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

Panacea Biotec Ltd. All Rights Reserved, Privileged & Confidential

Comparing the Opportunities and

Challenges in Thermostable Vaccines

with Conventional Vaccines

Rajesh Jain, Ph.D.

Joint Managing Director

Panacea Biotec Limited, New Delhi

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

• Pharmaceuticals

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

• Largest Vaccine Producer in India

• 3rd Largest Biotechnology Company in India (ABLE Survey 2011)

• Ranked 39th amongst Pharmaceutical Companies in India (ORG-IMS, 2011) Ranking

• 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

Infrastructure

• 3,800 Human Resource

• 291 in R&D

• 1,200 in Sales and Marketing

Manpower

• Turnover : INR 1,130 Cr. (~USD 250 Mn.), CAGR of 21%

• Listed in BSE and NSE Financials

Vaccine Business – Key Highlights

• Established presence of over 25 years in vaccines

• Reliable partner to WHO, UNICEF: largest supplier of

vaccines to UNICEF from India

• First Indian Company to launch innovative branded

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

other combination vaccines – Easyfour, Ecovac

• 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

• Innovative vaccines

• Universal Flu (Recombinant viral vectored)

• Dengue (Chimeric Recombinant Tetravalent )

• DTwP/DTaP- IPV combination (Fully Liquid

combination Vaccines)

• Recombinant Anthrax Vaccine

• Seasonal Flu (Quadrivalent seasonal Vaccine)

• Pneumococcal Conjugate (13 valent conjugate)

• Japanese Encephalitis (Inactivated cell culture

based viral vaccine)

• Meningococcal (Tetravalent conjugate vaccine)

• Adolescent Vaccines

• Tdap (AdTdap)

• Tdap- IPV (AdTdap -pol)

Established Presence

Robust Pipeline

Innovation Infrastructure

Drug Discovery: Small Molecules

• Target identification to development of pre-clinical

candidate

• Focus areas : Metabolic disorders, Anti-infectives, CNS

Laksh, Mohali OneStream, New Delhi

Drug Discovery: Novel Biologicals

• Target identification to development of pre-clinical candidate

• Focus areas for Novel Peptides: Metabolic Disorders

• Biosimilars & Vaccines

GRAND, Navi Mumbai Sampann, Lalru

NDDS Product Development

• Platform NDDS technologies : Nanoparticles,

Liposomes, Micro-particles, Depot Injections, SPORT,

Oral films etc.

• High barrier to entry generics

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

Generics NCE Biosimilars,

Peptides,

Vaccines

Pipeline

Generics NCE & Peptides

• 4 Best in Class

NCE

• 1 Atypical NCE

• 2 Novel Peptides

Biosimilars,

Peptides,

Vaccines

• 4 Biosimilars in

development

• 4 Peptides in

development

• 10 Innovative

vaccines in

development

US:

• 2 ANDAs filed

• 45 ANDAs in

development

Europe:

• 1 MAA

granted

• 20 products in

development

NDDS

NDDS

• 7 best in class

NDDS products

in development

(Scientific

advice from US

/ European

agencies has

been taken for

5 products)

Commitment to make Affordable Vaccines…

“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!”

The Pledge

June 2011,

London

Vaccine - Manufacturing Capabilities

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

Location Formulation Facilities

Capacities

(million doses/ annum

)

Built Up Area

Delhi 2 lines for Oral vaccines

1600

> 50,000 sq ft

Baddi

2 lines. 1 for Vials , 1 for PFS

switch able to lyophilized in Vials

One more filling line under

construction

PFS-16

Single dose -39

10 Dose -350

> 129,167 sq ft

Vaccine Formulation Facility

Vaccine - Manufacturing Capabilities

Indigenous & fully integrated bulk antigen manufacturing facilities

Location Bulk Antigen Facilities Built Up Area

Recombinant Vaccines

Bacterial Vaccines

Tetanus Vaccine

Cell Culture Vaccines

Lalru

> 40,000 sq. ft

> 18,000 sq. ft

> 20,000 sq. ft

> 30,000 sq. ft

(US-FDA / UK-MHRA compliant)

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 .

Source: Rappuoli R. Nature reviews. Immunology, Vol 11, Dec 2011, pp. 865-872.

In 21st century vaccines will be 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

Vaccines needed across all age groups

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 as well as low

temperature (freeze) 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 and

temperature-controlled 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

Zone Climate/Definition

Measured mean annual data Long-term stability

testing conditions 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, US bestowing

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

– 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 Preclinical Phase I/II Phase III Marketed

VeloThrax, rPA, Soligenix

Thermostable IM Measles

Microneedle patch for flu

HepB Formulations, PATH

Challenges in thermostable vaccine development • Commercial

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

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

– 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, Hep-

B & 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

Evolving Regulatory framework for Vaccines PQ

17 April 2013

Carmen Rodriguez Hernandez QSS/EMP/HIS/WHO

36 |

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


17 April 2013


37 |

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

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


17 April 2013


39 |

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)

40 |

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


17 April 2013



17 April 2013


41 |

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)

42 |

Implementation workshops- AFRO

1 Uganda 2 Ghana

3 Nigeria

4 Rwanda

5 Tanzania 6 Guinea Bissau

7 Kenya

8

9

10

11

Ethiopia

Cameroon

Gambia

Eritrea

1 Burkina Faso

2 Benin

3 Burundi

4 Central African Republic

5 Côte d'Ivoire

6 Gabon

7 Mauritanie

8

9

10

Sénégal

Tchad

Togo

1 Botswana

2 Ethiopia

3 Gambia

4 Kenya

5 Malawi

6 Namibia

7 Uganda

8 Sierra Leone

July 2011 July 2011 July 2012


17 April 2013


43 |

Implementation workshops- WPRO

1. CAMBODIA

2. LAO PEOPLE'S DEMOCRATIC

REPUBLIC

3. MONGOLIA

4. PAPUA NEW GUINEA

5. PHILIPPINES

6. SOLOMON ISLANDS

7. VANUATU

November 2012


17 April 2013


44 |

45 |

46 |


17 April 2013


47 |

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) Signed


17 April 2013


48 |

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


17 April 2013


49 |

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


17 April 2013


50 |

Regulatory networks: Discussions

DCVRN AVAREF

Meetings Bi-annual-Annual FtF

meetings + web meeting:

12 meetings held + 2

web meetings

Annual meetings: 7

Participants regulator from mature NRAs (USFDA, HC, EMA)

Other regulators from the region as observer

Sessions Scientific session

Closed session

QC labs- NETWORK OF WHO VPQ CONTRACTED LABORATORIES

ANSM France

Cantacuzino Romania

Department of Medical Sciences (DMSC) Thailand

Health Canada

Korea Food and Drug Administration

NCE Hungary

NIBSC UK

PEI Germany

RIVM

SIPH Belgium

South African National Control Laboratory

Swissmedic

TGA Australia


17 April 2013


51 |

Qualification of WHO contracted laboratories

Phase 1:

Review of SOPs and related documents

Validation protocol and report

Testing 3 batches in parallel with a contracted laboratory

Phase 2: Visit to the lab for evaluating

Organisation of the Laboratory

Quality System

Personnel

Premises and equipment

Handling of samples

Reagents and Reference material

Test methods

Test reports


17 April 2013


52 |

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

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


17 April 2013


53 |

Mali, polio campaign,

Photos: WHO/Olivier Ronveaux

Nati

on

al co

ld r

oo

m d

uri

ng

th

e c

am

paig

n

Contribution to development of Controlled Temperature

Chain Project Optimize: PATH/WHO

Transport to health centre

Nicaragua, rotavirus delivery, Photo: Gates Foundation

54 |

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

Studies to enable on label use of vaccines under

CTC and regulatory submissions

Regulators

Regulatory pathways

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)

55 |


17 April 2013


56 |

Capacity building in countries

NRAs

• NRA Observers in PQ evaluation procedures

• Joint reviews of PSF with NRAs of producing countries/NRA networks

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

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

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

Overview

Steps In Vaccine Manufacturing

Inactivated vaccine/toxoid final lot

Safety tests

Potency tests

Culture

Concentration

Detoxification/inactivation

Purification

Blending (adjuvant, antigens)

Production of the established

(inactivated) vaccines

Virulent micro-organism/toxin

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

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 : Extensive animal use

Practical aspects : Costs, time required

Limitations Of Current “In Vivo” Based Approaches

63

Ligand/receptor assays (not cell based)

SPR

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

Variability

Speed

Relevance

The Relevance Of Biological Assays

?

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?

Species Difference Can Be Unpredictable

• 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.)

Animal Usage In Human And Veterinary Vaccines

Sadhana Dhruvakumar, PETA, Feb 2005

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

The 3R’s Approach

Russell and Birch


testing

– The dependency on in vivo methods



methods



Conclusions

Overview

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 Center For The Validation Of Alternative Methods

(ECVAM) – Selected Examples

Replace:

• 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*)

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 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

European Directorate for Quality Medicine(EDQM)/European

Pharmacopeia (Ph. Eur.) – Selected Examples

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 non-

isotope method, MALDI-TOF, microarray hybridization, Massively

Parallel Sequencing –the ultimate solution for monitoring molecular

consistency of live viral vaccines?

WHO Animal Use Alternatives Example: Polio Neurovirulence

Testing

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

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: DTP 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: T Containing

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: Rabies 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 inter-

laboratory comparison

WHO Reduction And Refinement Examples: Yellow Fever Vaccines


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

WHO Continues To Evaluate Additional Opportunities


US-FDA Efforts on 3Rs

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)….”

US FDA Perspective on Potency

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

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 Vaccine Safety And Potency Testing


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 Perspective On Alternative Safety And Potency Tests


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 Process On Changing A Potency Or Safety Test


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

US FDA Conducts Research On 3Rs


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

Indian Authorities Continue to Embrace 3R’s - Examples

USP-MC monograph for Hepatitis B does not

include any “required” in vivo testing.

Haemophilus influenzae B vaccine monograph

under development applies same approach.

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

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

• Potency: multidilution vaccination challenge on

guinea pigs or mice

• 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

• Single dilution test (EU)

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

Diphtheria

• Safety: absence of toxin (5 guinea pigs for bulk

lot), irreversibility of toxoid, specific toxicity

•Potency: multidilution vaccination challenge on

guinea pigs with ~ 20 control animals

• Deletion of specific toxicity test (EU)

• Combined absence and irreversibility of toxin tests (EU)

• In vitro Vero cell test for toxin detection (WHO)

• Single dilution test (EU, WHO)

• Antibody estimation by Vero cell test (WHO) – ELISA and ToBI have

also been developed

Acellular

pertussis

vaccines

(ACPVs)

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

of toxoid (5 mice)

• Potency: multidilution vaccination + serology on

6 groups of mice

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

• Single dilution test (EU)

Bacterins

Whole cell

pertussis

vaccines

• Safety: mouse weight gain test with 10 mice for

testing specific toxicity

• Potency: Kendrick test - multidilution vaccination

and intracerebral challenge in 136 mice – large

numbers of animals, severe distress, poor

precision and reliability

• Modified to use 5 guinea pigs (EU)

• In vitro alternatives include LAL pyrogen test (WHO)

• Humane non-lethal endpoints (EU)

• Aerosol challenge instead of intracerebral

• Antibody estimation by whole cell ELISA (validated in 2000)

Cholera • Potency: multidilution vaccination + serology on

6 mice, guinea pigs, or rabbits

• This serology test is accepted by the EU

Haemophil

us type B

conjugate

• Potency: multidilution vaccination + serology on

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

Examples Animal test Alternatives (accepted by)

Rabies • Safety: extraneous agent testing

•Potency: NIH test (multi-dilution

vaccination + intra-cerebral challenge

test in up to 170 mice per batch)

• Cell culture test (WHO) + EU for vet vaccines

• Humane endpoints (EU)

• Single dilution (EU)

• Vaccination + antibody estimation using 5

mice (EU)

• Antigen quantification (WHO)

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

Status Of Alternatives In Common Vaccines: Viral Vaccines


testing




methods



Conclusions

Overview

Tetanus Toxoid Influence on

IFN-gamma levels

Tetanus Toxoid Influence on IL-4 levels

Biosensor Analysis of Diphtheria

Toxoids

Biomarkers for Pertussis Vaccine Toxicity


testing




methods



Conclusions

Overview

cGMP

QBD

Real-time fermentation data

In-process monitoring

In-process testing

Environmental monitoring

Testing using newer techniques

QA oversight and release

New Concepts Followed In Vaccine Manufacturing

‘…… 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)

What Is Process Consistency

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 Testing In Vaccine Quality Control: Procedure

Consistency Based Testing Is A Paradigm Shift

Traditional concept of vaccine

lot release testing

Each lot produced by a

manufacturer is considered to

be a unique product

Use of Reference preparation

Emphasis in quality control of

each vaccine lot is on final product

Quality control includes several

animal models and is animal

demanding

New paradigm: Consistency

testing

Each lot produced by a

manufacturer is one of a series

and is NOT unique

Use of clinical lot

Makes use of :

- strict application of quality

systems (GMP, QA)

- quality by design

- extensive in-process testing

- new innovative analytical tools

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

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 Offers Benefits

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!

Vaccine blending: adjuvant and antigen –

antigen interaction.

Validation: how to compare fundamentally

different approaches.

Consistency Approach Also Has Its Challenges

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

Conclusions

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 antigen binding

Immune cells antigen processing, B/T cell responses, cytokine

Novel Vaccine Adjuvants

Manish Gautam, Ph.D.

Serum Institute of India Limited

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

Outline of Presentation

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).

• 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

Novel Vaccine Adjuvants: Changing Paradigms

Reduces

booster

frequency

Boosts

immunogenicity

of sub-unit

vaccines

Boosts

immunogenicity

in neonates

and elderly

Rapid

seroprotection

Immune

directing

Reduces

vaccine

dose

Vaccine

adjuvant

Delivery system Immunomodulation (innate and adaptive

immunity ;B and T cell Immunity Inducers

Key to effective vaccines

Adjuvant Development

The 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.

liposomes

Europe (HAV, flu) AS04 (MPL)

(HPV, HBV)

MF59 AS03, MF59, AF03

Alum (flu elderly) (pan flu)

1900 1920 1940 1960 1980 2000 2020

Alum AS04 (MPL)

USA (HPV)

Taken from M.Friede 2011, WHO

Signal 1 only

= Tolerance

/Ignorance

MH

C-p

ep

tid

e

TCR

T cells

Innate immune

receptors

Co-stimulation

Cytokines Signal 1 + 2 (+3)

= Immune

response

Innate Immunity Adptive Immunity

Min Hours Days Months-Years

Antigen-specific

B and T cell responses

“Inflammatory responses’

Vaccine vehicle (vector)

Signal 2

= adjuvants

Signal-1

=Antigen

Antigen

Presenting

cells

Vaccine Adjuvants and Immune System Targets

Signal 3

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

Stimulates Th2 directed cytokines From DeGregorio, 2009

Formation of inflammasome

Alum induces cell death and the release of

not only uric acids but also host cell DNA

at injection sites

0

10

20

30

40

50

60

70

80

0h 4h 24h

Co

cen

trat

ion

(nm

ol/

mL)

0

500

1000

1500

2000

2500

3000

3500

4000

4500

0h 4h 24h

Co

nce

ntr

atio

n(n

g/m

L)

0.0E+00

1.0E+05

2.0E+05

3.0E+05

4.0E+05

5.0E+05

6.0E+05

0h 4h 24h 48h

Ce

ll n

um

be

rs

Uric acids dsDNA Dead cells

OVA / Alum

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

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

Delivery

systems Alum, MF59 etc

Immune

potentiators MPL, CpG,

Saponins etc

Antigens Recombinant

proteins

Long-lived B & T cell memory

New adjuvants: immune potentiators and

antigen delivery systems

Activates innate

immune cells by

mimicking “danger

signals” normally

provided by infection

to enhance immune

responses

Optimize delivery of

Ag (or other

adjuvants) to APC in

lymphoid tissues

Combo 2+ immune modulators

• Synergy through different

MOA (immune cell type or

pathways)

Combo: Immune modulator

+ delivery vehicle

• Enhance responses

through improved

delivery of antigen

and/or immune

modulator

• E.g., ISCOMS

Adjuvant in Development

Class component phase 1 phase II phase III licensed

TLR3 Poly I:C cancer

TLR4 MPL leish herpes malaria HPV, HBV

MPL pneumonia cancer Allergy

RC530 HIV

GLA flu

TLR5 flagellin influenza

TLR7 Imiquimod cancer

TLR8 Resiquimod cancer

TLR9 CpG, IC41 influenza Allergy HBV

TB cancer

Saponins QS21 pneumonia cancer malaria

QS21 HIV Alzheimer

O/W emulsion squalene HIV HBV, CMV Seasonal flu

tocopherol Pandemic flu

W/O emulsion squalene malaria

mineral oil cancer

Polysaccharides Inulin HBV, flu

Cationic liposomes DDA TB influenza

Virosomes malaria HAV, flu

poly-electrolytes Polyoxidonium influenza

T cell Immunity Adjuvants

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 IL-13

– Induced by helminthes parasites, allergens, immunization with soluble or alum-adsorbed antigens

– Immunity to extracellular parasites, bacteria

– Helper function in production of IgA, IgE, and neutralizing IgG to bacterial toxins

Adjuvants and Delivery Systems

.

TH1 Response

(Cytotoxicity) TH 2 Response

(Antibody

Production)

Adjuvant or

Delivery System

-

+

+

+

+++

+++

++++

+++

+++

+++

+++

+++

++

++

Alum

Chitosan

CT or LT (+/+)

CT or LT (mut)

PLG

Quil A/QS21

QS21 + MPL

Influence on Immune Response

Adjuvants and Delivery Systems

TH1 Response (Cytotoxicity)

TH2 Response (Antibody Production)

Adjuvant or Delivery System

++++

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

++

+

+ +

IL-12

Live vectors

Naked DNA

CpG-ODN

Influence on Immune Response

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

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

Proof of Concept Testing

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

Licensed product

Functional characteristics (non-clin)

Functional characteristics (clin)

Safety (very rare and long term)

Which model ?

What effects ?

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

Immunoadjuvant Study

with polysaccharide based

vaccine antigen (T cell independent antigen)

• Th1/Th2 immune responses (Flowcytometric studies )

• Ag. specific study: Humoral

& cellular immune response

Safety studies as per OECD guidelines

VCA: Diphtheria toxin neutralizing Abs.

Challenge associated Morbidity/ Mortality IFN-γ & IL-4 level (Th1/Th2 immunity) Sera cortisol level.

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.

B A C

Immunomodulatory

Potential using SRBC’s Immunoadjuvant Potential

Immunoadjuvant Study

against diphtheria in host

challenge model (T cell dependent antigen)

Total IgG level: Ab ELISA

Functional Ab. estimation: SBA

IFN-γ & IL-4 level (Th1 and Th2

immunity)

Immunomodulatory Study Key Trends….

Effect of diphtheria challenge on percent survival

34 35 37 38 39 40 41

20

30

40

50

60

70

80

90

100

28 29 30 31 32 33

Vaccine 1:160

V + ISHS-SIIL 2

V + ISHS-SIIL 3

V + ISHS-SIIL 1

***

**

Challenge

Unim

muniz

ed

36 42

Days post challenge

Pe

rce

nt

su

rviv

al

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 ‘DPT 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.

Immunoadjuvant potential ‘Men A vaccine’ Trends

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

0.00

100.00

200.00

300.00

400.00

500.00

600.00

700.00

800.00

900.00

Men A

Poly. 5 ug

Men A +

AlPO4

Men A +

MPL

Men A +

TMG

Men A +

QS21; 10

ug

Men A +

SIIL-F4-

6.25

Men A CJ 1

ug

To

tal I

gG

leve

ls (

IU/m

l)

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

０２４ｈ

Transcriptome, proteome, metabolome, micro RNAs

Efforts to detect any biological responses at molecular level

Comprehensive analysis: Microarray based Profiling

Patents and Publications

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).

Patents

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)

Vaccine Types

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

- Antigen-Antigen or Antigen-adjuvant interaction

-- 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

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

- Scalability of the process

- Choice of preservatives

antigens

preservatives adjuvants

Scalability

pH

stabilizers

excipients

Development of combination vaccine formulation may need

consideration of following:

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 DTaP-

based 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

Vaccine 0 Tetanus

Lf/dose Potency (IU/dose)

With 95 % CI

Hib conjugate Carrier

protein 24.619

( 14.7180 -39.0347)


protein 21.0581

(14.5986-31.7399)


protein

18.4641

(9.6346-29.6911)

DTP 7.5 69.20

( 45.305-110.45)

DTP 7.5 72.350

( 46.06 -111.4)

DTP 7.5 76.255

( 53.99-106.92)

DTPHBHIB 4 85.20

(57.84-108.29)

DTPHBHIB 4 96.77

(72.125-132.29)

DTPHBHIB 4 98.85

( 65.565 – 152.885)

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.

0

50

100

HIB

1

HIB

2

HIB

3

DTP

7.5Lf/d

ose

DTP

7.5Lf/d

ose

DTP

7.5Lf/d

ose

DTPH

BHIB

4Lf/d

ose

DTPH

BHIB

4Lf/d

ose

DTPH

BHIB

4Lf/d

ose

Profile of tetanus potency

Pote

ncy

Tco

mpo

nen

t(I

U/S

HD

)

Sr.

No Formulation

IgG

GMT of Anti Hib

Titres (ELISA)

1 Hib monovalent vaccine

(Reconstituted with Non-adjuvanted diluent) 1007.9

2 DTPHBHib liquid vaccine

5971.40

The presence of adjuvant and a stabilized formulation

demonstrate a significant difference in IgG response in pentavalent

vaccine with Hib-TT component.

Immunogenicity of Hib-TT conjugate in presence of other antigens (wP) in rats

Immunogenicity of Hib-Tetanus Conjugate

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)

Health & Medicine

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