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

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Presentation on theme: "USP Reference Standards for Biologics Tina S. Morris, Ph.D., Vice President Biologics & Biotechnology USP-NF User Forum January 17 th, 2013 Istanbul, Turkey."— Presentation transcript:

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

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 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)

7 Types of Reference Standard Identification, peak identification, system suitability Potency/Assay/Limit Collaborative Study Design Qualitative applicationQuantitative 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

8 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 study Pre-characterization of bulk

9 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 IdentificationIsoforms Limit of Triton X-100 General Chapter Protein A Quality Attributes SDS-PAGE IgG Binding* IEFHPLC * Used solely for system suitability

10 Collaborative Study – USP Enoxaparin Sodium for Bioassays R S 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 1 H, 13 C and HSQC NMR spectroscopy –Anti-factor IIa activity –Anti-factor Xa potency (Assay) –Stability studies Pharmacopeial applications: Monograph Anti-factor IIa activity Assay Enoxaparin SodiumSpectrophotometric Enoxaparin Sodium Injection Spectrophotometric

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 Cys 18 –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 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

14 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 Filgrastim Collaborative Study

15 The following statement will be included on the USP Certificate for Filgrastim Lot F0L526: Each ampoule contains 8.5 x 10 7 IU when assayed against the WHO 2 nd International Standard for Granulocyte Colony- Stimulating Factor. Potency Value

16 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) 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 Protein Determination

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 products 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 Monograph AssayLimit of trypsin Crystallized Trypsin Enzymatic with UV detection -- AprotininEnzymatic with titration-- Aprotinin InjectionEnzymatic with titration-- Chymotrypsin-- Enzymatic with pH indicator, visual detection Chymotrypsin for Ophthalmic Solution -- Enzymatic with pH indicator, visual detection

23 Tryspin Crystallized RS 23 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 N -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 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

26 Number of Biological RS in Active Portfolio: 123 Total Biological RS: 123

27 RS Released Since July RS NameRS TypeLot NumberStatusBallot Gonadorelin Hydrochloride NewF0K033AvailableQ3, 2012 D-Glucuronic AcidNewF0L340AvailableQ3, 2012 Dextran 40 CalibrationReplacementG0L325AvailableQ3, 2012 Galactosamine Hydrochloride ReplacementG0L378AvailableQ3, 2012 Heparin Sodium Identification New UseG0I116AvailableQ3 2012

28 RS Under Development – Next 6 Months 28 RS NameRS TypeLot NumberStatusBallot Trypsin CrystallizedReplacementJBallot StageQ4, 2012 FilgrastimNewF RSCEP in Preparation Q4, 2012 Heparin Sodium Molecular Weight Calibrant NewF RSCEP in Preparation Q4, 2012 Bile SaltsReplacementK Collaborative Testing Q AcarboseNewF Collaborative Testing Q1, 2013 Acarbose System Suitability Mixture NewF Collaborative Testing Q1, 2013 Dermatan SulfateReplacementG Collaborative Testing Q1 2013

29 RS Under Development – Next 6 Months 29 RS NameRS Type Lot Number StatusBallot Glucosamine Hydrochloride ReplacementG Collaborative Testing Q1, 2013 Pepsin for AssayNewF Collaborative Testing Q1, 2013 Hemoglobin Protease Substrate NewFPlanningQ1, 2013 Cosyntropin AcetateNewF Collaborative Testing Q2, 2013 Dextran 10 CalibrationReplacementGPlanningQ2, 2013 Low Molecular Weight Heparin Molecular Weight Calibrant NewF Waiting for Draft Collaborative Testing Q2, 2013 rAlbumin HumanReplacementG Bulk Procurement Q2, 2013

30 RS Under Development – On the Horizon 30 RS NameRS Type Lot Number StatusBallot VasopressinReplacementHPlanningQ2, 2013 Dextran 250 CalibrationReplacementG Bulk Procurement Q2, 2013 rHuman Interleukin 4NewF Collaborative Testing Q3, 2013 Fetal Bovine SerumNewF Production Fill Underway Q3, 2013 Monoclonal IgG System Suitability NewF Bulk Procurement Q2, 2013

31 Thank you!

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