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Laboratory of Immunobiochemistry. Allergenic Products Advisory Committee, April 8, 2003. Allergenic Products Advisory Committee, April 8, 2003. Lab overview Staffing Lot release Reference replacement Operational issues Sheep serum replacement issues ISO certification report

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laboratory of immunobiochemistry

Laboratory of Immunobiochemistry

Allergenic Products Advisory Committee, April 8, 2003

allergenic products advisory committee april 8 2003
Allergenic Products Advisory Committee, April 8, 2003
  • Lab overview
    • Staffing
    • Lot release
    • Reference replacement
  • Operational issues
    • Sheep serum replacement issues
    • ISO certification report
  • Research/regulatory update
    • Endotoxin studies
    • Cockroach antigens and antibodies
lab overview
Lab overview
  • Staffing
  • Lot release
  • Reference replacement
slide4
Principal Investigators
    • Jay E. Slater, MD, Lab Chief – Supervisory Medical Officer (4)
    • Ronald Rabin, MD - Senior Staff Fellow (2)
  • Post Doctoral Fellows
    • Jonny Finlay, PhD - IRTA (2)
    • Bo Chi, MD - Visiting Associate (<1)
slide5
Research Technicians
    • Albert Gam – Biologist (2)
    • Mona Febus – Microbiologist (3)
    • Marc Alston – Biologist (2)
    • Cherry Valerio – Biologist (2)
    • Katia Dobrovolskaia – Visiting associate (2)
routine regulatory activities
“Routine” regulatory activities
  • Lot release
  • Reference distribution
  • Reference maintenance
    • semiannual checks
    • replacement
slide8
Lot release activities
    • 357 protocols submitted and reviewed
    • 1 withdrawn
  • Reference distribution
    • 2002: 1978 vials in 107 shipments sent to manufacturers
operational issues
Operational issues
  • Replacement of cat and ragweed antisera
  • Transition to ISO compliance
ragweed and cat antisera need to be replaced
Ragweed and cat antisera need to be replaced
  • S2a cat
    • Released in 1998
  • S6 ragweed
    • Released in 2000
  • Replacement programs for both initiated spring 2002
immunization protocol15
Immunization protocol

Immunization doses

plasmapheresis

transmissible spongiform encephalopathies
Transmissible spongiform encephalopathies
  • Animal
    • scrapie (sheep and goats)
    • chronic wasting disease (mule deer, elk)
    • transmissible mink encephalopathy
    • bovine spongiform encephalopathy
      • feline spongiform encephalopathy
transmissible spongiform encephalopathies19
Transmissible spongiform encephalopathies
  • Human
    • kuru
    • Creutzfeldt-Jakob disease
      • classic sporadic
      • familial
      • iatrogenic
      • new variant (a/w bovine TSE)
    • Gerstmann-Sträussler-Scheincker
    • fatal familial insomnia
    • sporadic fatal insomnia
scrapie
Scrapie
  • In Europe for >250 years
  • In US since 1947

>1000 flocks

  • Vertical transmission
  • Horizontal transmission, presumably by contamination with placenta and blood during lambing season
  • Long incubation period
  • No human transmission
slide21
Prion polymorphisms associate with different susceptibility, especially at codon 171
    • QQ (glutamine/glutamine) susceptible
    • RR (arginine/arginine) resistant
    • QR (glutamine/arginine) intermediate
scrapie eradication program
Scrapie eradication program
  • Preclinical testing and surveillance
    • Live animal test (third eyelid biopsy)
      • May take months to years to turn positive
  • Tracking of infected and exposed animals
  • Cleanup strategies: identify and genotype exposed animals
    • Destroy QQ exposed
    • QR or RR exposed are tracked but may be slaughtered for human consumption
slide23
Not believed to pose any risk to humans
    • No recognized human transmission in three centuries of exposure in Europe
  • This serum is safe to use because
    • No documented transmission to humans
    • Contact with affected sheep was limited
      • Not in adjoining pens; >30 feet distant
    • Normal BSL 2 precautions in place for all work with animal sera
  • However, in order to maintain a serum reagent that is as safe as possible…
current approach
Current approach…
  • Begin immunizing two new sheep
  • Process plasma from ewes 6747 and 7777 now
  • Conserve current stocks
current approach25
Current approach…
  • If new sera are available before we run out, sera from 6747 and 7777 will be saved frozen for possible future use
  • If we have a shortfall, sera from 6747 and/or 7777 will be used until the new sera are available
current approach26
Current approach…
  • Advantages
    • Highest degree of safety
    • Large supplies for future
  • Disadvantages
    • Possibility of two serum switches in short period
    • Time
    • Expense
slide28

CBER Laboratory Quality Management Initiative

  • Seek ISO-17025 compliance for official product testing
    • ISO – International Organization for Standardization, Geneva, Switzerland
    • 17025: “General requirements for the competence of testing and calibration laboratories” (1999)
      • Set of guidelines for labs that do testing and calibration to show operation of a quality system to assure technical competence and production of valid results
cber laboratory quality management initiative
CBER Laboratory Quality Management Initiative
  • Establish policy with Center level Quality Manual
  • Audit for compliance with ISO-17025
  • Obtain Test/Lab Accreditation where appropriate
    • “Accreditation – successful 3rd party audit
why is cber becoming iso compliant
Why is CBER becoming ISO compliant?
  • Establish recognized competence, assure the trust and value of our data and processes
  • We require the manufacturers’ labs to be in GMP compliance
    • International efforts at harmonization are attempting to equate GMP and ISO requirements
  • ISO is an internationally recognized standard
why is cber becoming iso compliant continued
Why is CBER becoming ISO compliant? (continued)
  • Implement Laboratory Quality Management policies and practices for official testing activities
    • To document a high level of training, competence, and proficiency
    • To establish a consistent product testing process
elements of iso compliance
Elements of ISO compliance
  • Management of
    • People
    • Equipment
    • Documents
    • Processes
elements of iso compliance33
Elements of ISO compliance
  • Management of People
    • Defined roles
    • Appropriate training
    • Demonstrated proficiency
      • Initial and ongoing
    • Authorization process
elements of iso compliance34
Elements of ISO compliance
  • Management of Equipment
    • Program for equipment calibration
    • Maintenance
    • Process to assure that only calibrated and maintained equipment is used
    • Assurance of measurement traceability to accepted standards
elements of iso compliance35
Elements of ISO compliance
  • Management of Documents
    • Includes policies, procedures, specifications, equipment manuals, certifications
    • Must be reviewed, issued and controlled
    • Approved and issued prior to use
elements of iso compliance36
Elements of ISO compliance
  • Management of Processes
    • Approval of testing materials
    • Environmental specifications and monitoring
    • Handling of samples
    • Validation and suitability
    • Handling of data (including non-conforming data)
    • Corrective action, Preventive action systems
    • Internal Audits
    • Management review
    • Recording and handling complaints
cber s commitment to implementation of a lab quality system
CBER’s commitment to implementation of a Lab Quality System
  • Establishment of CBER Quality Board
  • Development of Quality Assurance structure within CBER
    • Appointment of Center Level Quality Manager
    • Hiring Office quality Managers
    • Appointment and hiring of Division Quality Coordinators
  • Preparation of CBER Quality Manual
  • Purchase of Integrated Quality Management computer software
what will this mean to lib
What will this mean to LIB?
  • No substantive protocol revisions
  • Documentation
    • Formatting
    • Substance
  • Formal separation of research and regulatory equipment
  • Internal audits
  • External audits
timetable tentative
Timetable (tentative)
  • Implementation in stages over the next 3 years… Seek accreditation in 2005
    • Software operational during 2003
    • Policies and Quality Manual issued 2003
    • Initiation of compliance audits (ongoing)
    • Training and process development (ongoing)
laboratory of immunobiochemistry40

Laboratory of Immunobiochemistry

Research/regulatory update

active research projects
Active research projects
  • PI Slater
    • Cockroach allergen standardization
      • Determination of optimal surrogate test
      • Depletion analysis of CR extracts
    • Cockroach IgE combinatorial library
    • Endotoxin in allergen vaccines
  • PI Rabin
    • MDR proteins in T cell activation
    • RSV responses in human tonsil
publications
Publications

Patterson ML, Slater JE. Characterization and comparison of commercially available German and American cockroach allergen vaccines. Clin Exp Allergy 2002;32:721-727

Sutherland MF, Drew A, Rolland JM, Slater JE, Suphioglu C, O'Hehir RE. Specific monoclonal antibodies and human IgE show Hev b 5 is an abundant allergen in high protein powdered latex gloves. Clin Exp Allergy 2002;32:583-589

Trivedi B, Valerio C,Slater JE. Endotoxin content of standardized allergen vaccines. J Allergy Clin Immunol 2003 (in press).

publications reviews
Publications (reviews)

Lockey RF, Slater JE, Esch R. Preparation and standardization of allergen vaccines. In Middleton’s Allergy: Principles and Practice, 6th ed. St. Louis: Mosby (in press).

abstracts
Abstracts

Rabin RL, Alston MA, Huang H, Slater JE. Cytokine secretion by activated T cells is dependent on multidrug resistance protein-1 (MRP-1). J Allergy Clin Immunol 2003; 111:S153.

Valerio C, Slater JE. The effects of lipopolysaccharide (LPS) on immune responses in C57Bl/6 mice. J Allergy Clin Immunol 2003; 111:S166.

Slater JE, Valerio C, Trivedi B. Endotoxin in standardized allergen vaccines. J Allergy Clin Immunol 2003; 111:S243.

Finlay WJJ, Rabin RL, Slater JE. Analysis of IgE heavy chain V-gene usage in human tonsil. J Allergy Clin Immunol 2003; 111:S313.

endotoxin content of allergen vaccines
Endotoxin content of allergen vaccines
  • Allergenic extracts are not required to undergo evaluation for the presence of pyrogens (21CFR 610.13(b))
  • Prior studies confirmed variable endotoxin content (Siraganian, et al. J Allergy Clin Immunol 1979; 64:526-533)
products exempted in 21 cfr 610 13 b
Products exempted in 21 CFR 610.13(b)
  • Blood products
  • Horse serum
  • Bacterial, rickettsial and viral vaccines
  • Toxoids
  • Toxins
  • Allergenic extracts
endotoxin
ENDOTOXIN

(1,3) b–D- glucan

Factor C

Act. factor C

Factor B

Act. factor B

Act. factor G

Factor G

Proclotting enzyme

Clotting enzyme

Coagulogen

Coagulin

endotoxin content of allergen vaccines possible interference
Endotoxin content of allergen vaccines – possible interference
  • Non-endotoxin (1,3)--D-glucans may induce clotting by an alternative pathway in the standard LAL assays
  • Proteases (especially in cat and mite extracts) may also induce clotting
endotoxin content of allergen vaccines approach
Endotoxin content of allergen vaccines - approach
  • Determine endotoxin content using gel-clot method
  • Assess the contribution of non-endotoxin components ((1,3)--D-glucans, enzymes)
study design
Study design
  • Standardized allergen vaccines
  • LAL gel-clot assay
  • Adsorption of selected allergens with ENP-silica resin, followed by LAL assay
    • Endotoxin neutralizing protein (ENP) is a ~12 kDa, cationic, amphipathic protein that binds to and neutralizes the biological activity of lipopolysaccharide.
  • Pre-treat selected allergens at 95°C for 15 min, followed by LAL assay
slide53

G

G

G

G

G

G

G

G

G

G

G

G

G

G

GP

GP

GP

GP

GP

GP

GP

GP

GP

P

slide54

P

P

P

G

G

G

G

G

GP

slide55

G

G

G

G

G

G

G

GP

GP

slide56

G

G

G

G

G

G

G

G

G

G

G

G

GP

GP

conclusions
Conclusions
  • The observed LAL gel-clot activity probably represents real endotoxin content, not -glucan or protease activity
is this amount of endotoxin physiologically significant
Is this amount of endotoxin physiologically significant?
  • Mean endotoxin content: 1,900 EU/mL (allergen immunotherapy dose 0.5 – 2.0 mL per month)
    • 40 – 50 EU/kg (2,800 to 3,500 EU) (administered IV) can elicit a rise in temperature, heart rate, and white blood cell count

Wolff SM. J Infect Dis 1973; 128:Suppl-64.

Michie HR, et al. N Engl J Med 1988 Jun 9 318;1481-6.

specific endotoxin limits
Specific endotoxin limits
  • Product-specific
  • Generally based on
    • Drug dose
    • 5.0 EU/kg limit
specific endotoxin limits64
Specific endotoxin limits

* Based on USP limits and estimated maximum therapeutic doses

the clinical consequences of endotoxin in allergen vaccines have not been studied
The clinical consequences of endotoxin in allergen vaccines have not been studied
  • No data that adverse events from IT are associated with endotoxin levels
  • No data to support a beneficial effect of endotoxins
  • Future studies of allergen IT should be controlled for endotoxin dose
  • Role of endotoxin in safety and efficacy of IT should be assessed
conclusions66
Conclusions
  • Pollens < cat and mite
  • Cat hair < cat pelt
  • D. pteronyssinus << D. farinae
    • Bioburden?
    • Endogenous heat-stable ENP-binding LAL activator in D. farinae?
    • Endogenous ENP in D. pteronyssinus?
slide67
Plan
  • Expand study of endotoxin content
    • Additional standardized and non-standardized extracts
    • Different methods (GC mass spec)
  • Investigate differences between D. farinae and D. pteronyssinus
cockroach allergen standardization
Cockroach allergen standardization
  • Clinical studies
  • Developing the appropriate surrogate
    • Correlation
    • Depletion studies
  • IgE combinatorial library to cockroach
cockroach allergen standardization69
Cockroach allergen standardization
  • Clinical studies
  • Developing the appropriate surrogate
    • Correlation
    • Depletion studies
  • IgE combinatorial library to cockroach
cockroach allergen standardization70
Cockroach allergen standardization
  • Clinical studies
  • Developing the appropriate surrogate
    • Correlation
    • Depletion studies
  • IgE combinatorial library to cockroach
current standardized allergens
D. farinae

D. pteronyssinus

Cat hair

Cat pelt

Short ragweed pollen

Hymenoptera

Honey bee

Wasp

Yellow jacket

Yellow hornet

White-faced hornet

Mixed vespid

Grass pollens

Bermuda grass

Red top

June (Kentucky blue)

Perennial rye

Orchard

Timothy

Meadow fescue

Sweet vernal

Current standardized allergens
allergen standardization
Allergen standardization
  • Establish a US standard, and
  • Establish a testing procedure
    • Manufacturers may use the established procedure, or may develop equivalent procedures
which allergens should be standardized impact criteria
Which allergens should be standardized? Impact criteria…
  • Availability of stable, preferably lyophilized material for use as long-term reference extracts.
  • Consistency of currently marketed product.
  • Widespread use as a diagnostic and/or therapeutic reagent in the U.S.
  • Number of manufacturers producing the product.
  • Potential use in immunotherapy or diagnostics.
  • Public health impact of correct diagnosis and/or adequate treatment.
allergens and asthma
Allergens and asthma
  • Indoor allergens
    • dust mites*
    • cat*
    • cockroach
    • molds
    • dog
  • Outdoor allergens
    • molds

* = already standardized

why is cockroach allergy important
Why is cockroach allergy important?
  • Ubiquitous
  • Difficult to control
  • Associated with asthma
why is cockroach allergen standardization important
Why is cockroach allergen standardization important?
  • To the patient
    • More accurate diagnosis
    • Safer and more effective immunotherapy
  • To the physician/scientist
    • Better science (if you can’t measure it, you can’t study it…)
      • Pathophysiology
      • Epidemiology
      • Environmental control
  • To the FDA
    • Safer, more effective product
phase i laboratory
Phase I - Laboratory
  • Develop/adapt methods for allergen determination
  • Compare allergen content of different lots
goals
Goals
  • Determine consistency of available US products:
    • protein content
    • specific allergen content
    • overall allergenicity
  • Determine best lot release measures
extracts used as reference
Extracts used as reference
  • E2-Cg and E2-Ca
  • Previously characterized
  • Limited skin test data
  • Lyophilized; available in large quantities
cockroach extracts studied
Cockroach extracts studied
  • From all nine allergen extract manufacturers
correlation of protein concentrations and elisa results
Correlation of protein concentrations and ELISA results

Protein concentration

Relative potency

R2 = 0.83

R2 = 0.92

R2 = 0.96

R2 = 0.92

R2 = 0.93

R2 = 0.92

Bla g 1 levels

Bla g 2 levels

conclusions89
Conclusions
  • Commercially available cockroach allergen extracts:
    • vary widely in protein content, Bla g 2 content, SDS-PAGE banding pattern, and overall allergenicity.
    • appear to be less potent and contain less Bla g 1 than the candidate reference extracts
conclusions90
Conclusions
  • Established that cockroach allergen vaccines need to be standardized
what we need now
What we need now…
  • New cockroach references
  • Characterize the references
  • ID50EAL testing (Intradermal Dilution for 50 mm Sum of Erythema Determines Bioequivalent Allergy Units)
  • Proceed to next phase
phase ii clinical
Phase II - Clinical
  • skin testing, histamine release, IT data
  • establish biological unitage and ideal dosing ranges
id 50 eal testing
ID50EAL testing
  • Proficiency
  • Recruitment
  • Testing
  • Analysis
recruitment
Recruitment
  • Inclusion criteria
    • 18 to 65 years of age
    • history of allergic disease, such as allergic rhinitis, related to exposure to the allergen of interest
    • puncture sum of erythema diameter responses (ΣE) to the allergen concentrate of ≥30 mm.
recruitment96
Recruitment
  • Exclusion criteria
    • Asthma with use of systemic steroids in the past 12 months
    • Peak flow < 75% predicted at the time of testing
    • Skin coloring or condition that would preclude the measurement of erythema responses
    • Dermographism (> 4 mm ΣE following saline skin test)
    • Immunotherapy – past or present - with the test allergen
    • Current use of antihistamines, tricyclic antidepressants, MAO inhibitors, and beta-blockers
how many study subjects
How many study subjects?

Rabin et al., Sample Size Considerations for Establishing Clinical Bioequivalence of Allergen Formulations. Arb.Paul Ehrlich Inst.Bundesamt Sera Impfstoffe Frankf.A.M, in press

d for d50 should be 10
dfor D50 should be 10%

BAU/mL= 3-(14 - mean D50) * 100,000

for d50 is about 10 20
 for D50 is about 10-20%

Smith et al. Annals Allergy Asthma Immunol 1995; 75:317-323

how many study subjects101
How many study subjects?
  • Estimate n = 40 to establish D50 for each extract (based on /d = 1.5): 80
  • Geographic diversity: 80 x 3 = 240
  • Overlap between American CR and German CR allergic subjects may permit reduction in n (150-200)
conclusions102
Conclusions
  • Established that cockroach allergen vaccines need to be standardized
  • Need to establish the potency of candidate US reference materials by bioassay (ID50EAL)
niaid inner city asthma consortium
NIAID Inner City Asthma Consortium

“…established in FY 2002 to explore and evaluate promising new strategies for the treatment of asthma among minority children residing in the inner city. This consortium of basic scientists and clinical investigators will conduct clinical studies to elucidate the immunopathogenesis and natural history of asthma in this population.”

From the FY 2003 Budget Justification Narrative, NIAID,

http://www.niaid.nih.gov/director/congress/2002/cj/narrative.htm

niaid inner city asthma consortium steering committee
Busse, William W., MDChair

Adams, Kenneth, PhD

Eggleston, Peyton A., MD

Gruchalla, Rebecca S., MD, PhD

Kattan, Meyer, MD

Kercsmar, Carolyn M., MD

Liu, Andrew H., MD

Malveaux, Floyd J., MD, PhD

Mitchell, Herman, PhD

Morgan, Wayne, MD, CM

O'Connor, George T., MD, MS

Pongracic, Jacqueline A., MD

Sampson, Hugh A., MD

Smartt, Ernestine, RN

Strunk, Robert C., MD

Szefler, Stanley J., MD

NIAID Inner City Asthma Consortium steering committee
timetable
Timetable
  • Steering committee approval - done
  • Study centers identified
  • Order extracts
  • IRB approvals
  • IND approval
  • Distribute materials
  • Proficiency testing
  • Proceed with study
conclusions107
Conclusions
  • Established that cockroach allergen vaccines need to be standardized
  • Need to establish the potency of candidate US reference materials by bioassay (ID50EAL)
  • Endotoxin issue to be studied in depth
will overall allergenicity measurements be sensitive to changes in specific allergen levels
Will overall allergenicity measurements be sensitive to changes in specific allergen levels?
  • In mite stability study (1998-1999), RP was stable at -20°C and 4°C for up to 12 months
  • Degradation of specific allergens (group 1 and 2; specific bands) was observed at 4°C

Soldatova LN, Paupore EJ, Burk SH, Pastor RW, Slater JE. J Allergy Clin Immunol 2000; 105:482-8.

rid with monospecific antiserum
RID with monospecific antiserum
  • Examples: cat, ragweed
  • Advantages
    • quantitative
    • monospecific
  • Disadvantages
    • need to identify relevant allergen(s)
competition elisa with pooled allergic human sera
Competition ELISA with pooled allergic human sera
  • Examples: mites, grasses
  • Advantages
    • quantitative
    • reflects spectrum of allergen recognition
    • does not require identification of relevant allergens
  • Disadvantages
    • use of pooled sera
    • effects of fluctuations in individual allergens difficult to measure
will overall allergenicity measurements be sensitive to changes in specific allergen levels112
Will overall allergenicity measurements be sensitive to changes in specific allergen levels?
  • Depletion analysis
    • Raise specific antibodies to Bla g 1, 2, 4 and 5
    • Selectively adsorb
    • Test for specific allergen levels
    • Test for overall allergenicity
selective adsorption of bla g 1

Sham adsorbed

Adsorbed with anti-Bla g 1

Bla g 1 ELISA

Selective adsorption of Bla g 1
conclusions116
Conclusions
  • Established that cockroach allergen vaccines need to be standardized
  • Need to establish the potency of candidate US reference materials by bioassay (ID50EAL)
  • Endotoxin issue to be studied in depth
  • Surrogate test may not be the competition ELISA
conclusions117
Conclusions
  • Standardized German and American cockroach allergen vaccines will
    • facilitate definitive studies on the role of cockroach allergens in inner city asthma, and on the best methods for eradication and treatment
    • make for safer and more effective products