PRODUCT QUALITY CHARACTERIZATION: RELEASE CRITERIA, POST THAW ASSESSMENT AND STABILITY TESTING Elina Linetsky, Ph.D. cGMP Facility, Cell Transplant Center Diabetes Research Institute University of Miami Miller School of Medicine May 2010
Cellular Therapy: Mode of Action • Replacement of deficient / defective tissues – Modified / Ex Vivo expanded bone marrow cells, mesenchymal stem cells • Tolerance induction protocols – Minimally manipulated selected CD34+ populations • Systemic / metabolic reconstitution and/or substitution – Replacement of metabolically inactive cells / tissues (e.g. islet cells) • Immunomodulation of the effector cell population – Natural Ex-Vivo expanded T-reg and NK cells; cellular-based vaccines)
Regulatory Background 361 Products 351 Products • PHSA, section 361 applies • PHSA, section 351 applies • Minimally manipulated, short • Manipulated so that biological & processing times functional characteristics are • Many use functionally “closed altered systems” • Include genetically modified, ex- • Intended for homologous use only vivo expanded & enzymaticaly • manipulated products, Exclude combination products • • Used for non-homologous Free of systemic effects & reconstitution independent of the metabolic • activity of living cells for its primary Combination products function, unless the product is for • Active systemically, or dependant – Autologous use on the metabolic function of living – Allogeneic use in first- or second- cells for their primary function degree blood relatives • cGMPs, cGTPs (21 CFR 210 & – Reproductive use 211, and 1271) & rules for • cGTPs apply (21 CFR 1271) biologics (21 CFR 610) apply
Regulatory Concerns • Control of the source material – Donor screening and testing (21 CFR 1271) – Source material testing for advantitious agents, retroviral species, sterility, Mycoplasma, purity, tumorigenicity (relevant guidance documents) • Control of the manufacturing process (establishment of in-process testing requirements and outcomes)
Lot Release Testing • Lot release testing a controlled process which evaluates the suitability of the product, defines how the product is tested and confirms the quality of the product according to the pre- established criteria, at distribution. – Identity – Safety (sterility and Mycoplasma testing) – Purity (Endotoxin level & other impurities) – Potency (dose, viability, composition, In Vitro & In Vivo biological activity, stability) – Effectiveness (reproducibility and consistency of the manufacturing process)
Lot Release Testing: Regulatory Requirements 21 CFR 210.165 (cGMPs) – For each product batch there shall be conformance to final specifications for identity & strength – Each product batch shall be tested for and be free of objectionable microorganisms – Method of sampling and number of units to be tested must be described in established SOPs (methods described in 21 CFR Part 610.12) – Acceptance criteria must be established to ensure each batch meets pre-established requirements – Sampling methods must be validated – Drug products failing to meet established criteria shall be rejected
Lot Release Testing 21 CFR 1271.265 (cGTPs) Prior to distribution • Each product must meet pre-established criteria to prevent communicable disease transmission • Manufacturing and tracking records must be reviewed; the fact that product release criteria are met must be verified; • Products that are contaminated, in quarantine, recovered from ineligible donor, or from donor for whom donor-eligibility determination is not completed, or those that deviate from procedures can not be distributed • Procedures, including those for product lot release criteria, must be established and followed.
Lot Release Testing Product Testing Methods Identity Morphological evaluation Staining / fluorescent staining methods, Flow Cytometry (differential staining) HLA ABO/Rh, Is this the correct type of cell? Genetic polymorphisms Safety • Sterility (including Gram stain) 21 CFR 610.12 (Gram stain: routine method); USP <71>; rapid detection methods* • Mycoplasma 21 CFR 610.30; PCR-based*; enzyme-based* • Adventitious Viruses (including In Vitro (cell lines) and In Vivo (animals) retroviruses) PCR-based methods (for retroviral elements) Is the product free from contaminating organisms; is it safe? *can be used during Phase I, equivalency in sensitivity & specificity to FDA-mandated methods have to be demonstrated by Phase III
Lot Release Testing Product Testing Methods Purity Endotoxin (Endosafe -PTS) for products intended for injection; assessment of extraneous materials (residual proteins, materials used in Is the product pyrogenic? What other type manufacture, etc) of cells, materials, proteins, antibiotics , etc. are present? Potency • Dose • Cell count (cell counter, hematology analyzer) • Viability • Flowcytometry, Trypan Blue, FDA/PI • Potency (assessment of functionality) • Tests of biological function / activity** (colony formation, cytokine release, activity suppression assays, stimulation index assay, etc) • Stability • Assurance that all release criteria are met after short- and long-term storage How many cells of the desired cell type are present? Are cells alive? Are they able to function as intended? **These should be developed by the manufacturer based on ability and capacity of the product to effect a given result
Lot Release Testing What types of products do we process?
Lot Release Testing Hematopoietic Cellular Products (361 products) CD34+ Products – Lot release testing • CD34+ cell dose: as determined by requesting physician • CD3+ log reduction: <1.0 x 10 6 /kg recipient BW or 2 log reduction • Viability (Trypan Blue; 7-AAD by Flowcytometry): 70% • Sterility (Gram stain): negative • LAL (Endosafe - PTS): ≤5 EU/kg body weight – Post-lot release testing • Sterility (aerobic, anaerobic, fungal organisms): no growth • CFU assay
Lot Release Testing CD34+ cells analysis • Assessed using FACS analysis • Single platform method / ISHAGE protocol • Standardized gating methods • Cell counts using sequential gating strategies • Cell viability using 7-AAD • Performed by CLIA accredited laboratory • Participation in the external accredited proficiency program with positive results
Lot Release Testing Sterility • Rapid detection methods such as BacT/Alert system are utilized for detection of aerobic / anaerobic / fungal organisms • 21 CFR Part 1271 does not specify sterility testing method • Positives are identified by sub-culture • Gram stain is performed using a routine laboratory method
Lot Release Testing Endotoxin • Can be detected by gel clot, endpoint, kinetetic assays and Endosafe -PTS (Charles River Laboratories) • Endosafe -PTS is a hand-held spectrophotometer with uses a test cartridge, preloaded with positive and negative controls • Different Endotoxin sensitivities • Quantitative results is in ~15 minutes • Reagents and system are FDA-approved
Lot Release Testing Colony Forming Unit Assay • CFU assay is commonly used to assess self-renewal capacity and to quantify committed hematopoietic progenitor cells (measure of functionality) • Results are not always co-related with engraftment • Difficult to standardize, although commercial kits are available (STEMCELL) • External proficiency testing programs are available (STEMCELL)
Lot Release Testing Colony Forming Unit Assay • HALO (HemoGenix ) is cell-based proliferation assay • Luminescence & instrument-based • More standardized compared to CFU assay • Changes are measured in concentration of intracellular ATP levels during cell proliferation / inhibition of proliferation. After incubation ATP is released from cells by lysis • The ATP acts as limiting substrate for a luciferin / luciferase reaction to produce bioluminescence • Bioluminescence is detected by plate luminometer • The readout is in Relative Luminescence Units are converted to ATP units by the use of standard dose response curve
Lot Release Testing Hematopoietic Cellular Products (351 products) CD34+ Products – Minimally manipulated – For non-homologous reconstitution: restoration of cell mass/function in patients with Type 2 diabetes – Lot release testing • CD34+ cell dose: all available • Viability (Trypan Blue; 7-AAD by Flow): 70% • Sterility (Gram stain): negative • LAL (Endosafe -PTS): 5 EU/kg body weight – Post-lot release testing • Sterility (aerobic, anaerobic, fungal organisms): no growth • CFU assay
Lot Release Testing Hematopoietic Cellular Products (351 products) Bone Marrow-Derived Stem Cell Products – Expanded Ex Vivo – Purpose: chronic wound healing – Lot release criteria • Cell dose: 2 x 10 6 cells or all available cells from individual harvest • Viability (Trypan Blue): 70% • Sterility (Gram stain): negative • LAL (Endosafe - PTS): ≤5 EU/kg body weight • Mycoplasma (PCR): negative – Post-lot release testing • Sterility (aerobic, anaerobic, fungal organisms): no growth
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