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6 th 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: Scope of Products Blood and Blood Products Cell, Gene, Tissue Therapies
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Fouad Atouf, Ph.D.
Director, Biologics and Biotechnology
Blood and Blood Products
Cell, Gene, Tissue Therapies
Therapeutic Proteins, Recombinant and Naturally-derived
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 assayBiological Medicines: Opportunities and Challenges
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 sitesBiotechnology Products, Subset of Biologicals
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)Biotechnology Products, Subset of Biologicals, cont’d.
Retention Time from chromatographic assay
HPLC (Reverse Phase)
Limit on High Molecular Weight Species (Size Exclusion)
Glycoforms (Isoelectric focusing)
Chromatographic when possible
To address secondary and tertiary structures
Cellular preferred over animal
Monographs also cover sterility, and other general requirements such as labeling, packaging and storageBiotech Products – Quality Testing and Monographs
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.coliexpression system is called FilgrastimFilgrastim: G-CSF?
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
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 slideFilgrastim Monograph: Identification
Pancreatin is a substance containing enzymes, principally amylase, lipase, and protease, obtained from the pancreas of the hog, SusscrofaLinné var. domesticus Gray (Fam. Suidae) or of the ox, BostaurusLinné (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.
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”
Free fatty acids
pH > pKa
Titration* by Na+OH-
*Principle of the USP PancrelipaseassaySlidecreated by FredericCarriere
The lipolysisreactioncatalyzed by pancreatic lipase
Control Unit / pH end point / NaOHdelivery
= µmoles FFA = Units
Water at 37°C
3. Release of FFA uponlipolysis and recording of FFA titration by NaOHat constant pH
TitrimetricLipase Assayby the pH-stat Technique
Adapted from Frederic Carriere
PancreaticLipase Specific Activities on VariousSubstrates
Carrière et al. Gastroenterology (2000) 119:949–960
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
less substrate vs. enzyme,
low enzyme turnover
Large excess of substrate,
high enzyme turnover
In Vitro / In Vivo Correlations: The Case Study of Pancreatic Lipase
(USP assay, fine emulsion with acacia)
(from butter, cooking oil, meat)
(synthetic short chain TAG,
fine emulsion under mechanical stirring)
One-dimensional separation, automated
Wide range of polarity by selection of stationary phase chemistry & mobile phase / gradient
Universal UV (210 nm), MS-detection
Sufficient dynamic range, linear, reproducible
Use of external standards
MS-coupling & fractionation for other techniques of identification (PMF, MS-MS, N-terminal sequencing), covers all ionizable speciesCharacterization of Pancreatin
Challenges: Cell Line, Cell Density, Cell Counting, Days in Culture