Vaccine

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Vaccine

USP Bioassay Workshop

August 12-13, 2009

USP Headquarters

Quality Standards for Medicines, Supplements, and Food Ingredients throughout the World


Regulatory perspective on

vaccine potency assays

USP’s 2 nd Bioassay Workshop

August 13, 2009

Phil Krause, MD

FDA/CBER


Potency

Specific ability or capacity of the product,

as indicated by appropriate laboratory

tests or by adequately controlled clinical

data obtained through the administration

of the product in the manner intended, to

effect a given result. -- [21 CFR §600.3

(s)]

2


Tests for 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) of this chapter. --

[21 CFR § 610.10]

3


Why do we do a potency assay?

• In development, to:

– Assure that safe potencies are not exceeded in clinical trials

– Obtain information that will support licensure‐including

correlation of potency with clinical response

• After licensure, to assure that lots behave similarly to

those tested in the clinical trials that supported licensure

– The potency should not be below the lowest potency believed to

be efficacious

– The potency should not exceed the highest potency believed to

be safe

• The potency assay thus provides a “bridge” between

licensed material and the clinical trials


What does a potency assay tell us?

• The assay estimates the mean potency value for a lot

• There are two sources of variability in the measured

potency of an individual vial from the same vaccine

lot– manufacturing variability and assay uncertainty

• Thus, we can never know the actual potency in an

individual vial that is given to a vaccine recipient

• We can know the characteristics of the lot of vaccine

from which that vial came (we routinely estimate the

mean potency)


Necessary attributes for

potency assays

• Predictive of clinical benefit

• Possess characteristics that are amenable to

validation

• Precision sufficient to meet goal of potency

assays, i.e., provide assurance that vaccine

is safe and effective throughout the dating

period

– Includes for use in stability studies

– Includes for use in the “bridge” between marketed

and clinical trial materials

• Stability indicating

6


Choosing “the right thing”

Every worker in biology

must know the temptation

to adopt a method

because it measures

something with

reasonable precision,

without waiting for

conclusive evidence that

what it measures is the

right thing

- Sir Henry Dale

Nobel Laureate in Medicine, 1936

7


Not everything that

counts can be

counted, and not

everything that can

be counted counts.

‐Albert Einstein


Building the Bridge

Clinical Lots Assay Results

Potency Assay Bridge

During clinical development, it is necessary to

establish a quantitative link between product efficacy

and laboratory measured parameters? What are the

relevant parameters to use? How do we choose the

“right thing.”

9


Building the Bridge

Clinical Lots Marketed Lots

Potency Assay

Bridge

An assay, or set of assays, is used to measure the

comparability of a given lot of product to those lots of

product that were used in the clinical trials to establish

efficacy. Were the relevant parameters established?

10


Building the Bridge

• Establishing a bridge between clinical outcomes

and laboratory test parameters is enabled by

– Knowledge of disease pathogenesis and mechanisms of

mediation

– Existence of surrogate markers

– Characterization of products (antigens and antibodies

for vaccines)

– Existence of appropriate animal models

– Data from clinical trials

11


Relationship between potency and

surrogate markers

• Potency refers to the ability to effect a clinical

outcome

• Surrogate markers, in clinical subjects, predict

actual clinical outcomes, and imply an

understanding of the mechanism of action

• Often, the best potency assays measure attributes

of the product that are likely to induce a response

measured by a surrogate marker

12


Surrogate Markers

“surrogate endpoint . . . is reasonably likely, based

on epidemiologic, therapeutic, pathophysiologic, or

other evidence, to predict clinical benefit or on the

basis of an effect on a clinical endpoint other than

survival or irreversible morbidity.”

[21 CFR 314.510 (Subpart H) Accelerated approval]

13


Building the Bridge

• In the absence of an existing laboratory

surrogate of protection, establishing the

ability to accurately assess potency, i.e.,

develop the potency assay, will rest on

clinical results with drug preparations of

varying measured quality.

14


Development of a potency test:

whole-cell pertussis vaccine

1940’s: Potency test

for whole-cell

pertussis vaccine

Kendrick & Pittman

Mouse protection

from a lethal

intracerebral

infection

15


Development of a potency test:

whole-cell pertussis vaccine

1950’s: UK MRC

trials showed a

correlation of the

MP test with

vaccine efficacy

Became the world

wide accepted

potency test for

whole-cell pertussis

vaccine

16


• Cell culture systems:

Potency bioassays

– For biologicals, potency assays often detect the ability of the

product to achieve a desired effect in a cell culture system

• Animal models.

– often more difficult to use

– are time-consuming

– frequently have greater variability than in vitro assays

– may have greater biological relevance

• These differences suggest the value of attempting to

identify in vitro predictors of efficacy and safety early in

product development.

17


Potency Assays and Vaccines: A Few

Examples

• Number of plaque forming units (e.g., mumps, measles,

rubella, smallpox)

• Number of colony forming units (e.g., S. typhi, TY21a)

• Chemical and Physical chemical characterization (e.g.,

polysaccharide and polysaccharide‐protein conjugate

vaccines)

• Serological response in animals (e.g., diphtheria)

• Animal protection against challenge (e.g., rabies, anthrax)

18


Situations where potency assays are

particularly difficult to develop

• Mechanism of action or surrogates of protection not

well understood

• Multi‐component products, where there is a

significant interaction among components

• Absent definitive tests that predict efficacy, product

evaluation is based on the demonstration that the

newly manufactured lots are comparable to those

shown in clinical studies to be safe and effective

19


Potency and product life cycle

• Each progressive phase of development is based upon

an ability to relate their respective measures of potency.

• Doses used in Phase 1 studies must be capable of

being related to those used in pre-clinical studies

• In turn, doses used in Phase 2 and Phase 3 studies

must be capable of being related both to each other and

to material used in Phase 1.

• After licensure, potency assays are used to help assure

that marketed product contains a quantity of active

ingredient similar to that shown to be safe and effective

in clinical trials.

20


Potency and the product life‐cycle

(additional points)

• As clinical development progresses, the

measure or measurement of potency may

change.

• At the time a product is first developed, it will

likely not be known what in vivo or in vitro

characteristics are likely to correlate with

clinical benefit. This understanding will evolve

during the course of clinical development.

21


Potency and the product life‐cycle

(additional points)

• The need to understand the performance of assays over

time and through clinical development points to the need

for maintaining stable reference preparations against

which potency results may be standardized

• There is also considerable value in retaining (under

conditions of high stability) samples of material used

during the clinical development process in order to

bridge between clinical studies, for example, if the retesting

of potency in updated assays becomes

necessary.

22


Setting Specifications on Potency Assays:

• How much do we need?

Two Questions

• How certain do we need to be of that value?

23


U.L.

Potency

L.L.

Calculation of vaccine minimum

release potency

•For vaccines, we usually set a minimum release spec to

assure mean potency exceeds a clinically acceptable lower

limit (L.L.) throughout the shelf life

Release spec

} Error term

Time

Expiry

} Expected stability loss

based on stability studies

(linear regression, when

feasible)


U.L.

Potency

L.L.

Calculation of minimum release

}

storage

Release spec

Error term

shipping

storage in clinic

potency

reconstitution

Time

Expiry

} Expected stability loss

should encompass all

anticipated conditions


U.L.

Potency

L.L.

Calculation of minimum release

LRL

} Error term

potency (LRL)

• Error term should account for:

• Variability in potency assay

• Error associated with stability modeling

Time

Expiry

} Expected stability loss


U.L.

Potency

L.L.

Calculation of minimum release

LRL

} Error term

potency (LRL)

•Goal of calculation:

•A lot with a potency test revealing a mean

potency at the minimum release specification

LRL has a 95% chance of retaining a mean

potency at or above L.L. at the time of expiry

Time

Expiry

} Expected stability loss


U.L.

Potency

L.L.

Calculation of minimum release

LRL

} Error term

potency (LRL)

•If variability in potency assay is too great, then it

may not be feasible to manufacture product such

that potency can be assured to remain between LL

and UL (highest clinically acceptable dose)

throughout shelf life

Time

Expiry

} Expected stability loss


U.L.

Potency

L.L.

Release potency window

} Error term

URL

LRL

} Error term

(LRL –URL)

•If product cannot be produced such that it an be

released at potency between LRL and URL , then

there is no consistently manufacturable product

Time

Expiry

} Expected stability loss


When is this easier?

• Assay precision is high

– When assay precision is high, it is easier to

construct a release model due to improved

understanding of release potencies and stability

– Must be careful that assay is measuring the right

thing

• Manufacturing variability is low

• Therapeutic index is wide

– At some point, somebody may be nonetheless

tempted to push limits!


Strategies to reduce effect of

variability in potency assays

• Multiple replicates

– Permits greater confidence in result

• Standardization (to house or international standard)

– Normalize results to a standard of known potency

– Reduces impact of inter‐run assay variability, but not

within‐run assay variability

– Need some way to assure potency value for standard is

and remains accurate

31


A single assay doesn’t always

suffice

• Complex mechanism of action or complex

characterization

• Consider multiple assays, both bioassays and

physical chemical assays

– Example: one assay could address upper safety limit, while

a different assay addresses lower limit

– Bioassay to indicate range of potency or minimum value

and chemical assay to provide quantity

• Need to beware of multiplicity issues

32


Stability‐indicating assays

• Identify clinically meaningful degradation in

product

• Implies that the assay predicts clinical benefit

not only at the beginning of the dating period,

but also at the end

• Supportive data can often be obtained in

accelerated or forced degradation studies


Necessary attributes for potency

assays

• Predictive of clinical benefit

• Possess characteristics that are amenable

to validation

• Precision sufficient to meet goal of

potency assays, i.e., provide assurance

that vaccine is safe and effective

throughout the dating period

• Stability indicating

34


Influenza Potency Assays

Mark S. Galinski

Vaccine Analytical Sciences

MedImmune

13-Aug-2009


� Influenza Background

� Drift and shift of virus antigenicity

� Commercial Vaccines

Outline

� Potency Assay Traditional Inactivated Influenza Vaccines

� Single Radial Immunodiffusion Assay

� Potency Assay Live Attenuated Influenza Vaccine

� Fluorescent Focus Assay

� FFA Automation

� Isocyte for Foci Enumeration


Fields Virology, 5 th ed, 2007

Influenza Virus – Many Reservoirs

– Many Subtypes

� Influenza A, B, & C (serotypes) can infect humans

� Seasonal influenza vaccines contain A and B serotype proteins

� Influenza A has (16) HA and (9) NA subtypes


� HA Hemagglutinin

� Receptor Binding Protein

Fields Virology, 5 th ed, 2007

Influenza A HA and NA subtypes

� NA Neuraminidase

� Receptor destroying activity


Antigenic Drift

Annual Influenza Epidemics

� HA undergoes amino acid changes at sites targeted by antibodies (genetic

mutation) to generate a “current” circulating strain

� Immunity to the “current” circulating strain is reduced (incomplete

protection)

� New strain emerges

> Selection for variant strain to spread due to incomplete protection


Antigenic Shift

Pandemic Influenza

Belshe, R.B. The Origins of Pandemic Influenza –Lessons from the 1918 Virus. The New

England Journal of Medicine 353:2209‐2211, 2005.


Seasonal Influenza Vaccines

FDA-Approved Trivalent Vaccines

Manufacturer Vaccine Type

CSL Limited Inactivated, split

GlaxoSmithKline Biologicals Inactivated, split

ID Biomedical Corp of Quebec Inactivated, split

Novartis Vaccines and Diagnostics Ltd Inactivated, split

Sanofi Pasteur, Inc Inactivated, split

MedImmune, LLC Live, intranasal


Embryonated Eggs H1N1

� Traditional inactivated vaccine (split)

Influenza Manufacturing

Drug Substance

� Each strain is harvested from allantoic fluids, clarified, centrifuged,

inactivated, membrane containing surface antigens HA and NA purified

(split using detergent) from the internal viral proteins, and sterile filtered

� Live attenuated influenza vaccine

H3N2

� Each strain is harvested from allantoic fluids, clarified, centrifuged, sterile

filtered

B


Influenza Vaccine Antigen Components

� Hemagglutinin (HA) and Neuraminidase (NA) are the

immunologically relevant proteins associated with vaccine

efficacy

Neuraminidase

NA

Matrix

M

Nucleocapsid

N

Hemagglutinin

HA

vRNA

Membrane

Polymerase

Complex

PA, PB1, PB2


� Traditional inactivated vaccine (split)

� Blend of H1N1, H3N2 and B

� Parenteral administration

� Virus components directly antigenic

Influenza Vaccines

Drug Product

� Live attenuated influenza vaccine

� Blend of H1N1, H3N2 and B

� Intranasal administration

� Virus components require

replication to present antigens to

immune system


Inactivated Vaccine Potency

SRID/SRD

� Inactivated influenza vaccine dosage based on clinical studies

following re-emergence of influenza H1N1 in the late 1970s

� Naive subjects (no pre-existing immunity) required two doses

� Subjects with some degree of pre-existing immunity needed only a

single dose

� Potency of inactivated split vaccines measures HA content per

dose using a single radial immunodiffusion (SRID/SRD) assay

� 15 mcg/0.5 mL dose for each virus component

� HA content correlated with antibody titers using whole-virus or split

vaccine

� Dose claim balanced maximum immune response in individual with

maximizing population coverage and minimization of adverse reactions


Seasonal Influenza Vaccines

Potency Measurements

� Potency measurements require reference immunoreagents

� Type/Subtype matched antisera

� Type/Subtype matched antigen (virus) HA reference standard

� SRID assay features

� Antiserum is incorporated into agarose matrix

� Samples are solubilized with detergent and allowed to diffuse

into the agarose

� A zone of immunoprecipitin forms where the antibody-antigen

complex is no longer soluble


Single Radial Immunodiffusion Assay

SRD/SRID

� HA potency measured by assessing immunoprecipitin diameter

� Requires reference virus and reference antiserum (CDC, NIBSC)

Ag Dilution

Reference

Vaccine (1) 1x

Vaccine (2) 4x

Concentrated Virus

Antiserum in

Agarose

1:1 1:2 1:4 1:8

Adapted from Wood et al., 1977 Develop, biol. Standard., 39:193-200

R

V 1

V 2

C

1:1 1:2 1:4 1:8 1:1 1:2 1:4 1:8

4x 2x 1x

R

V 1

V 2

C


Live Attenuated Influenza Vaccine

Influenza vaccine live, intranasal

� Potency of live attenuated vaccine

measures virus infectivity per dose

using immunostaining of infected MDCK

cells (fluorescent focus units)

� 6.5-7.5 log 10 FFU/0.2 mL dose for each

virus component


Fluorescent Focus Assay (FFA)

� Potency of live attenuated vaccine measures virus infectivity

per dose using immunostaining of infected MDCK cells

(fluorescent focus units)

� 6.5-7.5 log 10 FFU/0.2 mL dose for each virus component

� FFA assay features

� Quantitative assay enumerates influenza-infected cells following

immunostaining

� HA-specific primary antibodies followed by detection with a

fluorescent-labeled secondary antibody (e.g. Alexa Fluor 488)

� Primary antibodies specifically bind to one of each of the vaccine

components

> A/H1N1, A/H3N2 and B strain


Fix cells

Stain with 1 °

Antibody

Infect 96-well MDCK cell monolayer

with serial dilutions of virus sample

Incubate 17-20 hrs

Stain with 2 °

Antibody

Fluorescent Focus Assay

Enumerate fluorescent foci


� FFA Validated

FFA Validated Release Assay

Drug Substance & Drug Product Release

� Accuracy, precision, LOD, LOQ, specificity, linearity/range,

ruggedness, robustness, system suitability

� Potency is a quantitative measure of infectivity assessed at a

specific time range post-infection

� Precision for FFA is better than for a TCID 50 quantal assay

� Typically 0.1 – 0.15 log 10 FFU

� Typically >0.3 log 10 TCID 50

� Accuracy/specificity requires critical immunoreagents

� Potency testing of trivalent drug product needs to distinguish

between each vaccine component


FFA Focus Enumeration

� Manual counting of foci does not use the entire well

� Scan and count across a portion (e.g. ~33%) of the well

� Region of interest (ROI) dependent on optical magnification


FFA Focus Enumeration

� Foci counting can be challenging

� Counting range 15-200 foci

� Analyst fatigue and ergonomics

� Photobleaching reduces signal to

noise for dimly stained foci

� Sample/plate throughput

� Automation assessed as a

technical improvement


The IsoCyte

Blueshift Biotechnologies

MDS Analytical Technologies

� Single or Dual Laser

� Laser lines: 405, 440, 488, 532, or

635 nm excitation

� 4-color detection simultaneously

� 2-color polarization

� light scatter image

� Accepts 6 – 1536-well plates and

microscope slides

� High speed laser scanning

fluorimeter & scatterometer

� Capablity to combine with stacker


Isocyte Technology

� Laser is focused on a confined detection region at bottom of well

� Depth of field allows large objects to be visualized in 3D

� Fluorescence is collected by PMTs and digitized

� Laser has very short dwell time - does not cause photobleaching

PMT 1

PMT 3

Collection

lens

Dichroic mirror

Laser

Sample

Scan lens

Scanning mirror

Emission filters

PMT 2

PMT 4


Implementation in QC Lab

Technical & Regulatory Considerations

� 21CFR Part 11 compliant data collection and control

� Demonstrate equivalency to current manual method

� Instrumentation needs to duplicate an analyst (human)

� Art of staining and manual counting can influence comparability

� Analyst trained to enumerate foci that an instrument finds

problematic

� Instruments can easily enumerate more foci more rapidly than

any analyst

� Extend enumeration capabilities (foci numbers & ROI)

� Rectangle (e.g. 33% of well); 200 max foci

� Circular (e.g.~80% of well); up to ~1,800 max foci (sample

dependent)

� PQ and assay validation


Implementation in QC Lab

Technical & Regulatory Considerations

� 21CFR Part 11 compliant data collection and control

� Password Protection Procedure

� User Account Procedure

� User Modes of Operation

� Audit Trail Management Procedure

� System Calibration Procedure

� Data Protection


Art of staining and manual counting can

influence comparability

FITC

Nonspecific Staining

Alexa 488

Specific Staining


Manual

160

140

120

100

80

60

40

20

0

Foci Number Comparison

y = 1.0715x

R 2 = 0.9636

0 50 100 150

ISOCYTE

Demonstrate equivalency

Instrumentation Needs to Duplicate an Analyst

Manual

10.0

9.0

8.0

7.0

6.0

Titer Comparison

y = 1.0042x - 0.0032

R 2 = 0.9915

6.0 7.0 8.0 9.0 10.0

ISOCYTE


Extend Enumeration Capabilities

(Foci Numbers & ROI)

� Circular (e.g.~80% of well); >500 foci

� Rectangle (e.g. 48% of well); 200 max foci

Manual Rec ROI

600

500

400

300

200

100

H1N1

y = 1.4308x

R 2 = 0.9998

y = 1.0156x

R 2 = 0.9898

0

0

0 100 200 300 400 500 600

Isocyte Rec ROI

Manual vs Isocyte Rec vs Rec Isocyte Rec vs Circular

Linear (Manual vs Isocyte Rec vs Rec) Linear (Isocyte Rec vs Circular)

600

500

400

300

200

100

Isocyte Cirular ROI


Extend Enumeration Capabilities

(Foci Numbers & ROI)

� Circular (e.g.~80% of well); >500 foci

� Rectangle (e.g. 48% of well); 200 max foci

Potency Isocyte (FFU/mL)

4.0

3.8

3.6

3.4

3.2

3.0

2.8

2.6

2.4

2.2

H1N1

2.0

2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0

Potency Manual (FFU/mL)

Iso-Rec Iso-Cir


Potency Isocyte (FFU/mL)

4.0

3.8

3.6

3.4

3.2

3.0

2.8

2.6

2.4

2.2

Extend Enumeration Capabilities

(Foci Numbers & ROI)

H3N2

� Circular (e.g.~80% of well); >500 foci

� Rectangle (e.g. 48% of well); 200 max foci

2.0

2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0

Potency Manual (FFU/mL)

Iso-Rec Iso-Cir

Manual Rec ROI

600

500

400

300

200

100

H3N2

y = 1.4708x

R 2 = 0.9999

y = 0.9378x

R 2 = 0.9973

0

0

0 100 200 300 400 500 600

Isocyte Rec ROI

Manual vs Isocye Rec vs Rec Isocye Rec vs Circular

Linear (Manual vs Isocye Rec vs Rec) Linear (Isocye Rec vs Circular)

600

500

400

300

200

100

Isocyte Cirular ROI


Manual Rec ROI

800

700

600

500

400

300

200

100

y = 0.9625x

R 2 = 0.9998

Extend Enumeration Capabilities

(Foci Numbers & ROI)

B

y = 1.4478x

R 2 = 0.9997

0

0

0 100 200 300 400 500 600

Isocyte Rec ROI

Manual vs Isocyte Rec vs Rec Isocyte REc vs Circular

Linear (Manual vs Isocyte Rec vs Rec) Linear (Isocyte REc vs Circular)

� Circular (e.g.~80% of well); >500 foci

� Rectangle (e.g. 48% of well); 200 max foci

Potency Isocyte (FFU/mL)

4.0

3.8

3.6

3.4

800

3.2

700 3.0

2.8

600

2.6

500 2.4

2.2

400

2.0

300 2.0 2.5 3.0 3.5 4.0

200

100

Isocyte Cirular ROI

B

Potency Manual (FFU/mL)

Iso-Rec Iso-Cir


� PQ and assay validation

� Regulatory Approval

Implementation in QC


Summary

� Influenza potency measurements assure that seasonal trivalent

vaccine products will meet their intended clinical dose effect

� The virus surface glycoproteins (HA, NA) are the

immunologically relevant antigenic components of influenza

vaccines

� SRID assay is the standard methods for release of traditional

inactivated influenza vaccines

� FFA assay is used to measure the infectivity of live attenuated

influenza vaccines

� Influenza potency assays are critically dependent upon the

immunoreagents and reference standards for an accurate

assignment of potency


Influenza Vaccine Potency

Determination and Pandemic

Preparedness

The BARDA/IED Perspective

for

USP 2 nd Bioassay Workshop August 13, 2009

Armen Donabedian, PhD

Senior Program Manager, Vaccines Advanced Development

HHS/BARDA/IED


From BARDA/IED to the

USP Workshop

Let’s work together to improve or

replace the Single Radial

Immunodiffusion (SRID) assay and

facilitate seasonal and pandemic

influenza preparedness


What is BARDA/IED?

� Biomedical Advanced Research & Development Authority

(BARDA) established by PAHPA in Jun. 07 in HHS/ASPR

� Implements USG strategies & policies for MCMs from the Public

Health Emergency Medical Countermeasure (MCM) Enterprise

(PHEMCE) within the office of the ASPR

� Coordinates integrated product portfolio approach to planning and

executing research, development and acquisition of public health

emergency MCMs

� Supports MCMs for CBRN Threats, Pandemic Influenza, and

Emerging Diseases

– Advanced development

– Stockpile procurement & establishment

– Manufacturing infrastructure building

– Science and Technology Platforms


Immediate Office

of the ASPR

Chem/Bio

Rad/Nuc

(CBRN)

Biomedical

Advanced Research

& Development

Authority

ASPR/BARDA/IED Organization

Office of the Secretary of HHS

Regulatory/Quality

Affairs

Influenza &

Emerging Diseases

ASPR

Strategic Science

& Technology

Policy, Planning &

Requirements

Office of Medicine,

Science,

& Public Health

Computer

Modeling

Resources &

Program Operations

Office of

Preparedness &

Emergency

Operations

Acquisitions

Management


Vaccines

U.S. Pan Flu MCM Strategic

Current & Possible New Policy

Goals

– Goal #1: Establish and maintain a dynamic pre-pandemic influenza vaccine stockpile

available for 20 M persons (2 doses/person) or more persons depending on vaccine mfg.

capacity & results of dose-sparing adjuvant studies and prime-boost immunization studies:

H5N1 vaccine stockpiles

– Goal #2: Provide pandemic vaccine to all U.S. citizens within 6 months of a pandemic

declaration: pandemic vaccine (600 M doses)

� Antivirals

– Goal #1: Provide influenza antiviral drug stockpiles for pandemic treatment of 25% of U.S.

population (75 M treatment courses) and federal share of antivirals for outbreak

prophylactic usage as a community mitigation measure as shared responsibility

– Goal #2: Provide influenza antiviral drug stockpiles for strategic limited containment at

onset of pandemic (6 M treatment courses)

� Diagnostics

– Goal #1: Develop new high-throughput laboratory, point-of-care (POC), and home detection

influenza diagnostics for pandemic influenza virus detection

� Other Countermeasures

– Goal #1:Develop and acquire other MCMs including syringes/needles, masks/respirators,

ventilators, antibiotics, & other supplies (Pneumococcal & Streptococcal Vaccines?)

National Strategy for Pandemic Influenza (Nov 2005) and HHS Pandemic Influenza Plan (Nov 2005) www.pandemicflu.gov


32 contracts &

2 grants

totaling $6.0 B

Advanced

Development

Stockpile

Acquisitions

Infrastructure

Building

BARDA Pan Flu Integrated

Program Portfolio Approach

Vaccines Antivirals Diagnostics/

Respiratory

Devices

Cell-based

Antigen-sparing

Next Generation

Recombinant

H5N1 Pre-Pandemic

Vaccine Stockpiles

& H1N1 Purchases

Retrofit Existing Mfg

Facilities

Build New Cellbased

Mfg Facilities

Egg-based Supply

Peramivir

More to Come

Tamiflu & Relenza

Federal Stockpiles

State Stockpiles

AV MedKits

Diagnostics

Point of Care

Clinical Lab

Ventilators

Next Generation

Masks &

Respirators


Pandemic Influenza

Medical Countermeasure

Supply-Demand Supply Demand Gap Closure

Reduce Demand: Pre-Pandemic Vaccines, Community Mitigation, Antivirals, Vaccines, Masks

Increase Capacity: Ventilators, Oxygen, Antivirals, Pandemic Vaccines, Masks

Pre-Pandemic

Vaccines

Demand for

Healthcare Services

Recombinant

Vaccines

Egg- & Cellbased

Vaccines

Increase Supplies of Critical Materiel

Current Healthcare Capacity


Influenza Potency Assay

Time Line Considerations

SRID potency assay reagent availability is a key element

8-12 weeks est. during a pandemic emergency - Actual for 2009 H1N1 was 13 weeks

(Seed strains available at end of April – first reagents available at the end of July)

Seasonal SRID potency assay reagents typically available in 4 months

Pre-Pandemic

Vaccines

Demand for

Healthcare Services

Recombinant

Vaccines

12

weeks

20

weeks

Egg- & Cellbased

Vaccines

Increase Supplies of Critical Materiel

Current Healthcare Capacity


Ant

Assay

Based on Bull World Health Organ. 1975; 52(2): 223–231. 223 231.

Influenza Vaccine Single Radial

Immunodiffusion

Ab

http://www.gbiosciences.com/EducationalUploads/EducationalProductsImages

http://www.gbiosciences.com/EducationalUploads/EducationalProductsImages

PAGE

Protein Assay

Calibration

Densitometer

http://www.gmi-inc.com/BioTechLab/Bio2520Rad

http://www.gmi inc.com/BioTechLab/Bio2520Rad


� The Problem

Influenza Vaccine Potency

Determination and Pandemic

Preparedness

– Preparation and calibration of SRID potency reagents delays

production, formulation, clinical study and/or release of influenza

vaccine

� Considerations for replacement

– SRID is the influenza vaccine stability indicating assay

– The immunological component of the assay is considered to be

essential

� One or more of the three vaccine strains (H1, H3, B) change annually

� compelling evidence of manufacturing consistency is difficult to establish

– Irony is that the antigen standard is measured using physio-chemical

methods only


� Proposed Partial Solution

Influenza Vaccine Potency

Determination and Pandemic

Preparedness

– Accelerate and improve accuracy of reagent calibration

– LC-MS/MS

Quantification of Influenza Virus Hemagglutinins in Complex

Mixtures Using Isotope Dilution Tandem Mass Spectrometry

Vaccine 26, 2008, p2510

Tracie. L. Williams, Leah Luna, Zhu Guo, Nancy J. Cox, James L.

Pirkle, Ruben O. Donis, John R. Barr

National Center for Environmental Health

&

National Center for Infectious Diseases


Isotope Dilution LC-MS/MS

� Unique conserved sequences were identified

– Apply to 97+% of H1, H3, H5 and B influenza strains

� The peptides are used as a stoichiometric representative of the

specific protein they are derived from

� A known amount (concentration essential) of labeled C 13 and N 15

labeled peptide is spiked into the sample (e.g. 6 -10 daltons

heavier)

� The endogenous target peptide is released by detergent

treatment and trypsin cleavage (completeness essential)

� Quantification is performed by comparing the peak area of the

labeled peptide with that of the endogenous target


� LC-MS/MS

Isotope Dilution LC-MS/MS

– Long established principle used for years in reference labs

– Replaces carbon-12 with carbon-13 to create an internal standard

with nearly identical chemical properties but different mass

� Accuracy traces to NIST standard reference materials for amino

acids

– Amino acid analysis using ninhydrin with UV detection did not show

agreement among labs

– CDC developed a MS amino acid analysis method using NIST

accuracy standards

– CDC analyses of NIST aa estimates 149.85 (CV 2.20%) pmoles

compared with an expected value of 150 pmoles


� Compares well with SRID

Isotope Dilution LC-MS/MS

Accuracy Precision LOD Time to establish

SRID 10% 10% 10 ug One month

LC-MS/MS

?

A priori* 5% 1 ug Immediate

* 24.38, 16.52 and 38.8 ug HA/dose for the H1, H3 and B strains.

– Each strain in influenza vaccine is formulated to 15 ug/dose based on the

SRID assay


BARDA/IED LC-MS/MS LC MS/MS

Proposed Agenda

� Collaboration between CDC and CFSAN for method transfer

– Review of method and qualification data

– Lab evaluation

� Instruments, data collection systems, personnel and special techniques

� Reagents – identify a source for peptides

� Development of acceptance criteria

� Further clarification of the method procedure if necessary

� Further qualification/validation of assay parameters such as sample

storage or sample preparation

� Procedure to confirm complete digestion of each new strain

– Transfer Protocol

� collect data and write report


BARDA/IED LC-MS/MS LC MS/MS

Proposed Agenda

� Collaboration between CBER and CFSAN for comparative

studies

– Implementation proposal (examples)

� Preparation of standards for testing using current methods and LC-

MS/MS

� Comparison of past (H5) antigen standard values with LC-MS/MS

� Comparison of (H5) vaccine and intermediates SRID values with LC-

MS/MS

� Evaluate alternative approaches to measure potency.

– Immunoprecipitation

– Protein mass mapping

– The sialic acid - HA binding concept (NCIRD/CDC)

– Other approaches


Development Funding Opportunity

Science and Technology Platforms

Applied to MCM Development

Broad Agency Announcement (BAA)

BARDA-BAA-09-100-SOL-0010

Solicitation Posted July 8, 2009

www.MedicalCounterMeasures.gov


Purpose of SSTD BAA

Promote Innovations in Product Development

Improvements, Testing, and Manufacturing

� Platform technologies to promote:

– Improvements in product safety, efficacy, stability, and ease of

use

– Efficient and cost-effective technologies associated with

process development, testing, and manufacturing

– Technologies relevant to targets of interest

� Generate validated data sets for comparability purposes

with products already in late stages of development

� Integration with ongoing regulatory activities

� Complement and support CBRN and IED portfolio efforts


SSTD BAA: Areas of

Interest

1. Technologies to Accelerate Evaluation of Candidate

Vaccines and Therapeutics

2. Formulation Chemistry, Protein Stabilization, and

Vaccine Delivery Technologies

3. Innovative Methods in Bioprocess Development and

Manufacturing

4. Methods and Technologies to Advance Development

of Diagnostic Tests for Rapid Diagnosis of Human

Infections


1. Anthrax Vaccines and Anti-Toxins

SSTD BAA: Targets of Interest

2. Pandemic Flu Vaccines and Antivirals

3. Botulinum Anti-Toxins

4. Diagnostic Platforms Applicable to a Range of

Pathogens (Bacterial and Viral)

– Category A Bioagents

– Emerging Infectious Diseases


SSTD BAA:

Contact Information

Michael Younkins

Contracting Officer

DHHS, BARDA

Email: Michael.Younkins@hhs.gov

www.MedicalCountermeasures.gov


� Federally-sponsored conferences

� Funding opportunities

� Resource programs

� Regulatory guidance

� Federal strategies and reports

Contact Info

BARDA:

URL: www.hhs.gov/aspr/barda/

E-Mail: BARDA@hhs.gov

� Upcoming Events

� Acquisitions

� BioShield

� Influenza Programs


Assessment of Neutralizing Antibodies

to Biological Therapeutics

Susan L. Kirshner, Ph.D

Office of Biotechnology Products

Division of Therapeutic Proteins


• Introduction:

Outline

– Immunogenicity concerns for therapeutic

proteins

– Definitions

• Evaluating Immunogenicity

• Neutralizing assays


Introduction


Concerns for Antibodies in the Clinic

Clinical Concern Clinical Outcome

Safety

Efficacy

Pharmacokinetics

None

•Neutralize activity of endogenous

counterpart with unique function

causing

deficiency syndrome

• Hypersensitivity Enhancing or decreasing reactionsefficacy

by

extending or decreasing half life.

• Enhancing or decreasing efficacy by

changing biodistribution.

• Antibody production may dictate

changes

in dosing level due to PK changes.

• Despite generation of antibodies, no

discernable impact


There are cases in which immune responses

to therapeutic proteins have had devastating

consequences for healthy volunteers and

patients.

• rDNA Human MGDF

• Erythropoietin

• Glucocerebrosidase

• a-glucosidase(Pompe’s)

• Factor VIII

• Insulin


What is immunogenicity?


Definitions

• Antigen – any substance that can be recognized

by the adaptive immune system (Janeway et al.

Immunobiology, 6th Ed. Pg. 2)

• Epitope – structure recognized by an antibody or

T cell antigen receptor

– Conformational – the epitope is recognized by its 3D

structure. It may be composed of continuous or

discontinuous protein sequences.

– Linear – the epitope is recognized primarily based on

its amino acid sequence. T cell epitopes are

continuous, B cell epitopes can be continuous.


• Adaptive immunity – antigen specific

immune response that evolves over time

leading to increased efficiency in pathogen

recognition and the establishment of

immunological memory to a pathogen.

Memory is established by the presence of

long lived populations of antigen specific T

and B cells. This generally involves either

prolonged or multiple exposures to a

pathogen or to pathogen epitopes (e.g.

vaccines).


Antibody structure


Binding vs Neutralizing Antibodies

Cell

Activation

Receptor

Therapeutic

Protein


-Bind anywhere on

the molecule

-May or may not

inhibit protein

activity in a

bioassay

-Detected through

non-biologically

based assay (e.g.

ELISA, ECL, SPR)

Binding Antibodies (BAbs)


Neutralizing Antibodies (NAbs)

Cell

Activation

-NAbs are a subset if BAbs

-Binding inhibits

receptor/ligand interactions

-Inhibit protein activity in a

bioassay

-Detected using a bioassay


Clinical Significance

• In a patient both BAbs and NAbs can lead

to loss of efficacy and/or negatively impact

safety, therefore both may be clinically

important

• NAbs may be more effective in directly

impacting efficacy

– IL-2 and IFN-b higher titers of NAbs are

associated with loss of efficacy


Evaluating Immunogenicity


Evaluating Immunogenicity: A Tiered

Approach

Sensitive screening immunoassay

Negative

Negative

Negative

Reactive

Confirmatory assay

(e.g. cold competition)

Reactive

Neutralizing

Bioassay

Positive

IgG

IgM

IgE

IgA


NAb Assays

• The presence of NAbs should be

assessed using a biologically based assay

unless the MOA of the therapeutic has

been shown to be cell independent (e.g.

an enzymatic activity that occurs in serum)

• The bioassay should be relevant to the

MOA of the therapeutic

• More than one assay may be required for

a protein with multiple functional domains

that are relevant to its therapeutic activity.


Potency Assays used in NAb

Assays

• Most frequently the assay used to assess

potency for drug release is adapted to be used

in the NAb assay. Therefore any format found to

be appropriate for release is generally

appropriate for the NAb assay. This includes

assays with early (e.g., cAMP induction),

intermediate (e.g. transcription) and late (e.g.

proliferation, cytokine release) endpoints

• Some cell lines do not adapt well to a matrix that

includes human serum. Therefore the NAb

assay may be based on a potency assay that is

different from the release assay. The assay

should still reflect the drugs purported MOA.


NAb Assay Formats

• Most assays are based on a fixed amount of drug

and cells and test articles at a single fixed dilution.

Samples will be assessed as positive or negative if

they inhibit the response beyond the cut-point.

Alternatively the test article may be assessed at

multiple dilutions with the titer reported as the lowest

dilution giving a response below the cut-point.

• For fixed dose assays it is recommended that cell

lines be stimulated to 40% - 70% of their maximum

response in the linear portion of the dose response

curve. This dose is generally established during

development. Dose response curves should be

provided to the Agency to support the dose

selection.


NAb Assay Formats

• Some assays are based on shifts in EC50

based on complete dose response curves

in the presence and absence of test sera.


NAb Assay Formats: Reporting Results

• It is recommended that assay results be reported as

+/- or in titer

• Results may be reported as units of drug inhibited

per volume. It should be understood that this

method of reporting results is quasi-quantitative and

does not necessarily reflect the amount of drug

inhibited in vivo. This method of reporting results is

discouraged because the results are frequently over

interpreted.

• Assay results should not be reported in mass units

based on back calculation to a standard curve

because it is unlikely that dilutional parallelism exists

between the Ab reference standard and Abs in the

test serum for all test articles.


NAb Assays

• The incidence of NAbs is reported in section 6.2

of the label (PLR format). Therefore it is critical

to have a robust validated assay.

• Assay validation:

– Cut-point

–Precision

– Selectivity – matrix interference (patient sera, on

board drug)

– Robustness – sensitivity to changes in operating

parameters (e.g., incubation time/temperature)

– Reproducibility – differences between laboratories.

This is only necessary if the assay will be performed

in more than one laboratory


NAb Assay Sensitivity

• Assay sensitivity is generally assessed by

determining the smallest amount of

neutralizing Ab that tests positive in the NAb

assay. Results are usually reported in mass

units per volume.

• Sensitivity is generally assessed as part of

the validation exercise but may be assessed

outside of the validation exercise.

Regardless, results should be reported to the

Agency.


NAb Assay Sensitivity

• The assessment of assay sensitivity is

dependent on the positive control Ab used

• The development of positive control NAbs can

be very difficult

• If polyclonal Abs are used as a positive control

then it is likely that only a very small portion of

the Ab population will be neutralizing. This will

result in an apparent lack of assay sensitivity.

• Therefore we do not have a recommended

target for assay sensitivity


NAb Assay Sensitivity

• The determination of whether an assay

has appropriate sensitivity is assessed

based on overall assay performance in the

validation exercise, results of the

sensitivity assessment and information

gained during assay development


Specificity

• Due to the complexity of cell based NAb assays

it is usually impossible to validate the specificity

of the Abs

• Because the Ab response is usually shown to be

drug specific in the binding and confirmatory

assays the specificity of the assay is frequently

not confirmed or validated as with BAbs

• Some Sponsors immunodeplete NAb positive

serum samples to confirm that positive

responses are Ab dependent


Selectivity

• It is critical to assess the performance of the cell

line in target population matrix because cell lines

may respond to serum factors other than the

API. Such responses can confound data

interpretation

• Incorporation of Ab against serum factors has

been successfully used to overcome this

problem.

• On-board drug in the serum can also impact

assay performance

• Samples should be obtained at trough drug

levels to mitigate this problem. Samples

obtained after a sufficient washout period

(generally ~5 half lives) may also be needed


Summary

• Patients should be monitored for the presence of

NAbs because they can negatively impact drug

safety and efficacy

• NAb assays should be biologically based and

reflect the purported MOA of the drug

• Results of assay validation exercises as well as

critical development data (e.g. dose/response

curves) and the assay SOP should be provided

to the Agency for licensure.

• NAb incidence (and titer when known/relevant)

is reported in the product label

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