Laboratory of Plasma Derivatives Research Overview

Dorothy Scott, M.D. Chief, Laboratory of Plasma

Derivatives DH/OBRR/CBER

Blood Products Advisory CommitteeDecember 15, 2010

LPD Mission Statement

To meet the public health needs for safe and effective products by performing high

quality research that directly impacts the safety, effectiveness and availability of

plasma derivatives.

Research Projects Cross Sections

Regulated Products

Immune Globulin (10 Products) – Primary Immune Deficiency, ITP, CIDP, Kawasaki Disease, secondary immune deficiencies

Specific Immune Globulins – for HBV, CMV, HAV, tetanus, rabies, measles, vaccinia, infant botulism, prevention of newborn hemolytic disease

Regulated Products

Antivenoms and antitoxins – coral snake, rattlesnake, and black widow spider envenomation, botulism, digitalis intoxication

Anti-thymocyte globulins – treatment of transplant rejection

Alpha-1 Proteinase Inhibitor – treatment of emphysema in A1PI deficiency

Standards and Reagents for release testing

• Development of international standards and test methods to increase safety of plasma derivatives

• Ongoing:– Testing of new CBER potency standard for Immune

Globulins – Development and testing of new international B19 virus NAT

standards for plasma testing– Characterization of the first A1PI international standard– Research on potency testing and safety correlates

• HCV immune globulin • Vaccinia immune globulin• Influenza immune plasma • Characterization methods for aggregates in plasma derivatives

Developing Predictive Models for Preclinical Product Evaluation Of Safety

and Efficacy• HCVIGIV and SARSIGIV– predicting

product efficacy based on neutralizing activity

• Influenza – more rapid test methods for neutralizing antibodies

• Developing and evaluating test methods to characterize product aggregates and to assess aggregate immunogenicity

Evaluating Efficacy and Safety of Immune Globulins for Pandemic and

Counterterror Response

• Animal model evaluation of VIG products and therapies

• Evaluation of animal models for counterterrorism-related immune globulins

• Influenza antibody testing of plasma collected from donors receiving influenza vaccine or post-influenza infection– Predicting donor characteristics likely to be

associated with highest neutralizing antibody titers

Research Framework Enables LPD to Scientifically Address Regulatory

Issues • Evaluation of IG products reported to cause

clinically relevant hemolysis • Biochemical changes in A1PI products were

detected and analyzed, leading to changes in product manufacture

• Evaluation of thrombosis-associated IGIV product lots (LPD/LH)

• Identification of B19 virus transmission by a coagulation factor

• Identification of bacterial constituent contaminant in a product using TLR-transfected cells

Preparing for the Future • Immunogenicity – addressing the lack of

predictive animal models • Improving IGIV safety, efficacy, potency

– Enhancing neutralizing potency (HCVIGIV)– Characterizing aggregates and their impact on potency,

safety, and immunogenicity – Facilitating development of potency assays most

relevant to IGIV used for primary immune deficiency patients

– Rapid collection and testing of plasma for manufacturing specific immune globulins in pandemic or BT scenarios

• Developing preclinical models for inhaled A1PI efficacy and immunogenicity

Public Health Problem – Improving Treatment for Progressive Vaccinia

(D. Scott, M. Kennedy, R. Fisher) • If there is a smallpox outbreak, mass vaccination

is likely • In this setting, immune compromised people are

likely to be exposed to vaccinia– Inadvertent vaccination – Contact exposure

• Treatment and post-exposure prophylaxis for progressive vaccinia are not well-defined– High mortality rate/need for intensive support

Development and characterization of an animal model to study pathogenesis and

treatment of Progressive Vaccinia

1) Animal model can be used to assess new therapies and current products

2) Need to understand mechanism of VIG potency

- Improve product

- Improve potency assays

for product release

SCID mouse model results

• Lesions similar to human lesions • Slow progression, lethality • Responsive to VIGIV

– Early treatment eliminates infection

• VIGIV + topical cidofovir – long-term survival with delayed treatment

• Treatment effect does not require complement

Conclusions• SCID PV model simulates many features of

human PV*• SCID PV model should be useful for testing

combined treatments and new antibody treatments

• Studies support the likelihood that early PEP may prevent PV in susceptible hosts

• Ongoing studies – – Identify VIGIV mechanism(s) of action– Animal model studies of maternal/fetal vaccinia

complications (funded, FDA Office of Women’s Health)

* In press, Clinical and Vaccine Immunology, January 2011

Neutralization of Viruses Relevant to Immune Globulin Products (P. Zhang, Ph.D.)

HCV Infection:About 170 million people worldwide including 4 million in the U.S. are infected with HCV. HCV-associated cirrhosis is the leading indication for liver transplantation in the U.S.

Public Health Problem Lack of vaccineNo immunoglobulin product for prophylaxis or treatment Treatment (IFN + ribavirin) is only 50% effective

Addressing the ProblemIdentifying critical epitopes for HCV neutralization Unexpected finding of interfering antibodies

HCV E2 ---QLINTNGSWHINSTALNCNESLNTGWLAGLFYQHKF---

412 446434426Epitope I Epitope II

HCIGIV Eluates

Presence of Neutralizing and Non-Neutralizing Antibodies in Experimental HCIGIV Preparations

AE , BE

Interference?

0.0

0.5

1.0

1.5

2.0

2.5Peptide A

Peptide B

Peptide C

Peptide D

AE BE CE DEControl

IGIVHCIGIV

A4

50

n

m

Binding Assay (ELISA)

DE

HCV E2 Peptides

A

DC

B

Epitope-Specific Neutralization and Interference of HCV by HCIGIV Eluates

DE

Infe

cti

vit

y

(% o

f Neg

ativ

e IG

IV

Con

trol

)

0

50

100

150

Control IGIV

Infe

cti

vit

y

(% o

f Neg

ativ

e IG

IV

Con

trol

)

0

50

100

150

DEControlIGIV

AE+D

E

Epitope IIEpitope IIHCV E2

Epitope IEpitope I

NeutralizationNeutralization

DDEE

Interference Interference

Epitope IIEpitope IIHCV E2

Epitope IEpitope I

AAEEDDEE

AE

Additional Findings • Depletion of interfering antibodies increases

HCVIGIV neutralizing activity• Depletion of interfering antibodies in infected

chimpanzee sera reveals cross-genotype neutralizing activity

• Evidence that interfering antibodies found more in chronic than recovered HCV patients

• Evidence from serial samples from one chronic HCV patient that interfering antibodies arise prior to neutralizing antibodies

Public Health Impact

• HCVIGIV preparations may be improved by removing interfering antibodies and enriching neutralizing antibodies

• Epitope-based binding assays can be designed to measure HCV neutralizing antibodies in sera (for clinical trials) and HCVIGIV products

• Vaccine design may be improved by developing antigens that will provide neutralizing but not interfering antibody responses

Use of Toll-like Receptor (TLR) Assays to Detect and Identify Microbial

Contaminants in Biological Products

Basil Golding M.D.

Site VisitJune 15, 2010

Objective

To develop a panel of TLR-expressing

cell lines that can detect the presence of

microbial components in biological

products including cellular and acellular

vaccines, cells used in gene therapy,

plasma-derived, recombinant and

transgenic proteins.

A Case StudyA recombinant product made from E. coli linked to adverse events in early clinical trials.

Testing Plan using TLR-expressing Cell Lines• A panel of blinded product samples labeled as

A, B, C, and D• Two protein product samples made from two

different manufacturing methods – both samples had passed lot release testing: LAL test, Total residual DNA assay and Host cell protein ELISA.

• One LPS positive sample• One negative control• Various TLR ligands

Results

• Sample B was found to activate cell lines expressing TLR5

• The ligand for TLR5 is flagellin

• Sample B was shown to contain flagellin by Western Blot and confirmed by Mass Spectrometry

Conclusions

• We have established a sensitive human cell-based assay which enabled the detection of trace amounts of a microbial contaminant in a biological product that was not detectable by standard lot release tests (LAL, DNA, HCP).

• The TLR assay results suggested that the putative contaminant was flagellin.

• This was confirmed by Western Blot and Mass Spectrometry.

Public heath problem - in vitro and in vivo

aggregation of A1PI (E. Marszal, Ph.D.) • Protein aggregation

– Causes human disease– Affects quality of biological products

• Protein aggregates in biologics are generally not well characterized• Structural and biochemical understanding of A1PI polymers is

critical – To enable development of therapies for A1PI ZZ-associated liver

disease – To improve product stability (understanding polymerization of

“normal” A1PI)• To address scientific questions:

– Investigation of the mechanism of 1-PI polymerization in vitro and in vivo

– Assay evaluation and development; characterization of 1-PI products

Collaborative project FDA/academia/manufacturers

Applied Research – Heterogeneity of 1-PI products

Marszal &Shrake 2006

-4 0 0 0 0 0

-2 0 0 0 0 0

0

2 0 0 0 0 0

4 0 0 0 0 0

6 0 0 0 0 0

8 0 0 0 0 0

1 E +0 6

1 E +0 6

0 5 1 0 1 5 2 0 2 5 3 0 3 5

Marszal BPAC 2005

0

2

4

6

8

10

12

14

16

18

20

0 5 10 15 20 25 30

Hydrodynamic diameter (nm)

% In

tens

ity

IEF, desialylatedplasma products

Polymer presence Adverse events:

rash, chills, fever,flu-like symptomsimmunogenicity?

Characterization of protein aggregates in 1-PI products Evaluation of methods andmethods developmentFirst product class characterization

Wes

tern

blo

t

Silver stain

Abso

rban

ce 2

15 n

m

HPLC

DLS

Elution time

Marszal 2010

Investigation of the structure of 1-PI polymers

55ºC 65ºC pH 4.1 Gu 1.4M

Tet Tri

D

M

TetTriD

M

55ºC 65ºC 65ºC 55ºC

Load EluateLoad EluateTet

Tri

D

M

TetTri D

M

Methods used:Gel electrophoresisCDFluorescenceMass spectrometryAFM

native PAGE native PAGE

Marszal, 2009 Gaczynska, Osmolski, AFM

Outcomes and plans• A new model of 1-PI polymerization was proposed.• Proposed polymerization model of 1-PI had impact on the

selection of formulation buffer for a product. • Continued investigation 1-PI polymer structure using AFM

will help to establish:– If there are one or more polymer types in A1PI- deficient patients – If polymers formed in different tissues have the same structure– Physiological relevance of model polymers and of PiZZ transgenic

mouse model of liver disease• AFM method for polymer characterization may potentially be

used for personalized treatment (screening drugs that could be used to dissociate patient’s polymers).

• Methods used to characterize polymers will include H/D exchange mass spectrometry, which may have a role in assessing future products.

• Characterization of protein aggregates in 1-PI products (collaborative study) will increase understanding of the methods and the products; potential impact on measuring protein aggregates in biologics.

Alpha-1 Proteinase Inhibitor and Innate Immune Responses

(J. Reed, D. Scott)

Public Health Issue: • Intravenous A1PI is approved to treat emphysema in

patients with A1PI deficiency – Original licensure based on achievement of serum A1PI

levels – Long-term clinical studies challenging– Treated A1PI-deficient patients may continue to have

declining pulmonary function/exacerbations

• Need to better understand– Effects of A1PI in lung tissue beyond elastase inhibition– Dose and route of A1PI therapy – relationship to efficacy

•

A1PI CD68

A1PI CD68

A1PI CD68

cysticfibrosis

RSV LRIof infancy

normaladult

Pulmonary Macrophages Express Abundant A1PI

homeostaticphagocytic

proinflammatorysusceptible to apoptosis

recently recruitedmonocyte

in situ differentiation

ROS

chemokines

model cell line: THP-1 MM6

model cell line: U937 28SC

Phenotypes of Lung Macrophages

A1PI

A1PI expression is inversely related to markers of inflammation in human macrophage cell lines

Research Hypothesis/Ongoing Work 1. A1PI regulates macrophage function• Nitric oxide scavenging, inhibiting activating signal amplification

(ongoing)• Inhibition of inflammasome activation and/or apoptosis• Hepcidin scavenging and inhibition of iron utilization (ongoing)• Confirmation in primary monocytes/macrophages (pending)2. Recruitment and maturation of lung macrophages are dysregulated in

A1PI deficient patients3. Increasing A1PI expression in patients with normal A1PI expression

could modify macrophage responses to injury and infectious insult

Implications:• Delivery of A1PI to lungs could be optimized• Potency assays could be developed to reflect additional A1PI

activities • Improved understanding of pathogenesis A1PI deficiency• Identifying A1PI pathway as target in ameliorating lung disease

Thank You!

Extra slides

Post-exposure Treatment with VIG Causes Primary Lesion Regression

and Improved Survival

VIG 10 mg/mouse on days 2, 5, 10, and 15 post-infection

Long-term survival of mice receiving VIG + topical Cidofovir

SCID mouse treatments: Scarify with 106 PFU vacciniaVIG 10 mg/mouse on days 7, 10, 15, 20Topical cidofovir 1% in dermovan 2 x daily for 2 weeks starting on day 7

0 25 50 75 100 125 1500

102030405060708090

100110

untreatedVIGtopical cidofovirVIG+topical cidofovir

Day

Per

cen

t su

rviv

al

Immunology Section Activities • Regulation of Immune Globulins, antivenoms, antitoxins, A1PI• Investigating product safety/efficacy issues

– Allergic reactions – Thrombotic events– Product contaminations – Lack of efficacy reports for immune globulins

• Product potency testing and standards (A1PI, VIG) • Shortage management (Rabies Immune Globulin, Immune Globulin, Digibind,

DigiFab, Thymoglobulin, Coral Snake antivenom)• Policy Issues – guidances, workshops, cross-office/center policy

– Immunogenicity– Animal Efficacy Rule implementation – Measles antibody activity decline in immune globulins – Licensing orphan products: Varicella Zoster Immune Globulin, Coral Snake antivenom– Animal models and licensure – cross-Center policy for anthrax immune globulins– Aggregates and particulates in products

• Responses to Congressional and international regulatory agencies• CDC consultations (vaccinia, anthrax cases); ACIP working groups (rabies, RSV

mAb’s• Emergency responses (Haiti, hurricane Katrina, influenza pandemic)