence direction
of DC maturation.
– PAMPs (LPS, CpG, dsRNA) or host cell molecules (CD40L,
IL-1, TNF-a) modulate DC maturation and subsequent TH
response.
e.g. LPS drives DC1 maturation and TH1 response; PC-GP
(nematodes) drives DC2 maturation and TH2 response.

IL-4 vs IFN-g

• Antigen recognition
through TcR in the context
of MHC Class I or II
molecules.
• Co-stimulatory molecular
interactions between T-cell
and APC:

Helper T-Cell Subsets

• TH1:
– IFN-g, TNF-b
– Cellular immunity vs.
intracellular bacteria,
small parasites
– Induction of
neutralizing
antibodies of the
IgG2a subclass (in
mice)

• TH2:
– IL-4, IL-5, IL-10, and IL13
– Induced by helminthes
parasites, allergens,
immunization with
soluble or alumadsorbed antigens
– Immunity to extracellular
parasites, bacteria
– Helper function in
production of IgA, IgE,
and neutralizing IgG to
bacterial toxins

Adjuvants and Delivery Systems
.
Influence on Immune Response
Adjuvant or
Delivery System

TH 2 Response
(Antibody
Production)

TH1 Response
(Cytotoxicity)

Alum
Chitosan
CT or LT (+/+)
CT or LT (mut)
PLG
Quil A/QS21
QS21 + MPL

+++
+++
+++

+
+
+
+++
+++
++++

+++
+++
++
++

Adjuvants and Delivery Systems
Influence on Immune Response
Adjuvant or
Delivery System

TH2 Response
(Antibody
Production)

TH1 Response
(Cytotoxicity)

IL-12
Live vectors
Naked DNA
CpG-ODN

++
+
+
+

++++
++++
+++++
+++++

Preclinical Evaluation of Adjuvants

Proof-of-concept testing
•
•
•
•

Mechanism of action
Effects on the different arms of the immune system
Distribution. Local vs. Systemic effects
Different components/combinations

Safety
•
•
•
•

General toxicity
Inflammation/local effect
Pregnancy
Autoimmunity

Proof of Concept Testing

Major areas
• Physical presentation of the antigen in the
vaccine
• Optimisation of antigen uptake
• Targetting to specific cells (dendritic cells,
Langerhans cells, macrophages, and others)
• Immune potentiation and modulation
• intracellular transport and processing of
antigens
• association with MHC class I or II molecules
• expansion of T-cells with different profiles of
cytokine production

Pre-clinical Pharmacology of Adjuvant

• Screening and optimization
– Antibody titers alone can be misleading as don’t consider antibody
function (i.e., avidity)
– T cell responses complex to understand and kinetics brief

• Selection of animal model
– Testing anti-sera for functional antigen-binding capacity, opsonization etc.

– Disease models: infectious challenge, allergy, cancer,

• Points considered
– Use of antigen and adjuvant doses and routes that can translate to
humans
– Use of animal species with physiological response to novel adjuvant
similar to that known or expected in humans

Safety Testing of Adjuvants

• General toxicity
• Inflammation/local effect
• Pregnancy

- Th1/Th2 ratios vary during different stages of pregnancy
- Biased Th1 responses during pregnancy have reported
autoimmunity related risk disorders
- Interference may result in defective placentation and
pregnancy loss

Autoimmunity and Immunomodulatory Adjuvants

Some animal data have suggested a link
between vaccine/adjuvants and
autoimmunity
•Complete Freund’s adjuvants (mineral oil,
mycobacterium) induces Experimental Allergic
Encephalitis

•Squalene (adjuvant component of AS03, MF59,
AF03) can induce arthritis in rats and lupus in mice.

•Holm, Lorentzen, 2004. Dark Agouti rats (arthritis-prone);
Intradermal injection of 300 μl at the base of the tail. Satoh et al,
2003. Balb/c mice, i.p. squalene 0.5 ml. Induction of autoantibodies
to cellular proteins

Formulation Development (2)

• Analytics - must support required
specifications and stability
– Identity
– Quantification
– Purity
– Characterization
– Safety
– Potency

– Pyrogenicity
130

Formulation Development (3)
• Formulation - antigen(s) + adjuvant(s)
– Stability testing
• On individual components and final formulation, different temperatures & durations
• Inadequate stability data for complex formulation at time of Phase 1 trial may
necessitate a “mix and shoot” or “bedside mixing” approach
• Selection of buffers and excipients for DS and DP
• Development of methods for freeze-thaw and lyophilization if required

– Longer term stability requires compatibility of antigen-adjuvant with each
maintaining their integrity
• Dependent on solution conditions such as pH, buffer and other excipients

– Considerations
• Complexity of formulation
• Dose and desired volume
• Route
• Desired presentation (i.e., vial, pre-loaded syringe)

131

Critical Challenges During Development

What
parameters ?

Physicochemical characteristics

Functional characteristics (non-clin)
Which model ?
Functional characteristics (clin)
What effects ?

Licensed product

Safety (very rare and long term)

Development of Botanical Immunomodulators
as Adjuvants for Improving Vaccine Efficacy
A Collaborative Project under
DST Drugs and Pharmaceutical Research Program
Serum Institute of India Pvt Ltd (SIIL).
&
University of Pune(UoP)

Research Team
SIIL: Dr. S.S.Jadhav (PI), Dr. Sunil Gairola (Co-PI), Dr. K.Suresh, Dr. Yojana
Shinde
UoP: Dr. Bhushan Patwardhan (PI), Manish Gautam, Sanjay Mishra and Dada
Patil

Chemical Adjuvants: Triterpenoid Saponins

Test material extraction and its Chromatographic characterization

Safety studies as per OECD guidelines

A
• Th1/Th2 immune responses
(Flowcytometric studies )

• Ag. specific study: Humoral
& cellular immune response

B
Immunoadjuvant Study
against diphtheria in host
challenge model

Immunoadjuvant Study
with polysaccharide based
vaccine antigen

(T cell dependent antigen)

(T cell independent antigen)

Immunomodulatory
Potential using SRBC’s

 Th (CD4) and CTL (CD8) percentage
 Th1:IFN-γ, IL-2 & Th2: IL-4 cytokines
 Lym. proliferation: I) T- cells: CD3+
II) B - cells: CD19+
 Humoral and Cellular immune response.

C

Immunoadjuvant Potential

 VCA: Diphtheria toxin neutralizing Abs.

 Total IgG level: Ab ELISA

 Challenge associated Morbidity/ Mortality

 Functional Ab. estimation: SBA

 IFN-γ & IL-4 level (Th1/Th2 immunity)

 IFN-γ & IL-4 level (Th1 and Th2

 Sera cortisol level.

immunity)

Immunomodulatory Study Key Trends….

Immunoadjuvant Potential ‘DPT Vaccine’ Trends…
Effect of diphtheria challenge on percent survival
100

***

90

**

V + ISHS-SIIL 3
V + ISHS-SIIL 2

70
V + ISHS-SIIL 1
60
50
Unimmunized

Percent survival

80

Vaccine 1:160

40
30
20

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

Days post challenge

Challenge

Sr. No.

Humoral

Protection

Th1/Th2

ISHS-SIIL-1

++

++

Th2 (Toxoid based vaccines,viral vaccines)

ISHS-SIIL-2

+++

+++

Th1/Th2 (Polysaccharide, recombinant & Toxoid based vaccines)

ISHS-SIIL-3

++

++++

Th1 (Polysaccharide, recombinant and Tuberculosis, Malaria)

Immunoadjuvant potential ‘Men A vaccine’ Trends

 IgG level: ISHS-SIIL-2 and 3 showed a significant increase of IgG
levels as compared to Men A alone group. ISHS-SIIL-2 showed higher
modulatory effect as compared to SIIL-3.
ISHS-SIIL-2>SIIL-3>SIIL-1

 SBA titres: ISHS-SIIL-2 and 3 showed significant increased SBA titers
in Men. A immunized animals. ISHS-SIIL-3 showed higher SBA titer as
compared to SIIL-2.
ISHS-SIIL-3>SIIL-2.

SIIL-3 development and T cell independent antigens
• Alternative to conjugation technology currently used in
vaccine industry for T-cell independent
antigens(Polysaccharide based vaccines) in inducing
protective bactericidal antibodies.
• CMC conforming to EMEA/WHO guidance on adjuvants
• Pharmacokinetics of adjuvant was established when
administered alone and along with antigen.
SIIL-F4 with Standard adjuvants

Total IgG levels (IU/ml)

900.00
800.00
700.00
600.00
500.00
400.00
300.00
200.00
100.00
0.00
Men A
Poly. 5 ug

Men A +
AlPO4

Men A +
MPL

Men A +
TMG

Men A +
QS21; 10
ug

Men A +
SIIL-F46.25

Men A CJ 1
ug

Vaccine Adjuvants
• Vaccinated animals showed a
significant increase antibody
titers.
• Significant reduction in
mortality and morbidity was
observed
• Better efficacy/ safety over

QS

Evaluation of SIIL-3 as vaccine adjuvant
Comprehensive analysis: Microarray based Profiling
Transcriptome, proteome, metabolome, micro RNAs

０

２

４ｈ

Efforts to detect any biological responses at molecular level

Patents and Publications

Patents






1247/MUM/2003. Process for manufacturing immunoactive extracts from medicinal plants for
making vaccine adjuvants(Granted 2009)
1253/MUM/2003.A kit containing a vaccine and an immunological adjuvant (Granted 09)
1246/MUM/2003: Process for manufacturing immunoactive aqueous extracts (Granted 09)
1184/MUM/2009: Vaccine composition for improved efficacy (Granted 2010)
US Patent on vaccine composition for improved efficacy of T cell independent antigens (Grant
expected in 2013, two queries responses answered successfully since publication) in Year
2011).

Challenges

• SIIL-F-4 needs further purification and chemistry
support.
• Up-scaling of process and GMP manufacture of
adjuvant.
• Advanced studies on pharmacodyanmics and
immunotoxicity.
• Synthetic routes to synthesize withanolides with
correct conformations.

Encouraging Global Trends
• WHO comprehensive regulatory guidance document on
development of newer adjuvants in under publication. (For
the first time, immunomodulatory adjuvants are included in
ambit of adjuvant definition).
• Nanoparticles based delivery systems needs
immunomodulation for efficient mucosal delivery
especially at distant sites. (examples of alum and QS-21
recently published with tetanus and malaria antigens).
• WHO have launched a Global Adjuvant Development
Initiative with a focus on identifying newer vaccine
adjuvants for vaccines such as HIV, Malaria, tuberculosis,
HPV, etc.

Summary
 Newer

advances in antigen discovery brings newer hopes for
adjuvant adjuvant.

 Immunomodulation

emerging as central mechansim for adjuvant
activity and Immunopharmacology based approaches will be
important for newer adjuvant development and assessment

 Safety

assessment of immunomodulatory adjuvants will be
challenging especially autoimmunity, preganancy.

 Regulatory

framework and science needs to be developed in order
to cater to newer adjuvant development

 WHO

have taken an excellent initiative in this direction and
guideline will play an important role in harmonization.



Indian Industry look forward to regulatory support on newer
adjuvants.

Issues and Challenges for
Development of Combination
Vaccines: DPT Based Combination
Vaccines as a Case Study
Sunil Gairola, Ph.D.
Serum Institute of India Limited

Guiding Principles for Introduction of Newer Vaccine

– Disease burden
– Quality of available vaccine
– Affordability
– Cost effective
– Existence of a robust delivery system

Rationality
• Practical way to overcome the constrains of multiple
injections to infants and less distress to parents
• Improve timely vaccination coverage and less visits to
health facilities.
• Reducing the cost of stocking and administering separate
vaccines.
• Reducing the cost for extra health care visits.
• Facilitating the addition of new vaccines into
immunization program.
• May simplify transportation and storage problems
(logistics)

Combination Licensed in Recent Years

•
•
•
•
•
•
•
•
•
•

DTwPHib
DTwPHB
DTwPIPV
DTwP-IPV-Hib
DTwP-Hib-HB
DTaP
DTaP-IPV
DTaP-Hib
DTaP-HB-IPV
DTaP-Hib-HB-IPV

•
•
•
•
•
•

Hib-HB
Rotavirus vaccine
Hep A-HepB
MMR
Pandemic influenzae
23 valent Pneumococci
vaccine
• Quadravalent Men ACYW
vaccine
• HPV

Considerations for Combination Vaccines During
Development

• Product should be stable, each component of the
combination vaccine should be given at the same age, and
the requirement of booster for each component should be
similar.
• New combinations cannot be less immunogenic, less
efficacious, or more reactogenic than the previously
licensed uncombined vaccines.
• Free from Immunological, physical, and/or chemical
interactions between the combined components have the
potential to alter the immune response to specific
components.
• Many advantages of combination vaccines should not be
achieved at the cost of reduced product stability.
• From supply standpoint, uncommon transport and storage
conditions and complicated bedside mixing could hamper
the development of a combination vaccine.

Challenges During Product Development

• Formulation development
• Immunological interferences- case studies
• Manufacturing (scalability)
• Analytical methods
• Clinical
• Regulatory

Formulation Development
Development of combination vaccine formulation may need
consideration of following:
-

Antigen-Antigen or Antigen-adjuvant
interaction

antigens

Displacement of antigen from adjuvant leading
to reduced immunogenicity.
- Differential requirement of each antigen with
respect to achieving consistent adsorption
--

-

pH, selection of excipients and
stabilizers

adjuvants

Similarly buffers, stabilizers and similar
components may interfere with the components
of the other vaccine.

-

Scalability of the process

-

pH

preservatives
excipients

stabilizers

Scalability

Choice of preservatives

Any decision on the considerations is based on impact on Quality aspects including stability and shelf life ,
Safety (reactogenicity), Efficacy, Finally scalability of the process

Immunological Interferences
• Candidate Formulations are studied for appropriate
immunogenicity in an animal model.
• Immunogenicity induced by the combination vaccine was
compared with that induced by the separate but
simultaneously administered individual vaccines.
• If there was an already licensed combination, then the
current licensed formulation has to be used in the control
group for comparisons of immunogenicity
• The immune response to each of the antigens in the
vaccine is assessed, including
– the quality of response,
– the potential interference and
– Incompatibilities between combined antigens.

Case study 1: Immune Interference of Hib antigen with Acellular
Pertussis and Tetanus Antigens

•

Reduction in antibody titers to the Hib component of the vaccine
polyribosylribitol phosphate antigen. This has been reported for many DTaPbased vaccines, including the hexavalent vaccine DTaP-HBV-IPV/Hib.

•

The interference has not been reported to the same extent for DTwP-based
combination vaccines.

•

It has been suggested, due to the adjuvant effect of the whole-cell pertussis
(wP) component, this effect is masked in DTwP based vaccines.

•

Studies in a rat model looking at the interference of Hib and different aP antigens

-

Reduced anti-PRP response with FHA is in line with the finding that it is a
potent suppressor of IL-12 and IFN-γ production in vivo and in vitro,
suppressing immune responses to co-injected antigens.

-

Another explanation for the reduced Hib response when combined with DTaP
vaccines is incompatibility with the alum adjuvant (aluminum hydroxide).
Experiments in the rat model with Hib alone have reported 5- to 11-fold lower
levels of anti-PRP antibodies when adsorbed to aluminum hydroxide adjuvant.

Challenges of Combination Vaccines-formulation

• When different antigens are combined into one vaccine,
chemical incompatibility or immunological interference can
be seen.
Case study 1: Immune suppression
Case study 2: Immune enhancement

Impact of Antigen Concentration on Potency Parameter (Product:
DTPHBHIB)
Lot

Diphtheria
(Lf/dose)

Diphtheria
Potency (IU/Dose)

Tetanus
(Lf/dose)

Tetanus
Potency (IU/Dose)

A

25

31.185 (18.455 – 45.67)

7.5

High Survival

B

25

37.1323 (22.3396 – 53.0642)

7.5

High Survival

C

25

75.745 (52.485 – 105.195)

5.0

High Survival

D

25

74.140 (48.525 – 116.125)

5.0

High Survival

E

25

85.045 (57.45 – 123.0)

2.5

62.425 (43.809 – 99.266)

F

25

56.315 (36.675 – 80.115)

2.5

70.46 (44.48- 114.645)

G

25

97.365 (64.40 – 143.76)

2.5

68.71 (41.745 – 109.96)

H

25

87.21( 61.285 – 123.515)

4.0

78.84 (57.84 – 108.26)

I

25

92.575 (65.34 – 130.63)

4.0

96.77 (72.125 – 132.29)

J

25

106.065 (65.655 – 163.32)

4.0

98.85 (65.565 – 152.885)

Tetanus antigen when added at 5 to 7.5 Lf/dose resulted in high survival leading to absence of end point
in potency assay (WHO challenge method for tetanus component).
Tetanus antigen when added at 7.5 Lf/dose also resulted in suppression of immune response against
diphtheria component resulting in lower potency estimates.
 Tetanus antigen at 2.5 Lf/dose showed borderline conformance to 95 % CI
Tetanus at 4.0 Lf/dose concentration for tetanus antigen produced optimum results and hence selected.

Effect of HIB Conjugate (TETANUS TOXOID AS CARRIER
PROTEIN) on Tetanus Potency in Combination Vaccine

Potency (IU/dose)
With 95 % CI
24.619
( 14.7180 -39.0347)

96.77
(72.125-132.29)
98.85
( 65.565 – 152.885)

4L
f

DT
PH

BH

IB

IB
BH

/d
os
e

/d
os
e
4L
f

/d
os
e

e

4L
f

DT
PH

H

/d
os

4

IB

DTPHBHIB

BH

4

e

DTPHBHIB

Lf

85.20
(57.84-108.29)

DT
PH

4

/d
os

DTPHBHIB

7.
5

76.255
( 53.99-106.92)

DT
P

7.5

e

DTP

0

Lf

72.350
( 46.06 -111.4)

7.
5

7.5

/d
os

DTP

Lf

69.20
( 45.305-110.45)

DT
P

7.5

3

DTP

50

IB

18.4641
(9.6346-29.6911)

7.
5

Carrier
protein

H

Hib conjugate

100

DT
P

21.0581
(14.5986-31.7399)

2

Carrier
protein

IB

Hib conjugate

Profile of tetanus potency

H

Carrier
protein

1

Hib conjugate

IB

Tetanus
Lf/dose

Potency T component
(IU/SHD)

Vaccine 0

Potency of T component in DTPHBHiB formulation is considerably high as compared to DPT formulation even at 4Lf/dose. This might
be due to Hib-TT conjugates which contributed up to 18-24 IU/ml of tetanus potency.

Immunogenicity of Hib-Tetanus Conjugate
Immunogenicity of Hib-TT conjugate in presence of other antigens (wP) in rats
Sr.
No

1

2

Formulation

Hib monovalent vaccine
(Reconstituted with Non-adjuvanted diluent)
DTPHBHib liquid vaccine

IgG
GMT of Anti Hib
Titres (ELISA)

1007.9

5971.40

 The presence of adjuvant and a stabilized formulation
demonstrate a significant difference in IgG response in pentavalent
vaccine with Hib-TT component.

Role of Adjuvants

•

Method of adjuvant preparation
- Insitu or preformed

•

Method of adsorption
- Sequential or simultaneous

•

Adsorption profile
-Kinetics and maturation of adsorption process

•

Studies on desorption:
- Stability of adsorbed antigens

Antigen Adjuvant Interactions During Different Stages of
Formulation
• Adjuvant

can be readymade gel or insitu preparation of

adjuvant.
• Adsorption of antigens can be significantly change during
the process of blending.
•It is generally noted that every antigen have special
requirements for adsorption for instance at lower pH, D
and T antigens are tightly adsorbed.
•Monovalent Hepatitis B is 99% adsorbed whereas, in
combination vaccines, the adsorption is decreased due to
competition.
•Addition of Hib to combination blend needs to be
monitored for stability of conjugate.

Manufacturing Challenges / Vessels

• Combination vaccine can be blended in variety of
blending vessels such as
– Vessels with vibromixer
• Amplitude of operation?
– Vessels with magnetic stirrers
– Vessels with magnetic stirrers and baffles.
• RPM?
• Careful assessment has to be done so that the
antigens are not denatured based on the vessel
type due to shear forces.

Challenges-Regulatory
• Case example: Suppose a manufacturer has licensed and
qualified DTPHepB and also have monovalent Hib vaccine
qualified. What will be regulatory implications if
manufacturer decides to develop pentavalent vaccine(DTPHibHepB) formulation:
 If a manufacturer has monovalent vaccines licensed, the combination is
considered as new product.
 As a new product, the combination vaccine has to undergo preclinical and
clinical trials. Each stage is governed by regulatory submission (preclinical,
phase I, II and III tials).
 Inspection of manufacturing facilities by national regulatory authorities and
license to manufacture is provided.
 Separate regulatory pathways if one of the antigen is recombinant
 This escalates into huge development costs and big increase in time lines.
 If manufacturer decides to keep monovalent, quadra or penta combinations,
product efficacy equivalence needs to be proven.

Analytical Challenges
•

The assays that are developed for determining the potency in monovalent
vaccine may not work for combination vaccine.

•

New assay methods have to be developed and validated.
•

•
•
•
•

•
•

For eg,In DTPHBHibTetanus Lf cannot be determined by flocculation methods. ELISA has to be
developed for same.

The free polysaccharide assay in monovalent Hib is done by Orcinol.
In DTPHBHib, the same assay cannot be adopted, instead, an alternate
instrumental assay is used, making the assay more expensive.
WHO is conducting a collaborative study to harmonize Hib assay in pentavalent
vaccine, wherein, Serum institute is one of the participant.
In meningococcal conjugate vaccines, the individual polysaccharide or
conjugates can be analyzed by either phosphate or sialic acid assays
(colorimetric methods).
In combination of Men ACYW, the colorimetric assay does not help.
An alternate instrumental assays are employed making the assays more
complicated and expensive (Dionex AEC-PAD).

Challenges - References
•

•

•

•
•

International or National references are not available for
combination vaccines, though combinations are available for
more than 50 years.
For determining potency assays, monovalent standards are
used, which may be responsible for enhanced or suppressed
results due to interference of antigens.
Way to create an in-house reference is to chose the clinical
trial vaccine with proven efficacy, can be calibrated as
internal reference std.
New standard can be generated by calibrating against the
clinical std.
Criteria of adsorption is informative but adsorption of all
subsequent lots should not be less than the clinical trial lot.

Clinical Immunogenicity
Regulatory expectations
• Immunogenicity induced by the combination vaccine was
compared with that induced by the separate but
simultaneously administered individual vaccines.
• If there was an already licensed combination, then the
current licensed formulation has to be used in the control
group for comparisons of immunogenicity
• For each component of a combination vaccine, non-inferiority
had to be demonstrated against the licensed combination.
Challenges
• Such studies had to have sufficient power to rule out clinically
meaningful differences in GMTs and/or seroconversion rates
• Intrinsic variability in assays and subjects, the regulators may
not take them into account

Case example-Selection of Concentration of Antigens

DTP-HB-Hib with 2.5 mcg Hib
• Compared for immunogenicity against licensed DTP-HB-Hib
vaccine. (A multi-centric, randomized Phase III clinical trial)
• 100 % seroconversion was observed with respect to Hib (≥ 0.15
ug/ml of IgG).
• The non-inferiority criteria was met for all components except
Hib at ≥ 1ug/ml of IgG (87 % versus 93 % in 10 mcg/dose
comparison to comparator. Hence long term protection could
not be established.
• Another clinical trial was taken with new formulation with
increased concentration of Hib antigen (10 mcg/dose).

LABELLING: Primary Container
REQUIREMENTS
•
•
•
•
•
•
•
•
•
•
•
•
•
•
•

Product generic name and brand name if applicable.
Total number of ml in container (liquid) and number of doses in container (freeze-dried).
Units/dose or per ml or minimum titer.
Dose and route of administration.
Nature and amount of any preservative present.
Storage condition.
Warning/instructions if any i.e. Not to be frozen, shake well.
Statements for reconstitution, photo sensitivity, etc.
Manufacturing licence number.
Expiry date.
Name and address of manufacturer.
Vaccine Vial Monitor (VVM) if applicable on label for liquids.
Visual cue if any.
Overprinting / additional information if any.
Barcodes if any.

CHALLENGES
 Label size/space constraint.
 Font size/text legibility.
 VVMs: 7 x 7mm minimum area required, Visual cues: 5 x 5 mm.
 Barcodes: 12 x 12 mm (2D barcode).

To accommodate all the above is difficult on the primary label

Summary
• Combination vaccines offers opportunities, however development
path is complex and challenging.
• Increased reactogenicity of combination vaccines is not accepted.
Suitable preclinical correlates for toxicity assessment is not
available for most of the antigens. In other words, prediction of
toxicity or reactogenicity during preclinical assessment is
challenging.
• Immune interference is a phenomenon in combination vaccines.
Needs excellent study designs to predict the same.
• Analytics of final lot especially detection and quantification of
antigens in combination vaccine is challenging. Several
technologies such as label free ELISAs, platforms such as Gyros,
MSD, Luminex offers oppurtunities.
• Reference standard for evaluation of combination vaccines:
(monovalent versus multivalent standard)
• Clear guidelines on usage of in vivo potency tests during stability
testing: (w.r.t to testing intervals, number,etc)

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