USP Reference Standards for Biologics
Tina S. Morris, Ph.D., Vice PresidentBiologics & Biotechnology USP-NF
User ForumJanuary 17th, 2013Istanbul, 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:
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Uses of USP Reference Standards
There are two main types of USP Reference Standards:
Standards with Quantitative ApplicationsAssays (for drug substances and for formulations)Limit tests (e.g., Impurity Reference Standards)
Standards with only Qualitative Applications Identification testsElution markersSystem Suitability tests
Development of Reference Standards
The Steps for the Development of a Reference Standard are:
1. Source MaterialDonation from pharmaceutical industryPurchase/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 AssignmentMass 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)
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Typical Candidate Evaluation Components
Candidates are Evaluated on the Following: Appearance (Visual or microscopic evaluation)
Identification Tests (more for first lot)– e.g., IR, NMR, MS, UV, Chromatography, test for counter-ion/salt, etc.
Indirect purity tests – 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)
Types of Reference Standard
Identification, peak identification, system suitability
Potency/Assay/Limit
Collaborative Study Design
Qualitative application Quantitative application
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
Bulk Candidate
Formulation/Lyophilisation
Standard Development
Content of fill Homogeneity Stability studies
Conventional Non-routine Quantity limited proposed RS presentation different
from sponsor (liquid vs. solid)
Pilot Fill
Definitive Fill
Collaborative study
Definitive Fill
Collaborative studyPre-characterization of bulk
Collaborative Study Design – USP rProtein A RS
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
Pharmacopeial Applications:
Monograph Identification Isoforms Limit of Triton X-100
General Chapter <131> Protein A Quality Attributes
SDS-PAGEIgG Binding*
IEF HPLC
* Used solely for system suitability
Collaborative Study – USP Enoxaparin Sodium for Bioassays RS
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
Pharmacopeial applications:
MonographAnti-factor IIa
activity Assay
Enoxaparin Sodium Spectrophotometric Spectrophotometric
Enoxaparin Sodium Injection
Spectrophotometric Spectrophotometric
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 re-freeze
– Lyophilized Filgrastim (new formulation)
• Eliminated potential RS shipping and storage issues
• New formulation was developed by monograph sponsor
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
Background
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 totalo 6 returned results
• Bioassayo 13 collaborator totalo 11 returned results
Filgrastim Collaborative Study
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 Colony-Stimulating Factor.
Potency Value
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 (4 storage conditions)⁰
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
Protein Determination
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 TrypsinEnzymatic with UV
detection--
Aprotinin Enzymatic with titration --
Aprotinin Injection Enzymatic with titration --
Chymotrypsin --Enzymatic with pH
indicator, visual detection
Chymotrypsin for Ophthalmic Solution
--
Enzymatic with pH indicator, visual
detection
Tryspin Crystallized RS
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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
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 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
Trypsin
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
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:
Tryspin Crystallized RS: Release of a New Lot
Number of Biological RS in Active Portfolio: 123
Peptide28%
Protein10%
Enzyme9%
Glycosaminoglycan18%
Carbohydrate23%
Tissue (photomicrograph)8%
Cell Line2%
Other2%
Number of RS in Official Portfolio
ClassNumber of RS in Official Portfolio
Peptide 35
Protein 13
Enzyme 11
Glycosaminoglycan 22
Carbohydrate 28
Tissue (photomicrograph)
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Cell Line 1
Other 3
Total Biological RS: 123
RS Released Since July 2012
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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
RS Under Development – Next 6 Months
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RS Name RS Type Lot Number Status Ballot
Trypsin Crystallized 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
RS Under Development – Next 6 Months
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RS Name RS Type Lot Number Status Ballot
Glucosamine Hydrochloride 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
Dextran 10 Calibration Replacement G Planning Q2, 2013
Low Molecular Weight Heparin Molecular Weight Calibrant
New F
Waiting for <209> Draft
Collaborative Testing
Q2, 2013
rAlbumin Human Replacement G Bulk Procurement Q2, 2013
RS Under Development – On the Horizon
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RS Name 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
Fetal Bovine Serum New FProduction
Fill Underway
Q3, 2013
Monoclonal IgG System Suitability New F Bulk
Procurement Q2, 2013
Thank you!
