Presentation on theme: "Director, Biologics and Biotechnology"— Presentation transcript:
1 Director, Biologics and Biotechnology 6th Annual Science and Standards Symposium January 16, 2013 Istanbul Quality Attributes for Biological Medicines and USP StandardsFouad Atouf, Ph.D.Director, Biologics and Biotechnology
2 Biological Medicines: Opportunities and Challenges Biological Medicines: Scope of ProductsBlood and Blood ProductsCell, Gene, Tissue TherapiesTherapeutic Proteins, Recombinant and Naturally-derivedVaccinesMulti-components (e.g. raw materials) manufacturing:Potential supply chain issues (e.g. animal derived materials)Testing of quality of components before manufacturing beginsComplex manufacturing processes with impact on:Quality attributes of finished productsChallenging regulatory approval pathwaysControl of the quality, safety and efficacy of biologicals is difficult, despite technological advancesOrthogonal methods needed to address a single quality aspectHigher order structures, often addressed by a biological assay
4 Biotechnology Products, Subset of Biologicals, cont’d. Heterogeneity and other factors with impact on quality attributesProduct-related substances (molecular variants, aggregates, deamidation, oxidation, glycosylation, etc…)Immunogenic potential: difficult to predict -occurrence and effectsProcess 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)
5 Biotech Products – Quality Testing and Monographs IdentificationRetention Time from chromatographic assayPeptide MappingN-Terminal SequencingPurityHPLC (Reverse Phase)Limit on High Molecular Weight Species (Size Exclusion)Glycoforms (Isoelectric focusing)PotencyChromatographic when possibleBioassay-BioidentityTo address secondary and tertiary structuresCellular preferred over animalMonographs also cover sterility, and other general requirements such as labeling, packaging and storage
6 Official USP Biologics Monographs by Product Class Number of monographspeptide47enzyme12complex extract11carbohydrateglycosaminoglycan9other5Tissue product6IgG/serumBlood component/proteinVaccine3
7 Peptide/Small Protein Drug Substance Monographs SomatropinInsulin HumanGlucagonFilgrastimIdentification - HPLCXIdentification - Peptide MappingAssay - HPLCImpurities – related proteins: HPLC (Assay)Impurities – Charge variants, IEFImpurities – Limit of HMW proteins: SECSpecific Tests: bioidentity, <85>, <61>/<62>, <731>no bioidentity test for DSno <731>
8 FilgrastimDrug substance monograph published in PF 36(5), becoming official in USPFilgrastim is the recombinant form of human granulocyte colony-stimulating factor (G-CSF), marketed under the brand name Neupogen™C845H1339N223O243S9The 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.
9 Filgrastim: G-CSF? Granulocyte colony stimulating factor (G-CSF) 18-20 kDaHematopoetic 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 chemotherapy174 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.coli expression system is called FilgrastimProtein Data Bank data (PDB: 1RHG)Hill, C.P., Osslund, T.D., Eisenberg, D. The structure of granulocyte-colony-stimulating factor and its relationshipto other growth factors. Proc.Natl.Acad.Sci.USA v90 pp , 1993
10 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.”IdentityAssay (Potency)ImpuritiesAdditional RequirementsPackaging and Storage; LabelingReference Standards
11 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 detectionAcceptance criteria: next slide
12 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%.Previously, the acceptance criteria was based on the relative retention time relative to peak 8, and the criterion was greater than or equal to 2.0 min.NOTE: 8 major peaks will be defined in the USP Filgrastim RS Data Sheet.
13 Pancreatin – Drug Substance Monograph Definition:Pancreatin is a substance containing enzymes, principally amylase, lipase, and protease, obtained from the pancreas of the hog, Sus scrofa Linné var. domesticus Gray (Fam. Suidae) or of the ox, Bos taurus Linné (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 AssaysAmylase, Lipase, ProteaseFat Content TestGeneral Requirements: Labeling, Packaging and StorageIdentification will be addressed in revisionProducts must meet enzymatic assays (e.g. Lipase assay)Inclusion of identification test (HPLC-based)
14 Potency Determination USP Pancreatin Monograph, Assay for lipase activity“One USP Unit of lipase activity is contained in theamount of pancreatin that liberates 1.0 microequivalentof acid per minute at a pH of 9.0 and 37° underthe conditions of the Assay”
15 Pancreatin Lipase Assay The lipolysis reaction catalyzed by pancreatic lipaseSubstrate:TriglyceridesProducts:Free fatty acids(FFA)LipasepH > pKaIonized FFATitration* by Na+OH-*Principle of the USP Pancrelipase assay Slide created by Frederic Carriere
16 In Vitro / In Vivo Correlations: The Case Study of Pancreatic Lipase Titrimetric Lipase Assay by the pH-stat TechniqueAdapted from Frederic CarrierepH0.1NNaOHWater at 37°CStirrerControl Unit / pH end point / NaOH deliveryµmoles NaOH= µmoles FFA = UnitsViLipase3. Release of FFA upon lipolysis and recording of FFA titration by NaOH at constant pHTime (min)2. LipaseadditionEmulsification ofolive oil substrate + Buffer+ Bile Salts
17 In Vitro / In Vivo Correlations: The Case Study of Pancreatic Lipase Pancreatic Lipase Specific Activities on Various SubstratesCarrière et al. Gastroenterology (2000) 119:949–960LipaseSubstrateSpecific activity(µmole FFA.min-1.mg-1)Theoretical duration for complete lipolysis of meal TAG *Human pancreatic lipaseOlive oil(USP assay)30006 secTributyrin(synthetic short chain TAG)80002 secMixed solid-liquid meal TAG(Hamburger, Fries, Butter…)1520 minNaturalsubstrateSyntheticPhysiologicalOnly 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
18 S 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)Tributyrin(synthetic short chain TAG,fine emulsion under mechanical stirring)Less emulsification,less substrate vs. enzyme,low enzyme turnoverSESELarge excess of substrate,high enzyme turnover
19 Characterization of Pancreatin Advantages of RP-HPLC / ESI-MS SeparationOne-dimensional separation, automatedWide range of polarity by selection of stationary phase chemistry & mobile phase / gradientDetection/QuantificationUniversal UV (210 nm), MS-detectionSufficient dynamic range, linear, reproducibleUse of external standardsIdentificationMS-coupling & fractionation for other techniques of identification (PMF, MS-MS, N-terminal sequencing), covers all ionizable species
20 Fetal Bovine Serum (FBS)- USP FBS Standard RequirementsOsmolality: mOsm/KgTotal Protein: mg/mLpH:Endotoxin: Not more than 10 units/mLHemoglobin level: Not more than 30 mg/dLIdentification: Radial Immunodiffusion (RID): species ID, IgG levelsFunctionality Assays (Growth Curve and Clonal Assay)Associated Reference Standard (RS), under developmentLiquid frozen, 10 mLCollaborative 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)
21 How the FBS Standard is Used: Growth Curve Challenges: Cell Line, Cell Density, Cell Counting, Days in CultureThree 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 moreUse the selected cell density to assess the test FBS side by side with the reference standard FBSDoubling time is estimated using a growth curve that is linear over three or more time points.Acceptance Criteria: R2≥ 0.98Doubling time of test sample should be not less than 90% of doubling time of RS
22 SummaryA 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.