USP Reference Standards for Biologics

1. USP Reference Standards for Biologics Tina S. Morris, Ph.D., Vice President Biologics & Biotechnology USP-NF User Forum January 17th, 2013 Istanbul, Turkey

2. What Is a Reference Standard? • A Reference Standard is a highly characterized specimen of a drug substance, excipient, major impurity, degradation product, food ingredient, or performance calibrator • Most are intended for use in compendial methods; however some Reference Standards are available for customer convenience but are not required • When a Reference Standard is required within compendial methods it is used to ensure that products are of the appropriate identity, strength, quality, and purity

3. USP Reference Standards The reference materials relate directly to methods in the USP publications:

4. Uses of USP Reference Standards • There are two main types of USP Reference Standards: • Standards with Quantitative Applications • Assays (for drug substances and for formulations) • Limit tests (e.g., Impurity Reference Standards) • Standards with only Qualitative Applications • Identification tests • Elution markers • System Suitability tests 4

5. Development of Reference Standards The Steps for the Development of a Reference Standard are: 1. Source Material Donation from pharmaceutical industry Purchase/custom synthesis 2. Perform a Collaborative Study (3 or more labs involved; tests generally include identity, purity, volatility, hygroscopicity, functional group, and inorganic impurity) • USP laboratories (Rockville, India, China, and Brazil) • Agencies (FDA, Health Canada, Australia, and China) • Industry labs • Contract labs 3. Analyze Data/Value Assignment Mass balance approach100% - % sum of all impurities (w/w) Impurities including Organic impurities by chromatography (e.g., process impurities) Inorganic impurities (e.g., catalyst, salt, etc) Volatile impurities (residual solvents, water)

6. Typical Candidate Evaluation Components • Candidates are Evaluated on the Following: • Appearance (Visual or microscopic evaluation) • Identification Tests (more for first lot) • e.g., IR, NMR, MS, UV, Chromatography, test for counter-ion/salt, etc. • Indirect purity tests • e.g., Melting range, Specific rotation, Refractive index, etc. • Direct purity tests (for mass balance calculation) • Chromatographic purity • Inorganic contaminants determination • Volatiles (water, solvents) • Vapor sorption analysis (for direction for use) • Functional group analysis (titration, elemental analysis, UV absorptivity) • Assays against another well-characterized standard (previous lot, international standard) 6

7. Collaborative Study Design Types of Reference Standard Qualitative application Quantitative application Identification, peak identification, system suitability Potency/Assay/Limit • Establish identity of candidate material • Evaluate chemical identity by compendial and non-compendial techniques • Value assignment (mass-balance, bioassay, etc) of the RS candidate • Potency RS calibrated relative to the current International Standard • Establish identity of candidate material • Evaluate chemical identity by compendial and non-compendial techniques • No value assigned to the RS candidate

8. Standard Development Bulk Candidate • Non-routine • Quantity limited • proposed RS presentation different from sponsor (liquid vs. solid) Conventional Collaborative study Pre-characterization of bulk Formulation/Lyophilisation Definitive Fill Pilot Fill Definitive Fill • Content of fill • Homogeneity • Stability studies Collaborative study

9. Collaborative Study Design – USP rProtein A RS Pharmacopeial Applications: * Used solely for system suitability • Bulk material formulated in water as a frozen liquid • Evaluate chemical identity by compendial/official and non-compendial techniques • Molecular weight (ES-MS) • Molecular weight (ID-A: SDS-PAGE) • IgG Binding activity (ID-B) • Total protein content • Chromatographic purity • Triton content • IEF • Freeze-thaw study

10. Collaborative Study – USP Enoxaparin Sodium for Bioassays RS Pharmacopeial applications: • Bulk material formulated as a lyophilized powder • Evaluate chemical identity by compendial/official and non-compendial techniques • Molecular weight using Broad Standard method • Molecular weight using Discrete Calibrant method • Structure verification by 1H, 13C and HSQC NMR spectroscopy • Anti-factor IIa activity • Anti-factor Xa potency (Assay) • Stability studies

11. USP Filgrastim RS • Recombinant form of human granulocyte colony stimulating factor (r-metHuG-CSF) • 175 amino acids • Two disulfide bridges, one free thiol at Cys18 • 18,799 daltons • Expressed in Escherichia coli • Nonglycosylated

12. Background • Reference Standard presentation • Presentations available from supplier were not optimal • Liquid presentation was not stable for more than 12 months; shaking not tolerated • Frozen presentation (-70°C) stable for 5 years but once thawed only stable for 30 days, cannot re-freeze • Lyophilized Filgrastim (new formulation) • Eliminated potential RS shipping and storage issues • New formulation was developed by monograph sponsor

13. Background • Formulation: 10 mM L-glutamic acid, 4% mannitol, 2% sucrose, 0.01% polysorbate 20, pH 4, and 1 mg/mLfilgrastim • Sponsor prepared 5000 mL of the formulation and shipped to NIBSC, definitive fill successful, samples shipped to USP and 2 sponsor sites for stability studies • Physicochemical and potency analyses indicate that reference standard candidate material remains stable through 13 months at the proposed storage temperature

14. Filgrastim Collaborative Study • Two Components • Physicochemical tests • Peptide Mapping • Chromatographic Purity (RP-HPLC) • SE-HPLC • SDS-PAGE • IEF • Protein Determination • Bioassay • Collaborators (International Study) • 16 collaborators total (some collaborators did both bioassay and bioanalytical tests) • Physicochemical tests • 8 collaborators total • 6 returned results • Bioassay • 13 collaborator total • 11 returned results

15. Potency Value The following statement will be included on the USP Certificate for Filgrastim Lot F0L526: Each ampoule contains 8.5 x 107 IU when assayed against the WHO 2nd International Standard for Granulocyte Colony-Stimulating Factor.

16. Protein Determination • Each collaborator performing the bioassay was asked to determine the protein on the ampoules assayed • Protein was also determined on candidate ampoules assayed during stability studies (4⁰ storage conditions) • Altogether the protein content was determined on 78 ampoules • Average of all results = 0.9780 mg per ampoule Standard Deviation = 0.02 %RSD = 2.38 • The label text will claim 0.98 mg protein per ampoule

17. Label for Lot F0L526 of USP Filgrastim RS

18. Graftskin is a tissue engineered product containing living, bi-layered skin substitute derived from neonatal foreskins Upper epidermal layer-human keratinocytes Inner dermal layer-human fibroblasts in bovine collagen lattice Cell banks generated and screened for microbial and viral contaminants Monograph tests Histology (Unique type of Reference Standards) Gene expression profile Barrier integrity Metabolic activity Graftskin: Authentic Visual References (AVRs)

19. Qualitative assessment of product’s structural quality Epidermal coverage Epidermal development Keratinocyte aspect Dermal matrix thickness Fibroblast density Matrix aspect Reference Standards: Authentic Visual References Passing units Failed units Graftskin: Histological Analysis

20. Graftskin AVR: Example of Passing Unit USP AVR Standards are used to visually aid the analyst in determining whether the product under analysis passes the Histological evaluation test.

21. Graftskin: AVRs for Failing Morphology Samples

22. Tryspin Crystallized: One RS, Different USP Monographs

23. Tryspin Crystallized RS • Intended uses of this RS: • Crystallized Trypsin and Chymotrypsin monographs: • Determine the suitability of the substrates and check the adjustment of the spectrophotometer by performing the Assay using USP Crystallized Trypsin Reference Standard. • Aprotinin monograph: • The determination of activity by the Assay is based on the specific inhibition of trypsin • Prepare a solution of USP Trypsin Crystallized RS containing about 4300 USP Trypsin Units per mL • Tryspin Crystallized RS is a quantitative RS

24. USP Trypsin Assay Method • Substrate: N-benzoyl-L-arginine ethyl ester hydrochloride (BAEE). • Conditions: T = 25°C, pH = 7.6, A253nm, Light path = 1 cm • Method: Continuous Spectrophotometric Rate Determination BAEE + H2O Na-Benzoyl-L-Arginine + Ethanol • One USP Trypsin Unit is the activity causing a change in absorbance of 0.003 per minute under the conditions specified in the assay Trypsin

25. Tryspin Crystallized RS: Release of a New Lot • Collaborative Study (5 Laboratories) established a RS with a value of 3369 USP Tryspin Units of Trypsin Crystallized per mg of material on the dried basis. • Additional tests: limit of chymotrypsin , loss on drying, appearance, electronic absorption , vapor Sorption • Replacement lot is suitable for use in its compendial applications. • Proposed label:

26. Number of Biological RS in Active Portfolio: 123 Total Biological RS: 123

27. RS Released Since July 2012

28. RS Under Development – Next 6 Months

29. RS Under Development – Next 6 Months

30. RS Under Development – On the Horizon

31. Thank you!