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

http://midwifethinking.files.wordpress.com/20

11/02/cb1.jpg

•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

Collection

Transport

Processing

Cryo-preservation

Storage

TX CT

Transport

Manufacturing protocol Stability program

•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

1.10

1.20

1.30

1.40

1.50

0 15 30 40

TN

C (

x1

0e

9)

Average TNC (x10e9)

0.00

1.00

2.00

3.00

4.00

5.00

6.00

0 15 30 40

CD

34 (

x10e6)

Average CD34 (x10e6)

0.0

5.0

10.0

15.0

20.0

25.0

0 15 30 40

CF

U (

x1

0e

5)

Average CFU (x10e5)

75.0

80.0

85.0

90.0

95.0

100.0

105.0

0 15 30 40

Via

bil

ity (

%)

Average Viability (%)

Time after DMSO addition (minutes)

Pre-freeze

•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) CFU Control Control Exposed Time

Post-Thaw % 2 min. 4 min. 6 min. 8 min. % TNC Viability Pool 1 76 75 (1) 75 (1) 69 (7) 63 (13) (% Loss) Pool 2 87 81 (6) 79 (8) 71 (16) 67 (20)

% CFU Pool 1 450,000 100 100 94 78 67

Recovery Pool 2 650,000 100 72 69 65 46

CFU Control Control Exposed Time

Post-Thaw % 1 min. x 5 2 min. x 5 3 min. x 5

% TNC Viability Pool 3 72 63 (9) 62 (10) 58 (14) (% Loss) Pool 4 74 72 (2) 70 (4) 65 (9)

CFU Recovery Pool 3 533,333 100 100 94 87

(%) Pool 4 300,000 100 100 100 89

% CD34+ Viability Pool 3 87 82 (5) 81 (6) 82 (5) (% Loss) Pool 4 65 64 (1) 76 (0) 61 (4)

•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)

•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 N Mean Median Std Dev Min Max

Acceptable

Ranges

TNC Recovery % 25 82 83 5 68 93 >80

TB Viability % 25 80 82 5 70 88 >70

Viable CD34 Recovery % 25 71 70 18 25 115 >60

Sterility Frequency Percentage

Negative Culture 25 100

Positive Culture 0 0

25 Unit Working Group

Parameter N Mean Median Std Dev Min Max

Acceptable

Ranges

TNC Recovery % 25 83 83 4 73 95 >80

TB Viability % 25 76 77 4 68 83 >70

Viable CD34 Recovery % 25 64 63 20 32 127 >60

Sterility Frequency Percentage

Negative Culture 25 100

Positive Culture 0 0

•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 Purpose Product characteristics

Visual inspection after thawing Determine integrity of container and closure

and label identity

Integrity and identity

Total nucleated cell (TNC)

count

Measuring total nucleated cell count in the CBU Potency

Viability Measuring viability of nucleated cells Potency

Viable CD34+ cell count Measuring viable CD34+ cell number in CBU Potency

Microbiology Detection of microbial contamination Integrity, purity, safety

•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