1. 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

2. USP Standards—Biologicals

3. 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

4. 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

5. 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

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

7. Biologics with no official USP Monograph

8. 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

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

10. 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

11. 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

12. <1084> - Glycoprotein Analysis Strategies

13. <1084> Glycoprotein Analysis Strategies

14. 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

15. 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

16. 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

17. 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)

18. 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.

19. 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

20. 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

21. 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

22. 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.

23. 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

24. Fetal Bovine Serum (FBS)- USP
FBS Standard Requirements
Osmolality: mOsm/Kg
Total Protein: mg/mL
pH:
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)

25. 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

26. 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?

27. Thank you!
As you can see from the world map on the outside of USP headquarters building, it is a global pharmacopeia based in Rockville, Maryland. No borders.