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


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


• 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


• 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. 


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. 


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 


U.L. 

Potency 

L.L. 


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 


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 


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 


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





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


4.0 

3.8 

3.6 

3.4 

3.2 

3.0 

2.8 

2.6 

2.4 

2.2 



H3N2 



2.0 

2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6 3.8 4.0 


Iso-Rec Iso-Cir 


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 


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


800 

700 

600 

500 

400 

300 

200 

100 

y = 0.9625x 

R 2 = 0.9998 



B 

y = 1.4478x 

R 2 = 0.9997 

0 

0 

0 100 200 300 400 500 600 


Manual vs Isocyte Rec vs Rec Isocyte REc vs Circular 

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




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 


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 


Egg- & Cellbased 


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 


Demand for 

Healthcare Services 

Recombinant 


12 

weeks 

20 

weeks 

Egg- & Cellbased 


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 



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 



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 


– 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 


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.


• 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


• 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

Vaccine

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