U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES NATIONAL INSTITUTES OF HEALTH Working with FDA: Biological Products and Clinical Development Chemistry, Manufacturing.

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)


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


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


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)


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


Manufacturing Process


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


Expression Vector and Cell Banking System


Source Materials

Mice Humans

Mammalian cell-culture

Yeast

Bacteria

Transgenics

Viruses

TSE

agents

Bacteria

Mycoplasma

Fungi


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


MCB and WCB

A working cell bank (WCB) is derived from the master cell bank (MCB) and is used to initiate a production batch


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


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


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


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


Production and Purification


Upstream cell culture & fermentation

Isolation/Capture of protein

Purification

Drug substance

Formulation

Drug Product

Downstream Processing


Fermentation Process


Purification Process


Drug Substance and Drug Product Characterization


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)


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


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


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


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


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


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


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


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


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


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)


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


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)


Good Manufacturing Practices


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


Facilities and Practices

Closed systems whenever possible

Aseptic Processing CIP/SIP Disposable Systems Environmental

Monitoring Water/HVAC Good record-

keeping and documentation (phase 1)


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


Potential Show Stoppers?


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


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


Acknowledgments

Emily Shacter

Barry Cherney

Steven Kozlowski

Wendy Shores

Susan Kirshner

Emanuela Lacana

Patricia Hughes

Joe Kutza

All of OBP


Questions? Comments?

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES NATIONAL INSTITUTES OF HEALTH Working with FDA: Biological Products and Clinical Development Chemistry, Manufacturing.

Documents

biological products

research fda slide

working cell bank wcb

release slide

kozlowski slide

biosimilars slide

characterized slide

x slide