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USP Reference Standards for Biologics

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Presentation on theme: "USP Reference Standards for Biologics"— Presentation transcript:

1 USP Reference Standards for Biologics
Tina S. Morris, Ph.D., Vice President Biologics & Biotechnology USP-NF User Forum January 17th, 2013 Istanbul, Turkey

2 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

3 USP Reference Standards
The reference materials relate directly to methods in the USP publications:

4 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 4

5 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)

6 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) 6 6

7 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 Value assignment (mass-balance, bioassay, etc) of the RS candidate Potency RS calibrated relative to the current International Standard Establish identity of candidate material Evaluate chemical identity by compendial and non-compendial techniques No value assigned to the RS candidate

8 Formulation/Lyophilisation
Standard Development Bulk Candidate Non-routine Quantity limited proposed RS presentation different from sponsor (liquid vs. solid) Conventional Collaborative study Pre-characterization of bulk Formulation/Lyophilisation Definitive Fill Pilot Fill Definitive Fill Content of fill Homogeneity Stability studies Collaborative study

9 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

10 Collaborative Study – USP Enoxaparin Sodium for Bioassays RS
Pharmacopeial applications: Monograph Anti-factor IIa activity Assay Enoxaparin Sodium Spectrophotometric Enoxaparin Sodium Injection 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

11 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

12 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

13 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

14 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) 8 collaborators total 6 returned results 13 collaborator total 11 returned results

15 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 Colony- Stimulating Factor.

16 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 = mg per ampoule Standard Deviation = 0.02 %RSD = 2.38 The label text will claim 0.98 mg protein per ampoule

17 Label for Lot F0L526 of USP Filgrastim RS

18 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

19 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

20 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.

21 Graftskin: AVRs for Failing Morphology Samples

22 Tryspin Crystallized: One RS, Different USP Monographs
Assay Limit of trypsin Crystallized Trypsin Enzymatic with UV detection -- Aprotinin Enzymatic with titration Aprotinin Injection Chymotrypsin Enzymatic with pH indicator, visual detection Chymotrypsin for Ophthalmic Solution

23 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

24 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 per minute under the conditions specified in the assay Trypsin

25 Tryspin Crystallized RS: Release of a New Lot
Collaborative Study (5 Laboratories) established a RS with a value of 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:

26 Number of Biological RS in Active Portfolio: 123
Total Biological RS: 123 Class Number of RS in Official Portfolio Peptide 35 Protein 13 Enzyme 11 Glycosaminoglycan 22 Carbohydrate 28 Tissue (photomicrograph) 10 Cell Line 1 Other 3

27 RS Released Since July 2012 RS Name RS Type Lot Number Status Ballot
Gonadorelin Hydrochloride New F0K033 Available Q3, 2012 D-Glucuronic Acid F0L340 Dextran 40 Calibration Replacement G0L325 Galactosamine Hydrochloride G0L378 Heparin Sodium Identification New Use G0I116 Q3 2012

28 RS Under Development – Next 6 Months
RS Name RS Type Lot Number Status Ballot Trypsin Crystallized Replacement J Ballot Stage Q4, 2012 Filgrastim New F RSCEP in Preparation Heparin Sodium Molecular Weight Calibrant Bile Salts K Collaborative Testing Q1 2013 Acarbose Q1, 2013 Acarbose System Suitability Mixture Dermatan Sulfate G

29 RS Under Development – Next 6 Months
RS Name RS Type Lot Number Status Ballot Glucosamine Hydrochloride Replacement G Collaborative Testing Q1, 2013 Pepsin for Assay New F Hemoglobin Protease Substrate Planning Cosyntropin Acetate Q2, 2013 Dextran 10 Calibration Low Molecular Weight Heparin Molecular Weight Calibrant Waiting for <209> Draft rAlbumin Human Bulk Procurement

30 RS Under Development – On the Horizon
RS Name RS Type Lot Number Status Ballot Vasopressin Replacement H Planning Q2, 2013 Dextran 250 Calibration G Bulk Procurement rHuman Interleukin 4 New F Collaborative Testing Q3, 2013 Fetal Bovine Serum Production Fill Underway Monoclonal IgG System Suitability

31 Thank you! As you can see from the world map on the outside of USP headquarters building, it is a global pharmacopeia based in Rockville, Maryland. No borders.

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