CRITERIA, POST THAW ASSESSMENT AND STABILITY TESTING Elina - - PowerPoint PPT Presentation

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

• PHSA, section 361 applies
• Minimally manipulated, short

processing times

• Many use functionally “closed

systems”

• Intended for homologous use only
• Exclude combination products
• Free of systemic effects &

independent of the metabolic activity of living cells for its primary function, unless the product is for

– Autologous use – Allogeneic use in first- or second- degree blood relatives – Reproductive use

• cGTPs apply (21 CFR 1271)

351 Products

• PHSA, section 351 applies
• Manipulated so that biological &

functional characteristics are altered

• Include genetically modified, ex-

vivo expanded & enzymaticaly manipulated products,

• Used for non-homologous

reconstitution

• Combination products
• Active systemically, or dependant
• n the metabolic function of living

cells for their primary function

• cGMPs, cGTPs (21 CFR 210 &

211, and 1271) & rules for 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

• utcomes)

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

• bjectionable 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 Is this the correct type of cell? Morphological evaluation Staining / fluorescent staining methods, Flow Cytometry (differential staining) HLA ABO/Rh, Genetic polymorphisms Safety

• Sterility (including Gram stain)
• Mycoplasma
• Adventitious Viruses (including

retroviruses) Is the product free from contaminating

• rganisms; is it safe?

21 CFR 610.12 (Gram stain: routine method); USP <71>; rapid detection methods* 21 CFR 610.30; PCR-based*; enzyme-based* In Vitro (cell lines) and In Vivo (animals) PCR-based methods (for retroviral elements)

*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 Is the product pyrogenic? What other type

• f cells, materials, proteins, antibiotics ,
• etc. are present?

Endotoxin (Endosafe -PTS) for products intended for injection; assessment of extraneous materials (residual proteins, materials used in manufacture, etc) Potency

• Dose
• Viability
• Potency (assessment of functionality)
• Stability

How many cells of the desired cell type are present? Are cells alive? Are they able to function as intended?

• Cell count (cell counter, hematology analyzer)
• Flowcytometry, Trypan Blue, FDA/PI
• Tests of biological function / activity**

(colony formation, cytokine release, activity suppression assays, stimulation index assay, etc)

• Assurance that all release criteria are met after

short- and long-term storage

**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 106/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 106 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

Lot Release Testing

Mycoplasma

• Rapid detection methods such as PCR or enzyme

immunoassays are utilized for rapid detection of Mycoplasma species in cell culture-derived biologicals

• VenorGem

(Sigma) detection kit or comparable reagents can be used

• Sample read-out is using 1.5% Agarose gel &

appropriate size markers

• This method must be demonstrated to

be of equal sensitivity and specificity as that described in 21 CFR 610.30

Lot Release Testing

Hematopoietic Cellular Products (351 products) Mesenchymal Stem Cells

– Expanded Ex Vivo, through long-term culture – Purpose: repair of cardiac function and output, following myocardial infarction

Test Lot release Post lot release Cell dose N/A 20 x 106 or 200 x 106 Viability 70% N/A Safety: Sterility Gram Stain Mycoplasma N/A Negative Negative No growth N/A N/A Purity (Endotoxin) (Flow) 5 EU/kg N/A N/A CD105+/CD45+ analysis Potency CFU N/A Functional analysis

Lot Release Testing

Therapeutic Cellular Products (351 products)

Natural Expanded T-Regulatory Cells

– Isolated by immunomagnetic selection & expanded Ex Vivo – Purpose: investigation of immunomodulatory effects of T- regulatory cell population in liver transplant recipients

Test Prior to Cryopreservation Lot release (post-thaw / @ distribution) Post lot release Cell dose All cells available 0.4 – 1.2 x 106 cells/kg N/A Viability 80% 70% N/A Purity (Endotoxin) (Flow) 5 EU/kg 70% 5 EU/kg N/A N/A 70% Safety Sterility Gram Stain Mycoplasma No growth N/A Negative N/A Negative N/A Negative Potency N/A N/A In development

Lot Release Testing

Therapeutic Cellular Products (351 products) Allogeneic Islet Cells

– Extensive manipulation during isolation, purification and culture – For purpose of replacement of metabolically active tissue destroyed by autoimmune process

Test Lot release criteria Post-lot release Identity Visual inspection using DTZ N/A Cell Dose 5,000 IEQ/kg body weight N/A Viability 70% N/A Safety Sterility Gram stain N/A Negative No growth N/A Purity (Endotoxin) Islet Purity 5 EU/kg body weight 30% N/A N/A Potency: SI Cell compos.& fractional viab. 1 N/A 1 Cell assessment

Lot Release Testing

Therapeutic Cellular Products (351 products)

Cellular-Based Vaccine Products – Expanded Ex Vivo – Purpose: immuno-surveillance via stimulation

• f immune response by direct Ag presentation
• Cell dose: 50 x 106 cells / 0.5 ml
• Sterility (aerobic, anaerobic, fungal organisms): no growth
• LAL: <10 EU/ml
• Mycoplasma (21 CFR 610.30): negative
• Expression of HLA & B7.1 (FACS): 65% cells
• Expression of gp96 Ig (ELISA): >60 ng/106 cells in 24 hrs

– Follow-up testing (every 6 months)

• Expression of HLA & B7.1 (FACS analysis): 65% cells
• Expression of gp96 Ig (ELISA): >60 ng/106 cells in 24 hrs

Product Stability

Stability limitations of cells and tissues are

• ften a function of suboptimal bio-

preservation of source material, intermediates, and finished cell/tissue products

• Aby J. Mathew, PhD. Stability Testing and Optimization -Considerations for Cell Therapy
• Products. ISCT Webinar, 2009.

Product Stability

• Product release testing

– Evaluates the cell therapy product upon completion of manufacturing – Is product within acceptable limits for safety, purity, identity, potency?

• Final cell therapy products should remain within

the same specifications as indicated

– At the time of product distribution (lot release), i.e. specs should remain the same between product distribution (lot release) and administration

Product Stability

• Stability testing

– Establishes the time limit within which product remains within acceptable limits for clinical use – Should reflect duration and conditions of product storage and transport – Ensures the product retains consistency in safety, purity, identity & potency through to expiration date/time, i.e.

• Throughout manufacture & cryostorage
• From thawing / washing to distribution
• From distribution to the expiration date / time and/or

administration

Product Stability

Isolation of CD4+ cells

In-process testing

• Sterility : no growth
• Cell count
• Viability: 80%
• Cell Purity: 80%

Isolation of CD4+CD25+ cells Culture/expansion/cryo

In-process testing (prior to cryo)

• Sterility: no growth
• Cell count
• Viability:

80%

• Cell Purity: 70%
• Purity (Endotoxin): 5 EU/kg
• Mycoplasma: negative

Thawing / washing, lot release, preparation for administration

Lot release

• Cell dose
• Viability:

70%

• Purity (Endotoxin): 5 EU/kg
• Gram stain: negative

Post lot release

• Sterility: no growth
• Cell purity (by FLOW): 70%
• Potency: functional assay

Product Stability

• Stability studies are necessary during all phases
• f product development
• Early phases of clinical trials

– Should demonstrate that the product is stable during holding & shipping, & for the period required by the study, i.e. cryopreservation

• Phase II of product development

– Expiration dating

• Phase III of product development

– Validation studies supporting storage conditions, expiration date/time, & shipping conditions including conditions of stress

Product Stability

• IND submission should include

– Results, protocols describing test methodology & test conditions utilized – Protocols should include testing for identity, safety, purity, potency & integrity – Testing conditions should be based on conditions during product storage, at distribution, & during shipment

• In-process testing should be based on conditions for product

storage

• Final product testing should demonstrate product retaining it’s

identity, safety, purity & potency

– Testing frequency (sampling test points)

References

• Code of federal regulations. 21 CFR Parts 210 & 211, 312, 610, 1271.

Washington, DC: US Government Printing Office, 2008 (revised annually).

• United States Pharmacopeia. General chapter <71> sterility tests. USP
• 30. Rockville, MD: USP Convention, 2007.
• ICH Q5C: Quality of Biotechnological Products: Stability Testing of

Biotechnological/Biological Products (1996) http://www.fda.gov/downloads/RegulatoryInformation/Guidances/UCM1 29109.pdf

• ICH Guideline Q1A(R): Stability Testing of New Drugs and Products

(revised, 2003) www.fda.gov/RegulatoryInformation/Guidances/ucm128179.htm

• ICH Guideline Q1E: Evaluation of Stability Data (2004)

www.fda.gov/RegulatoryInformation/Guidances/ucm128092.htm

References

• Points to Consider in the Manufacture and Testing of Monoclonal

Antibody Products for Human Use http://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceCompl ianceRegulatoryInformation/OtherRecommendationsforManufacturers/ UCM153182.pdf