Transcript Quality Attributes of Biologics

USP Reference Standards for
Biologics
Tina S. Morris, Ph.D., Vice President
Biologics & Biotechnology USP-NF
User Forum
January 17th, 2013
Istanbul, Turkey
What Is a Reference Standard?
 A Reference
Standard is a highly characterized specimen of a
drug substance, excipient, major impurity, degradation product,
food ingredient, or performance calibrator
 Most
are intended for use in compendial methods; however
some Reference Standards are available for customer
convenience but are not required
 When
a Reference Standard is required within compendial
methods it is used to ensure that products are of the
appropriate identity, strength, quality, and purity
USP Reference Standards
The reference materials relate directly to
methods in the USP publications:
Uses of USP Reference Standards
 There
are two main types of USP Reference Standards:
 Standards
with Quantitative Applications
 Assays
(for drug substances and for formulations)
 Limit tests (e.g., Impurity Reference Standards)
 Standards
with only Qualitative Applications
 Identification
tests
 Elution markers
 System Suitability tests
4
Development of Reference Standards
The Steps for the Development of a Reference Standard are:
1. Source Material
Donation from pharmaceutical industry
Purchase/custom synthesis
2. Perform a Collaborative Study
(3 or more labs involved; tests generally include identity, purity,
volatility, hygroscopicity, functional group, and inorganic impurity)
– USP laboratories (Rockville, India, China, and Brazil)
– Agencies (FDA, Health Canada, Australia, and China)
– Industry labs
– Contract labs
3. Analyze Data/Value Assignment
Mass balance approach
100% - % sum of all impurities (w/w)
Impurities including
Organic impurities by chromatography (e.g., process impurities)
Inorganic impurities (e.g., catalyst, salt, etc)
Volatile impurities (residual solvents, water)
Typical Candidate Evaluation Components
 Candidates
are Evaluated on the Following:

Appearance (Visual or microscopic evaluation)

Identification Tests (more for first lot)
–

Indirect purity tests
–

e.g., IR, NMR, MS, UV, Chromatography, test for counter-ion/salt, etc.
e.g., Melting range, Specific rotation, Refractive index, etc.
Direct purity tests (for mass balance calculation)
–
–
–
Chromatographic purity
Inorganic contaminants determination
Volatiles (water, solvents)

Vapor sorption analysis (for direction for use)

Functional group analysis (titration, elemental analysis, UV
absorptivity)

Assays against another well-characterized standard (previous lot,
international standard)
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Collaborative Study Design
Types of Reference Standard
Qualitative application
Quantitative application
Identification, peak identification,
system suitability
Potency/Assay/Limit
Establish identity of candidate material
Evaluate chemical identity by
compendial and non-compendial
techniques
 No value assigned to the RS candidate
Establish identity of candidate material
Evaluate chemical identity by compendial
and non-compendial techniques
Value assignment (mass-balance,
bioassay, etc) of the RS candidate
Potency RS calibrated relative to the
current International Standard
Standard Development
Bulk Candidate
Conventional
Non-routine
 Quantity limited
 proposed RS presentation different
from sponsor (liquid vs. solid)
Collaborative study
Pre-characterization of bulk
Formulation/Lyophilisation
Definitive Fill
Pilot Fill
Definitive Fill
Content of fill
Homogeneity
Stability studies
Collaborative study
Collaborative Study Design – USP rProtein A RS
Pharmacopeial Applications:
Monograph
Identification
Isoforms
Limit of Triton
X-100
General Chapter
<131> Protein A
Quality Attributes
SDS-PAGE
IgG Binding*
IEF
HPLC
* Used solely for system suitability
Bulk material formulated in water as a frozen liquid
Evaluate chemical identity by compendial/official and non-compendial techniques
– Molecular weight (ES-MS)
– Molecular weight (ID-A: SDS-PAGE)
– IgG Binding activity (ID-B)
– Total protein content
– Chromatographic purity
– Triton content
– IEF
– Freeze-thaw study
Collaborative Study – USP Enoxaparin Sodium for Bioassays RS
Pharmacopeial applications:
Monograph
Anti-factor IIa
activity
Assay
Enoxaparin Sodium
Spectrophotometric Spectrophotometric
Enoxaparin Sodium
Injection
Spectrophotometric Spectrophotometric
 Bulk material formulated as a lyophilized powder
 Evaluate chemical identity by compendial/official and non-compendial techniques
– Molecular weight using Broad Standard method
– Molecular weight using Discrete Calibrant method
– Structure verification by 1H, 13C and HSQC NMR spectroscopy
– Anti-factor IIa activity
– Anti-factor Xa potency (Assay)
– Stability studies
USP Filgrastim RS

Recombinant form of human granulocyte colony
stimulating factor (r-metHuG-CSF)
– 175 amino acids
– Two disulfide bridges, one free thiol at Cys18
– 18,799 daltons
– Expressed in Escherichia coli
– Nonglycosylated
Background
 Reference
Standard presentation
– Presentations available from supplier were not optimal
• Liquid presentation was not stable for more than 12
months; shaking not tolerated
• Frozen presentation (-70°C) stable for 5 years but
once thawed only stable for 30 days, cannot refreeze
– Lyophilized Filgrastim (new formulation)
•
Eliminated potential RS shipping and storage
issues
• New formulation was developed by monograph
sponsor
Background

Formulation:
10 mM L-glutamic acid, 4% mannitol, 2% sucrose,
0.01% polysorbate 20, pH 4, and 1 mg/mL filgrastim

Sponsor prepared 5000 mL of the formulation and
shipped to NIBSC, definitive fill successful, samples
shipped to USP and 2 sponsor sites for stability
studies
– Physicochemical and potency analyses indicate that
reference standard candidate material remains
stable through 13 months at the proposed storage
temperature
Filgrastim Collaborative Study

Two Components
– Physicochemical tests
•
•
•
•
•
•
Peptide Mapping
Chromatographic Purity (RP-HPLC)
SE-HPLC
SDS-PAGE
IEF
Protein Determination
– Bioassay

Collaborators (International Study)
– 16 collaborators total (some collaborators did both bioassay and
bioanalytical tests)
• Physicochemical tests
o 8 collaborators total
o 6 returned results
• Bioassay
o 13 collaborator total
o 11 returned results
Potency Value
The following statement will be included on the USP Certificate
for Filgrastim Lot F0L526:
Each ampoule contains 8.5 x 107 IU when assayed against
the WHO 2nd International Standard for Granulocyte ColonyStimulating Factor.
Protein Determination

Each collaborator performing the bioassay was asked to determine
the protein on the ampoules assayed

Protein was also determined on candidate ampoules assayed
during stability studies (4⁰ storage conditions)

Altogether the protein content was determined on 78 ampoules

Average of all results = 0.9780 mg per ampoule
Standard Deviation = 0.02
%RSD = 2.38

The label text will claim 0.98 mg protein per ampoule
Label for Lot F0L526 of USP Filgrastim RS
Graftskin: Authentic Visual References (AVRs)
 Graftskin is a tissue engineered product containing
living, bi-layered skin substitute derived from neonatal
foreskins
– Upper epidermal layer-human keratinocytes
– Inner dermal layer-human fibroblasts in bovine collagen lattice
 Cell banks generated and screened for microbial and
viral contaminants
 Monograph tests
–
–
–
–
Histology (Unique type of Reference Standards)
Gene expression profile
Barrier integrity
Metabolic activity
Graftskin: Histological Analysis
 Qualitative assessment of product’s structural quality
 Epidermal coverage
 Epidermal development
 Keratinocyte aspect
 Dermal matrix thickness
 Fibroblast density
 Matrix aspect
 Reference Standards: Authentic Visual References
– Passing units
– Failed units
Graftskin AVR: Example of Passing Unit
USP AVR Standards are used to visually aid the analyst in
determining whether the product under analysis passes the
Histological evaluation test.
Graftskin: AVRs for Failing Morphology Samples
Tryspin Crystallized: One RS, Different USP Monographs
Monograph
Assay
Limit of trypsin
Crystallized Trypsin
Enzymatic with UV
detection
--
Aprotinin
Enzymatic with titration
--
Aprotinin Injection
Enzymatic with titration
--
--
Enzymatic with pH
indicator, visual
detection
Chymotrypsin
Chymotrypsin for
Ophthalmic Solution
--
Enzymatic with pH
indicator, visual
detection
Tryspin Crystallized RS

Intended uses of this RS:
– Crystallized Trypsin and Chymotrypsin monographs:
• Determine the suitability of the substrates and check the
adjustment of the spectrophotometer by performing the Assay
using USP Crystallized Trypsin Reference Standard.
– Aprotinin monograph:
• The determination of activity by the Assay is based on the
specific inhibition of trypsin
• Prepare a solution of USP Trypsin Crystallized RS containing
about 4300 USP Trypsin Units per mL

Tryspin Crystallized RS is a quantitative RS
23
USP Trypsin Assay Method
 Substrate: N-benzoyl-L-arginine ethyl ester hydrochloride (BAEE).
 Conditions: T = 25°C, pH = 7.6, A253nm, Light path = 1 cm
 Method: Continuous Spectrophotometric Rate Determination
BAEE + H2O
Trypsin
Na-Benzoyl-L-Arginine + Ethanol
 One USP Trypsin Unit is the activity causing a change in
absorbance of 0.003 per minute under the conditions specified in
the assay
Tryspin Crystallized RS: Release of a New Lot

Collaborative Study (5 Laboratories) established a RS with a value of
3369 USP Tryspin Units of Trypsin Crystallized per mg of material on
the dried basis.

Additional tests: limit of chymotrypsin , loss on drying, appearance,
electronic absorption , vapor Sorption

Replacement lot is suitable for use in its compendial applications.

Proposed label:
USP REFERENCE STANDARD
TRYPSIN CRYSTALLIZED 300 mg
CAUTION! Irritant, Allergen
Allow container to reach room temperature before opening. Determine the loss on drying on
a separate portion under vacuum at 60o for four hours. For USP applications, use a value of
3369 USP Trypsin Units per mg of material on the dried basis. Keep container tightly closed.
Hygroscopic. Protect from light and heat. Store in a freezer.
CAT NO. 1700002
USP ROCKVILLE, MD
LOT J0L380
Number of Biological RS in Active Portfolio: 123
Number of RS in Official Portfolio
Tissue (photomicrograph)
8%
Cell Line
2%
Other
2%
Peptide
28%
Total Biological RS: 123
Class
Carbohydrate
23%
Protein
10%
Glycosaminoglycan
18%
Enzyme
9%
Number of RS in
Official Portfolio
Peptide
35
Protein
13
Enzyme
11
Glycosaminoglycan
22
Carbohydrate
28
Tissue
(photomicrograph)
10
Cell Line
1
Other
3
RS Released Since July 2012
RS Name
RS Type
Lot Number
Status
Ballot
Gonadorelin
Hydrochloride
New
F0K033
Available
Q3, 2012
D-Glucuronic Acid
New
F0L340
Available
Q3, 2012
Dextran 40 Calibration
Replacement
G0L325
Available
Q3, 2012
Galactosamine
Hydrochloride
Replacement
G0L378
Available
Q3, 2012
Heparin Sodium
Identification
New Use
G0I116
Available
Q3 2012
27
RS Under Development – Next 6 Months
RS Name
RS Type
Lot Number
Status
Ballot
Replacement
J
Ballot Stage
Q4, 2012
Filgrastim
New
F
RSCEP in
Preparation
Q4, 2012
Heparin Sodium Molecular
Weight Calibrant
New
F
RSCEP in
Preparation
Q4, 2012
Bile Salts
Replacement
K
Collaborative
Testing
Q1 2013
Acarbose
New
F
Collaborative
Testing
Q1, 2013
Acarbose System
Suitability Mixture
New
F
Collaborative
Testing
Q1, 2013
Dermatan Sulfate
Replacement
G
Collaborative
Testing
Q1 2013
Trypsin Crystallized
28
RS Under Development – Next 6 Months
RS Type
Lot
Number
Status
Ballot
Replacement
G
Collaborative
Testing
Q1, 2013
Pepsin for Assay
New
F
Collaborative
Testing
Q1, 2013
Hemoglobin Protease
Substrate
New
F
Planning
Q1, 2013
Cosyntropin Acetate
New
F
Collaborative
Testing
Q2, 2013
Replacement
G
Planning
Q2, 2013
RS Name
Glucosamine
Hydrochloride
Dextran 10 Calibration
Low Molecular Weight
Heparin Molecular
Weight Calibrant
rAlbumin Human
Waiting for
<209> Draft
New
F
Q2, 2013
Collaborative
Testing
Replacement
G
Bulk
Procurement
Q2, 2013
29
RS Under Development – On the Horizon
RS Type
Lot
Number
Status
Ballot
Vasopressin
Replacement
H
Planning
Q2, 2013
Dextran 250 Calibration
Replacement
G
Bulk
Procurement
Q2, 2013
rHuman Interleukin 4
New
F
Collaborative
Testing
Q3, 2013
Q3, 2013
Q2, 2013
RS Name
Fetal Bovine Serum
New
F
Production
Fill
Underway
Monoclonal IgG
System Suitability
New
F
Bulk
Procurement
30
Thank you!