Fouad Atouf, Ph.D. Director, Biologics and Biotechnology

1. 6th Annual Science and Standards SymposiumJanuary 16, 2013Istanbul Quality Attributes for Biological Medicines and USP Standards Fouad Atouf, Ph.D. Director, Biologics and Biotechnology

2. Biological Medicines: Scope of Products Blood and Blood Products Cell, Gene, Tissue Therapies Therapeutic Proteins, Recombinant and Naturally-derived Vaccines Multi-components (e.g. raw materials) manufacturing: Potential supply chain issues (e.g. animal derived materials) Testing of quality of components before manufacturing begins Complex manufacturing processes with impact on: Quality attributes of finished products Challenging regulatory approval pathways Control of the quality, safety and efficacy of biologicals is difficult, despite technological advances Orthogonal methods needed to address a single quality aspect Higher order structures, often addressed by a biological assay Biological Medicines: Opportunities and Challenges

3. Scope of Products, Examples: Glucagon, Calcitonin ~ 30 Amino Acids Insulin - 2 Chains ~ 51 Amino Acids Somatropin - 1 chain, 192 amino acids, not glycosylated Epoeitin - 1 chain, 165 amino acids, 3 N-linked glycosylation sites, 1-O-linked glycosylation site MW ~ 30000. Factor VIII - 2331 amino acids 2 chains, 25 glycosylation sites Biotechnology Products, Subset of Biologicals

4. Heterogeneity and other factors with impact on quality attributes Product-related substances (molecular variants, aggregates, deamidation, oxidation, glycosylation, etc…) Immunogenic potential: difficult to predict -occurrence and effects Process related impurities (host cell DNA and proteins, endotoxins, reagents and ancillary materials) Process contaminants (leachables, adventitious agents) Potential for a variety of tertiary and quaternary structures, with a lack of validatable methods to measure 3-D structures and 3-D population profiles (Bioassay) Biotechnology Products, Subset of Biologicals, cont’d.

5. Identification Retention Time from chromatographic assay Peptide Mapping N-Terminal Sequencing Purity HPLC (Reverse Phase) Limit on High Molecular Weight Species (Size Exclusion) Glycoforms (Isoelectric focusing) Potency Chromatographic when possible Bioassay-Bioidentity To address secondary and tertiary structures Cellular preferred over animal Monographs also cover sterility, and other general requirements such as labeling, packaging and storage Biotech Products – Quality Testing and Monographs

6. Official USP Biologics Monographs by Product Class

7. Peptide/Small Protein Drug Substance Monographs

8. Filgrastim • Drug substance monograph published in PF 36(5), becoming official in USP • Filgrastim is the recombinant form of human granulocyte colony-stimulating factor (G-CSF), marketed under the brand name Neupogen™ • C845H1339N223O243S9 • The USP Nomenclature Expert Committee has finalized nomenclature for the official title of this drug substance, “filgrastim,” which is expected to be the “official title” on the monograph recognized in USP-NF.

9. Granulocyte colony stimulating factor (G-CSF) 18-20 kDa Hematopoetic cytokine that acts on cells of the neutrophil lineage causing proliferation, differentiation and activation of committed precursor and mature neutrophils. Used in treatment of neutropenia following chemotherapy 174 Amino acids, 2 intra-molecular disulfide bonds, one free Cysteine at residue 17 and one O-linked carbohydrate chain at Thr 133 (<4% of the molecular mass). Recombinant human G-CSF synthesized in an E.coliexpression system is called Filgrastim Filgrastim: G-CSF? Protein Data Bank data (PDB: 1RHG) Hill, C.P., Osslund, T.D., Eisenberg, D. The structure of granulocyte-colony-stimulating factor and its relationship to other growth factors. Proc.Natl.Acad.Sci.USA v90 pp.5167-5171, 1993

10. Filgrastim Drug Substance Monograph • Definition: • “It is a single chain, 175 amino acid nonglycosylated polypeptide produced by Escheria coli bacteria transfected with a gene encoding a methionyl human granulocyte colony-stimulating factor. When prepared as a drug substance, it contains NLT 1.0 mg/mL of Filgrastim. . . . It has a biological potency of NLT 80% and NMT 125% relative to the standard.” • Identity • Assay (Potency) • Impurities • Additional Requirements • Packaging and Storage; Labeling • Reference Standards

11. A. It meets the requirements described under Assay. Acceptance criteria: It has a biological potency of NLT 80% and NMT 125%. B. It meets the requirements described under Chromatographic purity. Acceptance criteria: NMT 1.0% of reduced Filgrastim is found and NMT 2.0% of total impurity is found. C. Peptide mapping with UV detection Acceptance criteria: next slide Filgrastim Monograph: Identification

12. Identification C: Peptide Mapping with UV Detection • Acceptance criteria:The difference in retention of each of the eight major peaks between the Test solution chromatogram and the average of the Standard solution chromatograms must be ≤ 0.5 min. The relative difference in peak height between the normalized sample peak height (normalized by total peak height versus the average total peak height of the Standard solution chromatograms) and the average standard peak height of each of the eight major peaks must be ≤15%. • NOTE: 8 major peaks will be defined in the USP Filgrastim RS Data Sheet.

13. Pancreatin – Drug Substance Monograph • Definition: Pancreatin is a substance containing enzymes, principally amylase, lipase, and protease, obtained from the pancreas of the hog, SusscrofaLinné var. domesticus Gray (Fam. Suidae) or of the ox, BostaurusLinné (Fam. Bovidae). Pancreatin contains, in each mg, not less than 25 USP Units of amylase activity, not less than 2.0 USP Units of lipase activity, and not less than 25 USP Units of protease activity. • Enzymatic Assays • Amylase, Lipase, Protease • Fat Content Test • General Requirements: Labeling, Packaging and Storage • Identification will be addressed in revision • Products must meet enzymatic assays (e.g. Lipase assay) • Inclusion of identification test (HPLC-based)

14. Potency Determination • USP Pancreatin Monograph, Assay for lipase activity • “One USP Unit of lipase activity is contained in the amount of pancreatin that liberates 1.0 microequivalent of acid per minute at a pH of 9.0 and 37° under the conditions of the Assay”

15. Pancreatin Lipase Assay Lipase Products: Free fatty acids (FFA) pH > pKa Ionized FFA Titration* by Na+OH- *Principle of the USP PancrelipaseassaySlidecreated by FredericCarriere The lipolysisreactioncatalyzed by pancreatic lipase Substrate: Triglycerides

16. In Vitro / In Vivo Correlations: The Case Study of Pancreatic Lipase Control Unit / pH end point / NaOHdelivery Stirrer µmoles NaOH = µmoles FFA = Units 0.1N pH NaOH Lipase V i Water at 37°C Time (min) 3. Release of FFA uponlipolysis and recording of FFA titration by NaOHat constant pH • Emulsificationof • olive oilsubstrate + Buffer • + Bile Salts 2.Lipase addition TitrimetricLipase Assayby the pH-stat Technique Adapted from Frederic Carriere

17. In Vitro / In Vivo Correlations: The Case Study of Pancreatic Lipase Natural substrate Synthetic substrate Physiological substrate PancreaticLipase Specific Activities on VariousSubstrates Carrière et al. Gastroenterology (2000) 119:949–960 Only this value is physiologically relevant *For a mixed solid-liquid meal (700 mL) containing 30 g TAG, a secretion of 200 mg HPL per meal, and 2 acyl chains out of 3 released per TAG molecule

18. E E E E E S S E E E E E E S S S E E E S E S E E E E E S E E S S Less emulsification, less substrate vs. enzyme, low enzyme turnover Large excess of substrate, high enzyme turnover In Vitro / In Vivo Correlations: The Case Study of Pancreatic Lipase Olive oil (USP assay, fine emulsion with acacia) Meal triglycerides (from butter, cooking oil, meat) Tributyrin (synthetic short chain TAG, fine emulsion under mechanical stirring)

19. Advantages of RP-HPLC / ESI-MS Separation One-dimensional separation, automated Wide range of polarity by selection of stationary phase chemistry & mobile phase / gradient Detection/Quantification Universal UV (210 nm), MS-detection Sufficient dynamic range, linear, reproducible Use of external standards Identification MS-coupling & fractionation for other techniques of identification (PMF, MS-MS, N-terminal sequencing), covers all ionizable species Characterization of Pancreatin

20. Fetal Bovine Serum (FBS)- USP • FBS Standard Requirements • Osmolality: 280-360 mOsm/Kg • Total Protein: 30-45 mg/mL • pH: 7.00 - 8.00 • Endotoxin: Not more than 10 units/mL • Hemoglobin level: Not more than 30 mg/dL • Identification: Radial Immunodiffusion (RID): species ID, IgG levels • Functionality Assays (Growth Curve and Clonal Assay) • Associated Reference Standard (RS), under development • Liquid frozen, 10 mL • Collaborative study to include several laboratories to test: • Identification (FBS sample positive for bovine IgG and content is < 500 mg/L) • Growth curve (doubling time in test sample is not less than 90% compared to RS)

21. How the FBS Standard is Used: Growth Curve Challenges: Cell Line, Cell Density, Cell Counting, Days in Culture • Three cell densities, determine viable cell counts on days 0,1,2,3,4, and 7. Select the cell density that exhibit a growth curve with 3 phases: Lag, Log, Stationary; and linear over 3 time points or more • Use the selected cell density to assess the test FBS side by side with the reference standard FBS • Doubling time is estimated using a growth curve that is linear over three or more time points. • Acceptance Criteria: R2≥ 0.98 • Doubling time of test sample should be notless than 90% of doubling time of RS

22. Summary • A pharmacopeial monograph provide tools to control the key quality attributes of a medicinal product in terms of identity, strength and purity. • For biological medicines key quality attributes may require multiple orthogonal tests methods. • Biological assays are often needed to address the function of biologics, however high variability may be an issue.