1. Chemistry, Manufacturing and Control Issues in Production of Therapeutic Biologic Protein Products
Ingrid Markovic, Ph.D., Biologist
Laboratory of Biochemistry, Division of Therapeutic Proteins
Office of Biotechnology Products, Office of Pharmaceutical Science
Center for Drug Evaluation and Research
FDA

2. OBP/OPS/CDER Office of the Director Steven Kozlowski, M.D., Director
Wendy Shores, Ph.D., Deputy
Division of Therapeutic Proteins
Amy Rosenberg, M.D., Director
Barry Cherney, Ph.D., Deputy
Division of Monoclonal Antibodies
Kathleen Clouse, Ph.D., Director
Patrick Swann, Ph.D., Deputy
Fabrazyme
Enzymes
Enbrel
Fc-Fusion Proteins
Interferons
Interleukins
Cytokines
Herceptin
Avastin
Erbitux
G-CSF
Epo
Monoclonal
Antibodies
Growth factors
Botox
Toxins
Parallel Office to ONDQA, which also reviews proteins (e.g., insulin, HGH, etc.)
(courtesy of Dr. S. Kozlowski)

3. 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

4. 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

5. 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)

6. 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
~400 Da * x
This slide uses animation when in slide mode. Try to fix.
Therapeutic protein ~5, ,000 Da

7. Manufacturing Process

8. 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

9. Expression Vector and Cell Banking System

10. Source Materials Bacteria Mycoplasma Fungi Mice Humans
Mammalian cell-culture
Yeast
Viruses
TSE
agents
Bacteria
Transgenics

11. 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

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

13. Characterization of Cell Banks
*dependent upon cell substrate and manufacturing process

14. Characterization of Cell Banks (cont.)

15. 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

16. 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

17. Production and Purification

18. Downstream Processing
Upstream cell culture & fermentation
Isolation/Capture of protein
Purification
Drug substance
Formulation
Drug Product
Protein produced in yeast, E.coli, mammalian cells (ex: 293, CHO)
Protein either secreted into media (ex: mammalian cells, yeast) or found in cells (ex: E.coli).
Centrifugation, clarification to remove insoluble debris, cells, etc.
Harvest is mixture of proteins, lipids, DNA, RNA, media components, etc
Can mix and match techniques throughout entire purification scheme

19. Fermentation Process

20. Purification Process

21. Drug Substance and Drug Product Characterization

22. Proteins Can be Heterogeneous Mixtures
pyro-E K
pyro-E O D G O
Pyro-Glu (2) D
Deamidation (3 x 2) O
Methionine oxidation (2 x 2) G
Glycation (2 x 2)
High mannose, G0, G1, G1, G2 (5)
This slide has animation when in slide mode. Try to fix
Sialylation (5) K
C-term Lys (2)
(9600)2≈ 108
(Courtesy of Dr. S. Kozlowski)
2 x 6 x 4 x 4 x 5 x 5 x 2 = 9600

23. 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

24. 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

25. 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

26. 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

27. 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

28. 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

29. 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

30. 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

31. 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

32. 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)

33. 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

34. 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)
Deleted Identity from stability protocol.

35. Good Manufacturing Practices

36. 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

37. Facilities and Practices
Closed systems whenever possible
Aseptic Processing
CIP/SIP
Disposable Systems
Environmental Monitoring
Water/HVAC
Good record-keeping and documentation (phase 1)

38. ICH Q7: Good Manufacturing Practice Guide For Active Pharmaceutical Ingredients
I don’t see the value of the table that is shown in this slide
Phase III
Phase I
Phase II
Provide greater assurance in linking product quality to commercial manufacture

39. Potential Show Stoppers?

40. 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

41. 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

42. Acknowledgments Emily Shacter Barry Cherney Steven Kozlowski
Wendy Shores
Susan Kirshner
Emanuela Lacana
Patricia Hughes
Joe Kutza
All of OBP

43. Questions? Comments?