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

USP Standards—Biologicals

Major B&B Themes and Initiatives for 2010-2015 Modernization of Monographs Outreach to Industry for New Monographs Procedural Chapters and Characterization/Measurement of Ancillary and Process Materials Growing the portfolio to support all biologics Modernization of analytical approaches Biological Potency Tests: Implementation of new USP Chapters Assay transitions: animal to cell, cell to binding, activity to amount of substance – questions of equivalence and units Product Class Chapters: Monoclonal antibodies Glycoconjugate vaccines

Vertical Standards – Monographs Role and Use: Clearly define identity, strength and purity, as well as other important quality attributes at the product level Allow independent testing and verification based on a public standard Considerations for Standard Development: Complexity of specifications and system suitability criteria Biological potency assignments and unit maintenance Across manufacturers Internationally Product- specific vs. common product class requirements For well-characterized and legacy products: inclusion and bridging to new analytical technology

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

Current Biologics in the US Market From Kozlowski et al., NEJM 265;5, 2011

Biologics with no official USP Monograph

Current Key New Monograph Development Projects BB1: Insulin Glargine Fondaparinux Dalteparin Sodium Epoetin Octreotide Soon in ballot: Filgrastim BB2: Factor IX, plasma derived Soon in ballot: Sipuleucel T

Current Key Monograph Modernization Projects BB1: Heparin Sodium, stage 3 Menotropins Pancreatin/pancrelipase Insulins

Horizontal Standards - Procedural Benefits: Access to validated procedures and procedure performance criteria early in development Solid anchor point for product characterization Facilitate comparability during all development stages Directly apply ICH guidance, including: Q5E Q6B Establish consistency in analytical expectations

From General to Specific - Procedures <1084> Glycoprotein and Glycan Analysis – Introduction and Choice of Analysis Methods Guidance & Information <212> Oligosaccharide Analysis <210> Monosaccharide Analysis Procedures & Performance Criteria Oligosaccharide mixtures from Human α acid Glycoprotein Fetuin RNAse B Human IgG Monosaccharide Mixes 1–4 Procedural (Horizontal) Standards for System Suitability & Validation Single Monosaccharides

<1084> - Glycoprotein Analysis Strategies

<1084> Glycoprotein Analysis Strategies

Test Chapters – Current Major Initiatives Impurities <30> Residual DNA Physicochemical Tests <212> Oligosaccharide Analysis <210> Monosaccharide Analysis <121.1> Insulin Physicochemical Analysis <209> Low Molecular Weight Heparin Molecular Weight Determinations <XXX> Collagen Potency Assays and Content Measurement <57> Protein Determination Procedures <123> Glucagon Bioidentity Tests <124> Epoetin Bioassays <126> Somatropin Bioassays <208> Anti-Factor IIa and Xa Assays for Unfractionated and Low Molecular Weight Heparins

Vaccine Chapters Possible PC Chapters <1235> Vaccines for Human Use (completed) <1238> Bacterial Vaccines (completed) <1239> Viral Vaccines (in progress) <XXXX> Toxins/Toxoids <XXXX> Live Attenuated <1234> Polysaccharide and Glycoconjugate Possible PC Chapters <XXXX> Killed Viruses <XXXX> Live Attenuated <XXXX> Subunit Vaccines <XXXX> Other Subunit Sub-<1000> Analytical Chapters for Key Quality Attributes and RS

Will be accompanied by USP MAb System Suitability RS <129> Analytical Procedures for Recombinant Therapeutic Monoclonal Antibodies Will contain a collection of validated compendial procedures with established system suitability criteria for therapeutic MAbs Will be accompanied by USP MAb System Suitability RS Will not contain product or class specific acceptance criteria Will be supported by a suite of >1000 Information Chapters that discuss quality attributes, manufacturing and quality control aspects for MAbs

Horizontal Standards– Ancillary & Process Materials Benefits: Quality specifications and standards for complex ancillary and process materials Give access to process ingredients of consistent quality Control process variability Facilitate testing of process intermediates and bulks Challenges in Standard Development: Fast evolution of process materials: Serum-free, proprietary custom media Engineered 2nd and 3rd generation materials, e.g. Protein A Defining key quality attributes for complex materials, e.g. Fetal Bovine Serum (FBS)

Raw/Ancillary Materials Raw materials may or may not remain in the final therapeutic product as active substances or excipients Ancillary materials are a subset of raw materials Ancillary products may exert an effect on a therapeutic substance (e.g. a cytokine may activate a population of cells), but they are not intended to be in final formulation Concerns related to raw materials qualification have been amplified by recent materials supply issues: Glycerin, Heparin, Melamine Animal derived material (serum) used in manufacturing of some biologics Some components are more critical than others! Risk assessment strategies are required to ensure quality A critical material will come in contact with cells with a potential to alter either the growth characteristics of the cells or the ability of the cell culture to meet lot release specifications.

Ancillary Materials Standards: USP Approach General Information Chapter (Guidance) <1043> Ancillary Materials for Cell-, Gene-, and Tissue-Engineered Products Specific AM Chapters <90> FBS Quality Attributes <92> Cytokines and Growth Factors Quality Attributes Ancillary Material Requirements for Specific AMs Reference Standards: FBS Interleukin-4 FGF-2 Transferrin G-CSF Ancillary Material Reference Standards

Risk-based Classification of Ancillary Materials Tier 1 – Low-Risk, Highly Qualified Materials with Intended Use as Therapeutic Drug or Biologic, Medical Device, or Implantable Material Tier 2 – Low-Risk, Well Characterized Materials with Intended Use as AMs, Produced in Compliance with GMPs Tier 3 – Moderate-Risk Materials Not Intended for Use as AMs (frequently produced for in vitro diagnostic use or reagent grade materials) Tier 4 – High-Risk Materials, Materials not Produced in Compliance with cGMPs and materials not intended to be used in cell manufacturing

Risk-based Qualification – USP <1043> Elements of Qualification and/or Risk Reduction Activities Tier 1 Tier 2 Tier 3 Tier 4 Master File cross reference X Certificate of analysis lot-to-lot effect on process performance Removal from finished product Stability as stored and used in specific process Confirm Certificate Analysis Test Vendor Audit Upgrade Manufacturing to GMP level Develop internal specifications Lot to lot comparability may be needed Testing for adventitious agents may be needed Traceability to country of origin, safety from animal diseases Adventitious agent testing for animal source-relevant viruses

Recombinant Human Interleukin 4 -USP Interleukin-4 Standard Requirement Specific Activity: Not less than 0.5 x 107 USP Units /mg of total protein Labeled potency of RS will be based on bioactivity using TF-1 cell line Purity: 98% (SDS-PAGE and silver stain) Identity: N-term protein sequencing (10 residues) and Western Blot Associated Reference Standard Lyophilized powder Calibrated against International Standard (WHO) How is the standard used? Preparation of IL-4 for which the activity was determined by calibration against the USP standard, will provide consistency in the manufacturing of the cell therapy product, by way of using the right amount of material.

USP General Chapter <90> Fetal Bovine Serum – Quality Attributes and Functionality Tests Official uses of the USP FBS Reference Standard Identification – Radial Immunodiffusion FBS Functionality Tests – Growth Promotion Curve Five cell lines are recommended for use Functionality tests are performed on 3 cell lines Two from the recommended cell line list Third is cell line relevant to the user’s application

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

Future Directions for USP Ancillary Materials Standards Growth Factors and Cytokines – additions to <92>. Next revision adds FGF Standards for enzymes as ancillary materials Trypsin, others? Second and third generation identification tests for complex, naturally derived materials: Application of modern immunology and proteomics approaches?

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