Aleksandar M. Babic MD, PhD

Aleksandar M. Babic MD, PhD •Cord Blood as a source of Hematopoietic Stem and Progenitor Cells for Transplantation •Basic Principles of Umbilical Co...
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Aleksandar M. Babic MD, PhD

•Cord Blood as a source of Hematopoietic Stem and Progenitor Cells for Transplantation •Basic Principles of Umbilical Cord Blood Manufacturing •Stability Program for Umbilical Cord Blood products •Stability Testing for Umbilical Cord Blood products •Development of Expiration Dating Protocol

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•Most recently identified source of HPCs •The first human sibling antigen-matched UCB transplantation performed in 1988 in France for Fanconi anemia: •26 years later, continued donor reconstitution •Over 600,000 UCB units stored for transplantation worldwide •Over 30,000 UCBHT performed

•Cellular •Hematopoietic stem and progenitor cells (HSPC) •Mature myeloid and lymphoid leukocytes •Nucleated and mature red blood cells •Platelets •Unrestricted somatic stem cells (USSCs) •Mesenchymal stroma cells (MSCs) •Endothelial colony-forming cells (ECFCs) •Others?

•Acellular •Plasma

• It should produce units (lots) containing defined amounts of active ingredient and contaminants • Maximize the mononuclear cell content • Minimize the content of red blood cells and other cells unrelated to therepeutic effect • Concentrate UCB units: • To minimize storage space for frozen products • To optimize freezing by allowing uniform freezing and rapid thawing • Processing can be performed using: • Manual methods: a. Hetastarch b. PrepaCyte-CB • Automated methods: a. Sepax b. AXP-AutoXpress

Required to ensure that the product maintains its characteristics from end of manufacturing to patient administration Includes tests to evaluate product safety, identity, purity, and potency. For each test conducted, describe: Test method and basis for use as a stability-indicating assay Sampling time points

Initial value, intermediate time point(s), and end of stability study Evaluate packaging, storage/transport conditions

• • •

• • • •

Testing program designed to assess the stability characteristics of HPC, Cord Blood is required (21 CFR 211.166(a)). The results of stability testing must be used in determining appropriate storage conditions and expiration dates (21 CFR 211.166(a)). Sample size and test intervals based on statistical criteria for each attribute examined to assure valid estimates of stability (21 CFR 211.166(a)(1)); Storage conditions for HPC, Cord Blood and other product samples retained for testing (21 CFR 211.166(a)(2)); Reliable, meaningful, and specific test methods must be used (21 CFR 211.166(a)(3)); Testing of the HPC, Cord Blood in the same container-closure system as that in which the HPC, Cord Blood is marketed An adequate number of HPC, Cord Blood units must be tested to determine an appropriate expiration date and a record of such data must be maintained (21 CFR 211.166(b)). •

U.S. Pharmacopeia: Stability of patient-specific therapy should be established using material from multiple donors and at least 3 lots

Manufacturing protocol

Stability program Cryopreservation

Transport

CT Collection

Processing

Storage

TX Transport

•Part of product characterization

•Essential for determination of acceptable storage time interval for UCB (e.g. expiry date) •Stability testing conditions should reflect intended storage, packaging, and transport conditions •It should assess: •Identity confirmation •Safety •Purity •Potency •It covers the period from completed post-processing evaluation to product administration

Prior to export From Integrally attached segment: Required: Identity: Confirmatory HLA typing Sometimes requested: Qualitative: Total Viability and Total Colony Forming Unit activity Poor correlation between total viability and CFU activity from segments compared with post-thaw data

On receipt Analysis of temperature data logger to monitor temperature range during shipment (…) Identity and Safety: Label verification Visual inspection of container and closure

Post-thaw Identity: ABO and Rh typing Safety: Aerobic, anaerobic and fungal culture Purity and Potency: TNC, Viability, Viable CD34 cell count, CFU assay

•Critical Raw materials •Process intermediates •Reference standards •Final product •Clinical outcomes

•Freezing Solution •For each lot:

•Conformance testing •Color check •Turbidity •Presence of macroscopic particulate matter •Volume •sterility, •endotoxin, •specific gravity •pH •Osmolarity.

•Performance testing •3 UCB units will be processed. •Full characterization (TNC, viability, CD34+, CFU) will be performed post-processing and post-thaw. • Evaluate post-thaw recovery data for potency characteristics against the stability control group data. •Compare results of new lot to current lot

•Critical parameters: •Time to initiation of cryopreservation-DMSO toxicity •Rate of freezing Average TNC (x10e9)

Average CD34 (x10e6)

CD34 (x10e6)

TNC (x10e9)

1.50 1.40 1.30 1.20

1.10 0

15

30

40

6.00 5.00 4.00 3.00 2.00 1.00 0.00

0

15

30

40

Pre-freeze Average CFU (x10e5)

Average Viability (%)

20.0

Viability (%)

CFU (x10e5)

25.0

15.0 10.0 5.0 0.0 0

15

30

40

105.0 100.0 95.0 90.0 85.0 80.0 75.0 0

15

30

Time after DMSO addition (minutes)

40

•Brief exposures of a frozen blood component to ambient air during post-freeze processing, storage, shipping and transfer between cryogenic containers may affect viability of cells. •NYBC study: UCB collections matched for ABO group, were combined into two separate pools (Pool 1 and Pool 2), each containing >10 9 total viable nucleated cells. •Units were retrieved from LN2 and kept in ambient air for either 2, 4, 6 or 8 minutes (21°C, 60% humidity) and then put back into LN2 (Table 1)

% TNC Viability (% Loss)

Pool 1 Pool 2

% CFU Recovery

Pool 1 Pool 2

CFU Control

Control

Exposed Time

Post-Thaw

% 76 87

2 min. 75 (1) 81 (6)

4 min. 75 (1) 79 (8)

6 min. 69 (7) 71 (16)

8 min. 63 (13) 67 (20)

450,000 650,000

100 100

100 72

94 69

78 65

67 46

•Units from two additional pools (Pool 3 and Pool 4) were retrieved from LN2 and kept for either 1, 2 or 3 minutes, at room temperature (21°C) and then returned to LN2 for 2 minutes. •The same units were again brought to air for the same time period and back to LN2 for 2 minutes for a total of five identical cycles. (Table 2) % TNC Viability (% Loss)

Pool 3 Pool 4

CFU Recovery (%)

Pool 3 Pool 4

% CD34+ Viability (% Loss)

Pool 3 Pool 4

CFU Control

Control

Exposed Time

Post-Thaw

% 72 74

1 min. x 5 63 (9) 72 (2)

2 min. x 5 62 (10) 70 (4)

3 min. x 5 58 (14) 65 (9)

533,333 300,000

100 100

100 100

94 100

87 89

87 65

82 (5) 64 (1)

81 (6) 76 (0)

82 (5) 61 (4)

•Viability loss was demonstrated in several of TWE modes •Viability losses upon repeated exposure are additive •Units that may be used for transplantation should be monitored for TWE and all incidents recorded •Documentation of TWE should include minimally the time and the temperature of each exposure.

Transient Warming Events and Cell Viability of Placental/Umbilical Cord Blood (“PCB”) Ludy Dobrila, Ph.D., Phil Coelho, Pablo Rubinstein, M.D. New York Blood Center & ThermoGenesis Corp. Presented at ISHAGE meeting in 06/2001

•External reference standard available for frozen Umbilical Cord Blood processing •Commercially available Frozen Cord Blood Survey (TNC, CFU, CD34 count) •Measured against peer group • Internal reference standard: •Control data sets of products frozen for short time periods (48 hrs, 6 months and 1 year) and evaluation of their post-thaw identity, safety (sterility), purity and potency •Establishment of acceptable cutoffs for post-thaw recoveries 25 Unit Control Group Parameter TNC Recovery % TB Viability % Viable CD34 Recovery % Sterility Negative Culture Positive Culture

N Mean Median 25 82 83 25 80 82 25 71 70 Frequency Percentage 25 100 0 0

Std Dev 5 5 18

Min 68 70 25

Max 93 88 115

Acceptable Ranges >80 >70 >60

25 Unit Working Group Parameter

N

Mean

Median

Std Dev

Min

Max

TNC Recovery %

25

83

83

4

73

95

TB Viability %

25

76

77

4

68

83

25

64

63

20

32

127

Viable CD34 Recovery %

Sterility Negative Culture Positive Culture

Frequency Percentage 25 100 0 0

Acceptable Ranges >80 >70 >60

•If the final formulation of product is performed at a clinical site, stability studies on the final formulation should examine conditions to retain optimal product performance at the time of administration. •Two methods for preparation of UCB product for infusion: •Thaw and infuse: not used •Wash: •The original method designed to remove DMSO, lysed red blood cells and stroma. •Additional product manipulation and potential for contaminantion •May yield a significant cell loss depending on the lab and methodology •Published evidence (TRANSFUSION 2005;45:1909-1916.) suggests potential for up to 20% TNC loss due to washing and additional cell loss due to delay of infusion after washing. •Albumin-Dextran dilution without centrifugation •Faster •Improved TNC recovery •Less product manipulation compared to wash procedure •Should be performed in the lab •Not suitable for red blood cell replete units •Although considered safe for red blood cell depleted units, the rates of infusion recations are higher than in washed units

•Expiration date is used to determine shelf life after which the product may not function as intended due to degradation of active ingredient •The following criteria can be used in assessment of UCB shelf life: Test Visual inspection after thawing Total nucleated cell (TNC) count Viability Viable CD34+ cell count Microbiology

Purpose Determine integrity of container and closure and label identity Measuring total nucleated cell count in the CBU

Product characteristics Integrity and identity

Measuring viability of nucleated cells Measuring viable CD34+ cell number in CBU Detection of microbial contamination

Potency Potency Integrity, purity, safety

Potency

•Historical data from short term cryopreserved control sets of products should be used to define acceptance criteria. At SLCBB: •No cracks, leaks, breaks, clear legible labeling, TNC recovery>80%, Viability>70%, % Viable CD34+ recovery >60% •Based on the performance of control sets of different ages the appropriate expiration date should be proposed and verified using data obtained after thaw of working product group using descriptive statistics •Expiration date should be verified using clinical outcome data generated during clinical outcome stability analysis •Processing method specific