1. 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

2. 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

3. 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 ~
Factor VIII amino acids 2 chains, 25 glycosylation sites

4. 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)

5. 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

6. 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

7. 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>

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. 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 , 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. 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

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%.
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.

13. 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)

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
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

16. 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

17. 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

18. 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

19. 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

20. 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)

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 not less 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.