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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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6th Annual Science and Standards SymposiumJanuary 16, 2013Istanbul 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

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 assay

Biological Medicines: Opportunities and Challenges
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
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)

Biotechnology Products, Subset of Biologicals, cont’d.
biotech products quality testing and monographs

Retention Time from chromatographic assay

Peptide Mapping

N-Terminal Sequencing


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 storage

Biotech Products – Quality Testing and Monographs
  • 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.
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.coliexpression system is called Filgrastim

Filgrastim: 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

filgrastim drug substance monograph
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
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

Filgrastim Monograph: Identification
identification c peptide mapping with uv detection
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
Pancreatin – Drug Substance Monograph
  • Definition:

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.

  • 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)
potency determination
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
Pancreatin Lipase Assay



Free fatty acids


pH > pKa

Ionized FFA

Titration* by Na+OH-

*Principle of the USP PancrelipaseassaySlidecreated by FredericCarriere

The lipolysisreactioncatalyzed by pancreatic lipase



in vitro in vivo correlations the case study of pancreatic lipase
In Vitro / In Vivo Correlations: The Case Study of Pancreatic Lipase

Control Unit / pH end point / NaOHdelivery


µmoles NaOH

= µmoles FFA = Units







Water at 37°C

Time (min)

3. Release of FFA uponlipolysis and recording of FFA titration by NaOHat constant pH

  • Emulsificationof
  • olive oilsubstrate + Buffer
  • + Bile Salts



TitrimetricLipase Assayby the pH-stat Technique

Adapted from Frederic Carriere

in vitro in vivo correlations the case study of pancreatic lipase1
In Vitro / In Vivo Correlations: The Case Study of Pancreatic Lipase







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 emulsification,

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

Olive oil

(USP assay, fine emulsion with acacia)

Meal triglycerides

(from butter, cooking oil, meat)


(synthetic short chain TAG,

fine emulsion under mechanical stirring)

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


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 species

Characterization of Pancreatin
fetal bovine serum fbs usp
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
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 notless than 90% of doubling time of RS
  • 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.