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

Biological Medicines: Opportunities and Challenges 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

Biotechnology Products, Subset of Biologicals 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, cont’d. 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)

Biotech Products – Quality Testing and Monographs 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

Official USP Biologics Monographs by Product Class Number of monographs peptide 47 enzyme 12 complex extract 11 carbohydrate glycosaminoglycan 9 other 5 Tissue product 6 IgG/serum Blood component/protein Vaccine 3

Peptide/Small Protein Drug Substance Monographs Somatropin Insulin Human Glucagon Filgrastim Identification - HPLC X Identification - Peptide Mapping Assay - HPLC Impurities – related proteins: HPLC (Assay) Impurities – Charge variants, IEF Impurities – Limit of HMW proteins: SEC Specific Tests: bioidentity, <85>, <61>/<62>, <731> no bioidentity test for DS no <731>

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.

Filgrastim: G-CSF? 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.coli expression system is called Filgrastim 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

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

Filgrastim Monograph: Identification 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

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%. Previously, the acceptance criteria was based on the relative retention time relative to peak 8, and the criterion was greater than or equal to 2.0 min. NOTE: 8 major peaks will be defined in the USP Filgrastim RS Data Sheet.

Pancreatin – Drug Substance Monograph Definition: Pancreatin is a substance containing enzymes, principally amylase, lipase, and protease, obtained from the pancreas of the hog, Sus scrofa Linné var. domesticus Gray (Fam. Suidae) or of the ox, Bos taurus Linné (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)

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”

Pancreatin Lipase Assay The lipolysis reaction catalyzed by pancreatic lipase Substrate: Triglycerides Products: Free fatty acids (FFA) Lipase pH > pKa Ionized FFA Titration* by Na+OH- *Principle of the USP Pancrelipase assay Slide created by Frederic Carriere

In Vitro / In Vivo Correlations: The Case Study of Pancreatic Lipase Titrimetric Lipase Assay by the pH-stat Technique Adapted from Frederic Carriere pH 0.1N NaOH Water at 37°C Stirrer Control Unit / pH end point / NaOH delivery µmoles NaOH = µmoles FFA = Units V i Lipase 3. Release of FFA upon lipolysis and recording of FFA titration by NaOH at constant pH Time (min) 2. Lipase addition Emulsification of olive oil substrate + Buffer + Bile Salts

In Vitro / In Vivo Correlations: The Case Study of Pancreatic Lipase Pancreatic Lipase Specific Activities on Various Substrates Carrière et al. Gastroenterology (2000) 119:949–960 Lipase Substrate Specific activity (µmole FFA.min-1.mg-1) Theoretical duration for complete lipolysis of meal TAG * Human pancreatic lipase Olive oil (USP assay) 3000 6 sec Tributyrin (synthetic short chain TAG) 8000 2 sec Mixed solid-liquid meal TAG (Hamburger, Fries, Butter…) 15 20 min Natural substrate Synthetic Physiological 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

S 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) Less emulsification, less substrate vs. enzyme, low enzyme turnover S E S E Large excess of substrate, high enzyme turnover

Characterization of Pancreatin 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

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)

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 not less than 90% of doubling time of RS

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.