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Division of Monoclonal Antibodies Overview

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					Division of Monoclonal Antibodies
             Overview


      ACPS Presentation 10/19/04




             Steven Kozlowski
            DMA/OBP/OPS/CDER
                   Overview
• Quality Deconstruction & Dot Connection
• Biological Characterization
• Research Reviewer Model
• DMA Organization & Products
• DMA Ongoing Research
• Critical Path
• Critical Pathways & Directions
 Pharmaceutical Quality System
                Drug Safety           PAT            Process Capability

                               Continuous Learning
              Clinical           and Improvement
                           Clin Pharm                         CGMPs
                              & Bio Controls

           Pharm/Tox                                             CGMP “Design &
                                                                 Knowledge Space”
Clinical “Design
& Knowledge Space”
                         Chemistry           Manufacturing

             CMC “Design and Knowledge Space”

                                                                      Ajaz Hussain
      A Slice of the Polygon
                       Clin Pharm
        Clinical          & Bio
                                 Controls      PAT


          Pharm/Tox                           CGMPs




                      Chemistry             Manufacturing




• Chemistry to classic analytical controls
• Characterization to release specifications
           ICH Q6B Specifications
  TEST PROCEDURES AND ACCEPTANCE CRITERIA
 FOR BIOTECHNOLOGICAL /BIOLOGICAL PRODUCTS

• Characterization of a biotechnological or biological
  product…is necessary to allow relevant specifications to be
  established.
• Specifications are chosen to confirm the quality of the drug
  substance and drug product rather than to establish full
  characterization and should focus on…
• … those molecular and biological characteristics found to
  be useful in ensuring the safety and efficacy of the product.
         The Weakest Links
         Clinical
                                Controls




                    Chemistry              Manufacturing


• Chemistry to clinical
   – Relevant structural attributes (safety & efficacy)
• Controls to clinical
   – Relevant attributes or processes to control
                  Overview
• Quality Deconstruction & Dot Connection

• Biological Characterization
• Research Reviewer Model
• DMA Products
• DMA Ongoing Research
• Critical Path
• Critical Pathways & Directions
 Structure             Fab = ~1/3 mAb
of complex
molecules
• 1o sequence                           Statin
• higher order
  structure
                                                 o
• post-translational                        11 A
  modifications
• heterogeneity                              o
                                         87 A

Statin MW ~400 Da

Monoclonal Ab MW ~150,000 Da            PDB 2IG2, 1HW8
                  ICH Q6B
Specifications for Biotechnology Products
         “for complex molecules, the
       physicochemical information may
          be extensive but unable to
           confirm the higher-order
                 structure…:
   …which, however, can be inferred
     from the biological activity”
   :
        Biological Activity (Q6B)
• the specific ability or capacity of the product to
  achieve a defined biological effect.
• Potency is the quantitative measure of biological
  activity
• Bioassays
  – Animal based
  – Cell culture based
  – Biochemical based
• Other procedures, such as ligand/receptor
  binding assays may be acceptable
Lot release utility
  Low variability
                      Bioassay Continuum




                                                        Mechanistic relevance
                                                           true "potency"
              Ligand Cell-culture
              binding               Animal
                      bioassay               Clinical
                                    model
                                              study
 How do we choose the relevant biological
               activity?

                Q6B 2.1.2 Biological activity


• Assessment of the biological properties constitutes
  an equally essential step in establishing a
  complete characterization profile.


          Biological Characterization
             Biological Characterization
• Characterization --> classical specifications (Q6B)
• … those molecular and biological characteristics found to
  be useful in ensuring the safety and efficacy of the product.
• Defining those characteristics relevant to:
   – Risk management                                   Continuous Learning
                                                        and Improvement
   – CGMPs for the 21st century            Drug Safety         PAT
                                                                           Process Capability
   – PAT                             Clinical                           Clin Pharm & Bio
                                                                                   CGMPs
                                                            Controls
• Relevant to small molecules       Pharm/Tox

                                               Clinical “Design
                                               & Knowledge Space”
                                                                    Chemistry              Manufacturing
                                                                                                           CGMP “Design &
                                                                                                           Knowledge Space”
                                                       CMC “Design and Knowledge Space”

• Molecular mechanism of action
• Biological Plausibility
  Molecular Mechanism of Action

• Therapeutic Proteins: potency assays
• All CDER Products: facilitate prediction
  of
   • Relevant physiochemical properties
   • Toxicity
   • Drug interactions
   • Efficacy
• May suggest appropriate animal models
  and clinical monitoring
        Biological Plausibility

 • Biomarker development/validation
 • Interpretation of
    • Pharmacogenomic data
    • Proteomic data

    If Biological Characterization is so
important why is there such variability and
       limited guidance on this topic?
        Biological Characterization
   Guidance for Industry: IND Meetings for Human
        Drugs and Biologics: EOP2 Biotech
• Adequacy of physicochemical and biological
  characterization (e.g., peptide map, amino acid
  sequence, disulfide linkages, higher order structure,
  glycosylation sites and structures, other post-
  translational modifications, and plans for completion,
  if still incomplete)
• Bioassay (e.g., appropriateness of method,
  specificity, precision)
• Bioactivity of product-related substances and
  product-related impurities relative to desired product
           Biological Characterization
• Levels
   –   Binding; conformational changes
   –   Signal transduction
   –   Cell culture effects
   –   Tissue studies
   –   In vivo studies
• Multiplicity
• Relevant models
   – Receptor/pathway/cells/tissues/species
   – Molecular mechanism of action (circular)
• No linear algorithm
   – Systems approach
• Product specific
• Greater variability in methods
• Expense
                                                                           Purified/induced variants
     Biological




                                                   Clinical lot extremes




                                                                                                                       Developmental lots
   Activity Matrix




                                                                                                       Stressed lots
          One to some lots




                                   Clinical lots
           Many to all lots


Multiple binding/cellular assays

Small Animal/Complex Bioassay

Clinical/Clin Pharm

Validated bioassay
               Biology Expertise
• A biological characterization is only as good as the
  experimental data supporting it
   – If regulatory decisions are impacted by biological
     characterization
   – a framework for an appropriate interpretation of cell &
     molecular biology information is needed

• This expertise will become more important over time
   – Guidance for biological characterization
   – Mechanism for consultation

• With the recent consolidation, CDER now has
  additional cell & molecular biology expertise in place.
                   Overview
• Quality Deconstruction & Dot Connection
• Biological Characterization

• Research Reviewer Model
• DMA Products
• DMA Ongoing Research
• Critical Path
• Critical Pathways & Directions
     Researcher Reviewer Model
                        Pleiotropy

• Awareness of the nuts & bolts of many review issues
• Multiplicity is a challenge
   – Research producivity
   – PCE
   – Regulatory workload
• Investigator independence & relevance
    Researcher Reviewer Model
                       Catalysis & Synergy
• Not all reviewers can have active research programs
• A small nucleus can encourage
   – biochemical/biological characterization
   – process understanding
   – mechanistic consideration
• This group can consult when a key decision is dependent
  on biological characterization
   – This may include evaluating the validity of experimental data used
     to support the molecular mechanism of action
• The research reviewer group can network
   – NIH and other academic experts
   – OTR staff
   – Full time review staff
 Research




                                Cell Adhesion

                                                Differentiation

                                                                  Signal Trans.
                    Cytokines
Organization
 A Tapestry

      Immunology

    Tumor Biology

    Neuroscience                                                                  Research

                                                                        NIH/Acad.
     Dev. Biology                                                       Conferences
                                                                        Journal Clubs        Articles
                   Overview
• Quality Deconstruction & Dot Connection
• Biological Characterization
• Research Reviewer Model

• DMA Organization & Products
• DMA Ongoing Research
• Critical Path
• Critical Pathways & Directions
                                     DMA Organization



Lymphocyte and monocyte biology
Tumor suppressors and oncogenes

Cell-cell and cytokine-receptor interactions

Signal transduction

Antibody interactions and effector functions
Manufacturing process validation
                      # Licensed Products
                                                        7




                 0
                      1
                           2
                               3
                                   4
                                            5
                                                    6
    Infection

  Inflammatory
       Disease

Transplantation

     Psoriasis

      Asthma

      Cardiac

      Imaging

    Oncology
                                                                 Monoclonal Antibodies
                                                            Initial Indications for Licensed




                     Tumor
                     Biology
                                       Immunology
Approved Products
Biological Response Modifiers




     Anti-Infective Agents
          Approved Products
           Oncological Therapeutic Drugs




Product reviewers participate in PAIs & in biennials
             Approved Products
              Radio-immuno-imaging Agents




•   PTC for MAb                   •   Scientific Considerations
•   Plant Transgenic Products         for Follow on Proteins
•   Orphan Drug Status MAb             – Stakeholder mtg
•   Use of MAb as a Reagent for        – DIA mtg
    Further Manufacture           •   Q5E
                   Overview
• Quality Deconstruction & Dot Connection
• Biological Characterization
• Research Reviewer Model
• DMA Products

• DMA Ongoing Research
• Critical Path
• Critical Pathways & Directions
                 Research Areas
             Clinical
                                      Controls




                          Chemistry              Manufacturing


• Structure
     • TNF multimerization effects (Dr. Kathleen Clouse, Ph.D.)
     • Use of SPR in protein thermodynamics (Dr. Steven Kozlowski, M.D)

  – Antibody Structure
     V region CDRs
                                    IgG Structure
        Fab                                Jefferis et al.
                                        Immunology Letters
                                         (2002) 82; 57-65.
                                        & PDB 2IG2, 1FC1


                     FcgRI,II,III
hinge                C1q
                                              Man - GlcNAc - Gal
                      GlcNAc - GlcNAc - Man
          Asn
          297                                 Man - GlcNAc - Gal
                 FcRn
                 MBL
Fc               MR
  Antibody Structural Diversity
       Dr. Marjorie Shapiro, Ph.D.
• A number of molecular processes are
  involved to generate a mature specificity
  –   V(D)J Recombination
  –   Switch Recombination
  –   Somatic Mutation
  –   Positive and negative selection in a normal
      microenvironment
• New technologies
  – Phage display
  – Transgenic animals expressing human ab
    genes
Antibody Structure & Immunogenicity
• Murine n=8           Antibody gene usage
  – Whole antibodies   f display versus in vivo
     • 55 to >80%
                       generated antibodies
  – Fab or Fab’
     • <1 to 8%
• Chimeric, n=5, 3/5 whole mAbs, 1 Fab
  – <1 to 13%
• Humanized, n=7, whole antibodies
  – <1 to 8%
• Human (f display), n=1
  – 12%
         Antibody Effector Interactions
                               Dr. Mate Tolnay, Ph.D.
         • Complement receptors play a critical role
           in antibody function and production
         • A new family of FcR-like genes may play
           a role in antibody effector function
                       Dr. Gerald Feldman, Ph.D.
• Immune complex-mediated regulation of cell
  function as a side effect of Ab treatment
                                                                         Y




                                                                  Y
               IFNg




                                                              Immune Complex
                                                               Y Y Y Y Y Y


                                                                               YYY
         a             a
     b                     b            NEGATIVE




                                                                               YY Y
  JAK2   JAK1
                   JAK1    JAK2


             Stat1-Y                                    FcR
                   Research Areas
               Clinical
                                      Controls




                          Chemistry              Manufacturing


• Biological Characterization
  – In vitro
  – In vivo
         Adhesion & Costimulation
          Steven Kozlowski, M.D.
         ICAM-1                       MHC
                                                       B7.1
                                                       B7.2
                         LFA-3
                                          Ag

                                                        CD28
                           CD2
                                                 CD8
                                                 CD4
                                      TCR/CD3

              LFA-1

 Selective Effects on
                                  Migration
CD8 versus CD4 T-cells
                                 Proliferation
                                  Cytokines                    bead

                         Keratinocyte proliferation            T-cells
                                                              CD3/CD28
                                                              Activation
                 Lymphocyte Signaling
                Barbara Rellahan, Ph.D.
                                 TCR/CD3          Ag
                       Lat


                                                                       Lck
                                 Gads
             c-Cbl
PLCg1
                                                                       Zap70
                                   Slp 76
        Ub
                        PLCg1                                          TCR stimulated cells
                 ?




                                            Inositol Phosphate
    PLCg1                                                         20


                                               TCR-induced
                     70Z/3 Cbl                                    10
                                                                   0
                                                                 -10                          pCI-neo
                                                                 -20
                                                                                              pCI-Cbl
                                                                 -30
                                                                 -40                          pCI-70Z/3
                                                                 -50
Cbl participates in
                                                                        c-Cbl is a regulator of Ag-
Herceptin response                                                       receptor induced PLCg1
                                                                                activation.
  Cytokines Sustain HIV Reservoir
        Dr. Kathleen Clouse, Ph.D.

M-CSF                   HIV             HIV infected
                                        macrophage
        HIV infected
        macrophage
                       M-
                         CS
                            F
Chemokines
                                 Increased
                                susceptibility
                                   to HIV
     Uninfected                                     PDI
     macrophage
                                                 Viral entry
GTPase Cycle Targets & Neoplasia
         Wen Jin Wu, M.D. Ph.D.
  Extracellular stimuli             Soft agar
               EGFR                 growth


                                                Vector
         GEF

 Cdc42           Cdc42                          RasG12V
                          Targets
  GDP             GTP


         GAP                                    Cdc42F28L

    Erbitux                  Cellular transformation
Epithelial Differentiation & Neoplasia
      Wendy Weinberg, Ph.D.
CELL LAYER                            MARKER          PROTEIN mRNA
                                      Cornified
Cornified                             Envelopes
                                     Loricrin, TG,
 Granular                              Filaggrin

 Spinous                            Keratins 1 & 10


  Basal                             Keratins 5 & 14


 p53 family members play distinct roles in regulation of cell growth,
               differentiation and cancer pathology
 0.05mM Ca2+       0.12mM Ca2+         decrease in S phase
                                           in 0.12mM:        rNp63a



                                             43%               ----
            Research Areas
        Clinical
                               Controls




                   Chemistry              Manufacturing


• Contaminants
• Process Understanding
   Prion Peptide & Cell Signaling
          Dr. Gerald Feldman, Ph.D.

                  • NF-kB pathway

                  • Cytokine Induction

      On Monocytes                       On DCs
• Chemotactic                 • Activation and maturation
• Induction of calcium flux   • Lack of calcium flux
• Sensitivity to PT           • Resistance to PT
• Involvement of the FPRL-1   • No involvement of FPRL-1
Contamination & Process Control
             Kurt Brorson, Ph.D.

• Retroviral testing
  – implementation of Q-PCR based assays


• Process understanding
  – unit operation robustness & predictability-
    chromatography, membrane adsorbers,
    fermentation, virus inactivation
Cell Culture Process Changes
                                   Which of these can impact
                                   endogenous retrovirus expression?
                                   NO- Scale, nutrients

                                   Yes- Inducing agents, temperature




Photo courtesy of Applikon, Inc.   Brorson et al., Biotech Bioeng 80:257, 2002
                   Overview
• Quality Deconstruction & Dot Connection
• Biological Characterization
• Research Reviewer Model
• DMA Products
• DMA Ongoing Research

• Critical Path
• Critical Pathways & Directions
Critical Path
                   Critical Path
A. The technical hurdle or “problem

B. Dimensions of product development

C. Unique vantage point of FDA

D. Industry segments facing the problem
      - role of partnerships/collaborations

E. The government role in solving this problem

F. Solution impact on product development
      - breadth of solution on products/classes/sectors
Critical Path: David Frucht, M.D., LCB
A. Problem: Limited bioassays for anthrax lethal
   toxin activity and neutralization
  A. questionable relevance to the in vivo mechanism
  B. widely accepted assays involve analysis of the viability of two
     murine macrophage cell lines
  C. murine but not human cell lines undergo apoptosis in response to
     anthrax lethal toxin
  D. appropriate bioassays are critical since that it is not feasible to
     assess the efficacy of anthrax therapeutics in humans directly
     (Animal Rule).
  E. in vitro potency assays involving human cells or cell lines are
     more likely to be of predictive value for efficacy in humans


B. Dimensions: Medical Utility & Industrialization
   Critical Path: David Frucht, M.D.
C. FDA unique vantage: the FDA is
  A. evaluating several INDs for anthrax therapeutics
  B. involved in the discussions regarding the stockpiling of anthrax
     therapeutics for Project BioShield.
  C. in a unique position to compare cell-based bioassays for anthrax
     lethal toxin


D. Industry segments/partnerships:
  A. small & large biotechnology and pharmaceutical companies are
     developing products targeting anthrax
  B. academic collaborations+internal expertise; CDC NIH
     interactions; additional public-private partnerships can be
     developed.
    Critical Path: David Frucht, M.D.
E. The government role:
  A. BioShield: regulator & stakeholder
  B. IND product awareness
  C. internal expertise:
     A. anthrax lethal toxin activates a pro-inflammatory cytokine
          pathway involving the enzyme caspase-1 and its downstream
          effectors IL-1b and IL-18. This assay is more sensitive than
          existing assays examining cell viability.
     B. although human macrophages and macrophage cell lines are
          resistant to anthrax LT-induced cell death, the caspase- 1/IL-
          1b/IL-18 cytokine pathways are highly activated and readily
          detectable.


F. Impact: a relevant toxin assay helps to assure:
      A. development and quality of efficacious products
      B. recognition of more suitable biomarkers for infection
      C. & impacts the companies currently developing anthrax
         therapeutics, other toxin therapies and the public health
Molecular Mechanism of Anthrax Toxin
          David Frucht, M.D.
                                                                  LF
                        PA20         PA63                                    LF=Lethal factor
PA83




                                       H+
                                 H+          H+

                                H+                H+

                                H+              H+
                                     H+ H+ H+
                 LF


              MAPKKs                        Human: Blockade of Specific Cytokine Release
                                            In Cell lines and Primary Cells without Cell Death
                                            (Frucht lab)

               Cell death
           IL-1b and IL-18 Release
       In vitro and in vivo (Frucht lab)
Critical Path: Wendy Weinberg, Ph.D.

A. Problem: the need for animal models with
   higher predictive value for drug effectiveness
  A. models applied to development and evaluation of effective cancer
     treatments should reflect the molecular changes contributing to
     cancer evolution.
  B. transgenic mice with inducible expression of commonly mutated
     genes provide a more physiologic context of tumor development
     that is representative of the human situation than models
     commonly utilized by drug developers.

B. Dimensions: Medical Utility
Critical Path: Wendy Weinberg, Ph.D.

C. FDA unique vantage: the FDA is
  A. evaluating numerous INDs/NDAs/BLAs for cancer therapeutics
  B. in a unique position to compare in vivo and in vitro assays used
     to demonstrate product activity


D. Industry segments/partnerships:
  A. numerous biotechnology and pharmaceutical companies are
     developing products targeting cancers
  B. failed phase 3 studies for oncology products may have been
     prevented by better animal models
  C. academic collaborations+internal expertise; additional public-
     private partnerships can be developed.
 Critical Path: Wendy Weinberg, Ph.D.
E. The government role:
  A. product awareness
  B. internal expertise:
     A. experience in animal models of epithelial cancers that
          appropriately reflects the genetic changes observed in human
          cancer pathogenesis.
     B. the most frequently altered pathway in human cancers is that of
          the nuclear transcription factor p53; recently a related protein
          p63 has been identified that can modulate p53 activity and also
          act independent of p53 to alter growth properties of cells.
     C. the contribution of specific p63 isotypes will be determined in
          combination with additional changes such as p53 mutations (as
          observed in lung cancer), the presence of human papilloma
          virus (HPV) (as seen in cervical and some oral cancers), and
          her2 neu overexpression (as in breast cancers)
F. Impact: this project helps to assure:
      A. development of animal models reflective of human cancers
      B. & impacts a very large segment of the industry
   Critical Path: Kurt Brorson, Ph.D.
A. Problem: the need for less expensive
   strategies to assure viral safety
  A. Mammalian cells used in the production of monoclonal antibodies
     (mAbs) produce endogenous type C retrovirus particles and are
     capable of being infected by adventitious agents.
  B. Regulatory agencies require a demonstration that mAbs intended
     for human use are free of virus with an adequate margin of
     safety.
  C. Small scale viral clearance studies can be prohibitively expensive
     for small biotech firms and academic investigators
B. Dimensions: Safety & Industrialization

C. FDA unique vantage: This problem has been
  A. communicated to the agency at conferences
  B. noted in review of multiple INDs.
   Critical Path: Kurt Brorson, Ph.D.

D. It impacts multiple sponsors of biotech INDs,
   particularly small biotech firms and academic
   investigators.

E. The government role:
  A. government can provide a broad perspective for identifying
     acceptable solutions and approaches that comply with GMP
     standards
  B. industry-government collaboration may facilitate solution of this
     issue by pooling resources and expertise
  C. work on membrane absorbers and viral clearance will be part of a
     larger collaborative study concerning virus: membrane
     interactions with Genentech, the University of Maryland,
     Baltimore County and the University of Wisconsin, Madison
      Critical Path: Kurt Brorson, Ph.D.
F. Impact: this project helps to assure:
    A. lower barriers against innovation by biotech companies
    B. regulatory relief for these organizations



•   In earlier collaborative studies, Genentech Inc. and the
    Division of Monoclonal Antibodies performed two
    matrix/bracket studies of robust viral clearance steps (e.g.
    low pH inactivation and anion exchange chromatography;
    Brorson et al., Biotechnol Bioeng 82:321-9; Curtis et al.
    Biotechnol Bioeng 84:179-186, 2003).
PR772- suitability for testing large virus filters
                              Genome sequence resolved- AY441783
                              Highly similar to other Tectiviridae




 150,000 x


Lute et al., Appl. Environ.                             CsCl preps are
                               Phage size: 64 nm        monodispersed
Microbiol. 70:4864, 2004
   Critical Path: Kurt Brorson, Ph.D.

       Dimensions: Safety & Industrialization


• Gene arrays, cell culture process changes, and
  comparability

• Chromatography media decay and virus
  removal validation

• Databases of viral clearance experience and
  TSE risk (with Gerry Feldman, Ph.D.)
                   Overview
• Quality Deconstruction & Dot Connection
• Biological Characterization
• Research Reviewer Model
• DMA Products
• DMA Ongoing Research
• Critical Path

• Critical Pathways & Directions
             Critical Pathways


 Cell &
Molecular
                        Industrialization
 Biology

 • Relevant physiochemical                      CGMP & PAT
   properties
                                            • May suggest
  • Toxicity                                  appropriate animal
  • Drug interactions                         models and clinical
  • Efficacy                                  monitoring

         Manufacturing process validation
       Regulatory relief & fewer failed studies
              Critical directions
• Better define “biological characterization”
   • eventual guidance
   • role for follow on proteins
• Maintaining OBP biological expertise and
  research across relevant areas of biology for our
  products (critical pathways)
• Facilitating access to & between OBP biologists
   • interactions with other OPS offices, pharmacology and
     clinical review groups
   • Biotech Rounds
   • MAJIC/ Bioprocessing Journal Club
   • mechanism for consults
              Critical directions

• Extending some OBP research into defined
  Critical Path projects
• Relational databases
   •   viral clearance (Kurt Brorson)
   •   review management, USAN, targets (Patrick Swann)
   •   specifications (Patrick Swann)
   •   TSE (Gerry Feldman & Kurt Brorson)
   •   internal meetings
   •   workload
   •   structural/sequence information
• Enhancing safety & facilitating regulatory relief

				
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