Transcript Introduction

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
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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 3D 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
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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
B&B Overall Monograph Distribution by Product
Class
peptide
enzyme
complex extract
carbohydrate
glycosaminoglycan
other
Tissue product
IgG/serum
Blood component/protein
Vaccine
Other
Product Class
1%
4%
3%
8%
Number of
monographs
peptide
47
enzyme
12
complex extract
11
carbohydrate
11
5%
39%
4%
8%
9%
9%
10%
glycosaminoglycan
9
other
5
Tissue product
6
IgG/serum
9
Blood
component/protein
5
Vaccine
3
Peptide/Small Protein Drug Substance Monographs
Somatropin
Insulin
Human
Glucagon
Filgrastim
Identification - HPLC
X
X
X
X
Identification - Peptide
Mapping
X
X
X
X
Assay - HPLC
X
X
X
Impurities – related
proteins: HPLC (Assay)
X
X
X
Impurities – Charge
variants, IEF
X
X
Impurities – Limit of
HMW proteins: SEC
X
X
Specific Tests:
bioidentity, <85>,
<61>/<62>, <731>
X
X
X
no bioidentity
test for DS
no <731>
Filgrastim
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Drug substance monograph published in PF 36(5),
becoming official in USP
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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?
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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.”
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Identity
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Assay (Potency)
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Impurities
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Additional Requirements
– Packaging and Storage; Labeling
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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%.
NOTE: 8 major peaks will be defined in the USP Filgrastim RS Data Sheet.
Pancreatin – Drug Substance Monograph
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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.
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Enzymatic Assays
– Amylase, Lipase, Protease
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Fat Content Test
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General Requirements: Labeling, Packaging and Storage
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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
Lipase
Products:
Free fatty acids
(FFA)
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
Control Unit / pH end point / NaOH delivery
µmoles NaOH
= µmoles FFA = Units
Stirrer
pH
0.1N
NaOH
Lipase
Vi
Time (min)
Water at 37°C
1.
Emulsification of
olive oil substrate + Buffer
+ Bile Salts
2. Lipase
addition
3. Release of FFA upon lipolysis
and recording of FFA titration by
NaOH at constant pH
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
Human
pancreatic
lipase
Substrate
Specific activity
(µmole FFA.min-1.mg-1)
Theoretical
duration for
complete lipolysis
of meal TAG *
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
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
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)
E
E
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E
S
S
S
E
E
S
E
S
E
S
E
E
E
E
S
E
S
E
E
E
S
E
E
E
E
E
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S
Large excess of substrate,
high enzyme turnover
Less emulsification,
less substrate vs. enzyme,
low 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
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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.