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U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICESNATIONAL INSTITUTES OF HEALTH
Working with FDA:Biological Products and Clinical Development
Chemistry, Manufacturing and Control Issues in Production of Therapeutic Biologic Protein Products
Ingrid Markovic, Ph.D., BiologistLaboratory of Biochemistry, Division of Therapeutic ProteinsOffice of Biotechnology Products, Office of Pharmaceutical ScienceCenter for Drug Evaluation and ResearchFDA
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
OBP/OPS/CDER
Division of Therapeutic ProteinsAmy Rosenberg, M.D., DirectorBarry Cherney, Ph.D., Deputy
Office of the DirectorSteven Kozlowski, M.D., DirectorWendy Shores, Ph.D., Deputy
Division of Monoclonal AntibodiesKathleen Clouse, Ph.D., Director
Patrick Swann, Ph.D., Deputy
Fc-Fusion Proteins
Monoclonal Antibodies
Enzymes
Cytokines
Growth factors
Toxins
Fabrazyme
InterferonsInterleukins Herceptin
AvastinErbitux
Enbrel
G-CSFEpo
Botox
Parallel Office to ONDQA, which also reviews proteins (e.g., insulin, HGH, etc.)(courtesy of Dr. S. Kozlowski)
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Majority of Biotech Products Use Living Cells to Produce a Protein Product
Insert gene encoding the protein of interest
Cells require proper conditions for optimal growth (temp, pH, oxygen, feeds, etc.)
Culture and fermentation can take weeks
Complex Purification Steps
Safe product with desired potency
Bar Charts, Inc. 2003
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Protein Therapeutics
Protein Therapeutics are licensed through Biologics License Application (BLA) under provisions of both Public Health Service (PHS) and Food Drug & Cosmetic (FD&C) Acts
Protein therapeutics are also regulated through New Drug Application (NDA) under provisions of FD&C Act (e.g., insulin & HGH)
BLA under PHS act lacks an abbreviated pathway for follow-on biologics or biosimilars
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
How are Protein Therapeutics different from Small Molecule Drugs?
Contain intrinsic infectious agents Aseptic techniques required during production
(terminal heat or gamma sterilization rarely applied)
Usually have heterogeneous composition• Numerous process and product-related
impurities• Change in the manufacturing process can
cause change in product composition Exact structure may be unknown (e.g., all
possible variants often not fully characterized)
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Structure of Small Molecule vs. Protein Drugs
Proteins have expected: Size, charge,
hydrophobicity Correct folding (S-S bonds) Subunits Glycosylation Bioactivity
& Unexpected: Aggregation (side effects) Incorrect folding Amino acid modifications
– ox, deam, cys Truncation, proteolysis
Statin
Therapeutic protein ~5,000 - 300,000 Da
~400 Da*
x
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Manufacturing Process
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Components of the Manufacturing Process
Expression vector (plasmid)
Cell banking system
• Master Cell Bank (MCB)
• Working Cell Bank (WCB)
• End of Production Cells (EOP)
Drug substance manufacturing and release
Drug product formulation and release
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Expression Vector and Cell Banking System
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Source Materials
Mice Humans
Mammalian cell-culture
Yeast
Bacteria
Transgenics
Viruses
TSE
agents
Bacteria
Mycoplasma
Fungi
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Expression Vectors (Plasmids)
Used for transfer of genes from one organism to another
Used for production of large amounts of protein
Description of origin of the construct
Plasmid mapping (e.g., restriction sites, integration sites, promoter, copy number etc.) and stability
Sequencing of gene of interest
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
MCB and WCB
A working cell bank (WCB) is derived from the master cell bank (MCB) and is used to initiate a production batch
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Characterization of Cell Banks
Test MCB WCB EPC
Viability X X X
Identity X X
Purity X X X
Stability X X
*Karyology X X
*Tumorigenicity X X
*dependent upon cell substrate and manufacturing process
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Characterization of Cell Banks (cont.)
Test MCB WCB EPC
Sterility (bacterial & fungal cont) X X X
Mycoplasma (cultivable/noncultivable)
X X X
Adventitious viruses in vitro – cell lines in vivo – mice, guinea pigs, eggs
X X
Species-Specific (MAP, HAP, RAP)
X
Retrovirus (TEM, RT, infectivity)
X X
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Sources of Adventitious Agents
Cell Substrate• Endogenous viruses• Exogenous microbial contamination• Source material screening:
– Human (HIV, HBV, HCV, CJD, etc.)– Animal (TSE sources, species-specific viruses)
Raw Materials• Cell culture reagents (animal and non-animal
derived) Environment
• Water• Air• Humans/technicians
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Viral Clearance for Phase 1 IND
Demonstration of viral clearance may be required. Exceptions: certain source materials (e.g., E. coli, yeast) or in the event of unmet medical need
Perform small scale clearance study that mimics the clinical purification process
• Spike Drug Substance with a model virus to demonstrate viral removal by several logs beyond the potential load
• CHO cell substrate – demonstrate retroviral clearance
• Human cell substrate - demonstrate clearance of enveloped and non-enveloped viruses (e.g., parvoviruses)
• Design the process upfront to adequately assess potential risks
Two orthogonal robust steps (e.g., low pH, nano-filtration, solvent/detergent treatment, heat) typically included in the purification process
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Production and Purification
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Upstream cell culture & fermentation
Isolation/Capture of protein
Purification
Drug substance
Formulation
Drug Product
Downstream Processing
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Fermentation Process
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Purification Process
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Drug Substance and Drug Product Characterization
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
2 x 6 x 4 x 4 x 5 x 5 x 2 = 9600
K
pyro-E OD
G
G
D
OD
(9600)2≈ 108
O
O
Methionine oxidation (2 x 2)
pyro-E Pyro-Glu (2)
High mannose, G0, G1, G1, G2 (5)
Sialylation (5)
D
D
D
G
G
Glycation (2 x 2)
K C-term Lys (2)
(Courtesy of Dr. S. Kozlowski)
Proteins Can be Heterogeneous Mixtures
Deamidation (3 x 2)
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Drug Substance Characterization
Drug Substance should be positive for identity and have specified criteria for purity, potency and microbial contamination
Acceptance criteria for release and stability attributes should be established
• Often broader early in the development and subject to revisions (e.g., narrowed down) as manufacturing process develops
Results from release and stability testing should be provided in the IND
Raw data supporting Drug Substance characterization should be provided in the IND
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Drug Substance Characterization (cont.)
Safety
• Ensured by the specified limits for bioburden and endotoxin, misc. process-related contaminants
Purity & Characterization
• Assesses capability of purification process to remove process-related impurities (e.g., endogenous viruses, host-cell proteins, DNA, leachables, anti-foam, antibiotics, toxins, solvents, heavy metals, etc.)
• Product-related impurities (e.g., aggregates, breakdown products, product variants due to: oxidation, deamidation, denaturation, loss of C-term Lys in MAbs etc.)
• Product substances (product variants that are active)
Identity
• Unique for protein of interest, especially relevant for closely related proteins manufactured in the same facility
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Drug Substance Characterization (cont.)
Potency• Required to assess biological activity of the product
• Assay should be relevant for protein mechanism of action
• For MAb or Fc fusion proteins - a binding assay may be sufficient for early development, but a functional assay relevant for the mechanism of action should be developed
• If mechanism of action unknown - multiple bioactivities plus elucidating higher order structure may be required
Strength• Protein content
Stability• Drug Substance stability should be demonstrated with
appropriate stability-indicating assays
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Drug Substance Characterization – Methodology
Safety• LAL test, rabbit pyrogen test, bacterial culture methods
Purity & Characterization including but not limited to:• Reversed-phase HPLC, Peptide mapping, MS • SDS-PAGE, Western analysis, capillary electrophoresis• SEC, AUC, FFF, light scattering• Ion Exchange Chromatography • Carbohydrate analysis (capillary electrophoresis, HPAEC = high-pH
anion-exchange chromatography, IEF for sialic acid) Identity
• N-terminal sequencing• Peptide mapping • Immunoassays (ELISA, Western blotting)
Potency• Animal-based assays, cell-based assays, reporter gene, biochemical
(e.g., enzyme activity) Protein content
• RIA, ELISA, UV absorbance, Bradford
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Drug Product Characterization
Safety• Final Drug Product for injection should be sterile • Within specified limits for endotoxin • Immunogenicity should be screened and monitored
– Successfully reduced in MAb by replacing murine with human sequences
Purity & Characterization • Product and process-related impurities & product-
related substances should be within specified limits Identity
• Unique for protein of interest, especially relevant for closely related proteins manufactured in the same facility
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Drug Product Characterization (cont.)
Potency
• Assay should be relevant for protein mechanism of action
• For MAb or Fc fusion proteins, a binding assay may be sufficient for early development, but a functional assay relevant for the mechanism of action should be developed
• If mechanism of action unknown - multiple bioactivities plus elucidation of higher order structure may be required
Strength
• Protein content
Stability
• Drug Product should maintain stability for the duration of the clinical trial
Container closure compatibility
• Primary function - barrier to microbial ingress
• Extractables/Leachables studies – requirement for licensure
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Extractables
Migrate from a c/c system and/or other packaging components in DP vehicle or solvent under extreme T°C and time conditions
exaggerated conditions
Helpful in the predicting potential leachables and in selecting the appropriate c/c system
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Leachables
Migrate spontaneously from a c/c system and/or other packaging components
normal conditions of use and storage
Often a subset of extractables, or derived by their chemical modification
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Sources of leachables in the product
Syringes/prefilled syringes, ampoules, vials, bottles
IV bags
Storage bags for product intermediates
Closures (screw caps, rubber stoppers)
Container liners (e.g., tube liners)
Processing equipment:• stainless steel storage tanks/bioreactors
• tubing
• gaskets, valves, rings
• filters
• purification resins
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Examples of leachables impacting on safety and product quality
Example #1 – Impact of patient safety: Change: from HSA formulation to a polysorbate
Unchanged container closure system (pre-filled syringes with the uncoated rubber stoppers)
Source: vulcanizing agents leached from rubber stopper over time
Outcome:
• no detectable changes in product quality
• safety: serious adverse event (PRCA)
Hypothesis: leachables acted as adjuvants triggering immunogenicity
Example #2 - Impact on product quality: Change from a lyophilized to a liquid formulation Divalent cation leached from the rubber stopper Caused activation of metalloprotease (a process-related impurity co-
eluted with the API) Impact: product degradation at the N-terminal site (stability study)
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Stability Program
Drug Substance and Drug Product, real-time and accelerated stability data with several time points under upright and inverted conditions used to establish the expiration period
Stress studies (e.g., UV, exaggerated light, temperature and pH) useful to elucidate product degradation pathways and for defining acceptance criteria
Limited time stability studies may be acceptable if short-term trial is anticipated
Stability data generated from engineering lots also acceptable
Failure to demonstrate product stability is a potential hold issue
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Stability Program (cont.)
The following testing should be included at a minimum:
Safety• Bioburden/sterility
Purity• Product and process-related impurities & product-
related substances Sialic acid - if appropriate Potency Protein content/strength pH Appearance Leachables (separate study, not part of routine stability
testing)
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Good Manufacturing Practices
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Inspectional Activity
Three types:• Pre-licensed (PLI) - announced, generally required for approval• Pre-approval (PAI) – announced, could be waived• Surveillance (biennial post-licensure) – unannounced• No formal inspection requirement for sites manufacturing
biologics under clinical investigation• Manufacturing and testing sites are subject to inspection
Inspection system undergoing revision for OBP products Currently inspections of facilities manufacturing CDER BLA products:
• PAI led by TFRB, Office of Compliance, with Product Reviewer(s) sometimes part of on-site team
• Biennial post-licensure inspections led by Team Biologics with Product Reviewers which can be part of the on-site team involved in the inspection
NDA Products:• Pre-approval and post-licensure inspections led by district
personnel
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Facilities and Practices
Closed systems whenever possible
Aseptic Processing CIP/SIP Disposable Systems Environmental
Monitoring Water/HVAC Good record-
keeping and documentation (phase 1)
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Phase III
Phase IPhase II
Provide greater assurance in linking product quality to commercial manufacture
ICH Q7: Good Manufacturing Practice Guide For Active Pharmaceutical Ingredients
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Potential Show Stoppers?
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Potential CMC Hold Issues for Phase 1 IND
Comparability between preclinical and clinical lots not demonstrated
Insufficient characterization of cell banks (e.g., adventitious agents testing, identity, etc.)
Inadequate product characterization with regards to purity, identity, potency and safety
Lack of final product release testing Lacking or inappropriate specifications for release and stability
testing Lacking or inadequate potency assay Data supporting product stability have not been shown for the
planned duration of clinical studies Lack or inappropriate immunogenicity assays for high risk
products Lack of evidence for final Drug Product sterility
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Guidance Documents
Guidance for Industry: Content and Format of Investigational New Drug Applications for Phase I Studies of Drugs, including Well-Characterized, Therapeutic, Biotechnology derived Products (1995)
Guidance for Industry for the Submission of CMC Information for a Therapeutic Recombinant DNA-Derived Product or a Monoclonal Antibody Product for In Vivo Use (1996)
Guidance for Industry: IND for Phase 2 and 3 studies of Drugs, including Specified Therapeutic Biotechnology-Derived Products – CMC Content and Format (Draft, 1999)
FDA Guidance Concerning Demonstration of Comparability of Human Biological Products, including Therapeutic Biotechnology-derived Products (1996)
Guidance for Industry: INDs - Approaches to Complying with cGMP's for Phase 1 Drugs (Draft, 2006)
Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use (1997)
Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals (1993)
International Conference on Harmonization (ICH) documents
21 CFR 200’s, 600’s
PHS Act, FD&C Act
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Acknowledgments
Emily Shacter
Barry Cherney
Steven Kozlowski
Wendy Shores
Susan Kirshner
Emanuela Lacana
Patricia Hughes
Joe Kutza
All of OBP
Working with FDA: Biological Products and Clinical Development Ingrid Markovic
Questions? Comments?