Cord Blood Abnormal Freezing Curves: Potential

Impact on Quality

Salem Akel , PhD Scientific/Laboratory Director

AABB Annual Meeting – San Diego © 2011

Objectives • Compare current CB freezing methods (controlled rate freezing and the mechanical freezing) and their impact on CB quality (unit post-thaw findings)

• Evaluate Validity and use of product-attached segment, as sample source, to confirm frozen CB quality

• Examples of common and uncommon abnormalities of freezing curves: our evaluation for degree of injuries associated with such abnormalities

• Our investigational model of product quality for units with various abnormal freezing curves

CB production in SLCBB• Processing methods :Manual Plasma reduction/RBCs depletion using Prepacyte reagent or HES (old and backup)

• Freezing solution 10% DMSO/Dex

• Product cryobags are overwrapped before freezing

• Freezing using CRF system (primary method) and mechanical freezing (backup method)

• Thawing: Alb/Dex 1:1 reconstitution method

• Confirmatory testing before unit export include identity, viability (TB) and potency (CFU-C) using unit integral segment

Control Rate Gradual Freezing (CRF) CRF system utilizes a freezing chamber and computer-controlled freezing cycle with an established sequence of steps where timing, target temperatures and cooling rates are defined

….Product temp. is monitored by placing a monitoring probe on the unit surface within the cassette

….CRF graph shows, in parallel, target, chamber and product (sample) freezing curves

…..The current practice for the freezing run to include up to 6 products individually tagged with separate probes

ISlow cooling (-1 OC/min) + 20 (chamber) to -8 OC (sample) ( almost -15 OC, chamber)

(30-40 min)

II : Heat of fusion -Rapid cooling of the chamber (-25 OC/min)From -8 OC (sample) to -65 OC (chamber Temp.) (1.5- 2 min)-Fast warming of chamber (+ 15 OC/min) -65 OC to -14 OC (chamber) (3-4 min)

III

Slow cooling (-1 OC/min)-14 to -45 OC

(35 min)

IV

Fast cooling of frozen product (-10 OC/min) -14 to -90 OC

(5 min)

Subsequent Phases of normal freezing cycle (80 min)

I

IIIII IV

Three curves: Sample (green) is always above the target (black) curve which is expected to completely overlap with the chamber (blue) curve

Is performed by placing CB products directly into an ultra-low freezer (-80 oC) and leaving them in place and undisturbed and monitored by temperature data logger

Frozen product removed when the target unit temperature is reached (- 80 oC).

Sample Freezing curve is downloaded and printed from the data logger

Mechanical or Passive Freezing

Kinetic analysis of Passive Freezing Curves

In comparison with

CRF curves

Phase I II heat of fusion III IV

Average (n =12) calculated product cooling rate OC/min

-1.8 * Faster than CRF

Started at -11 to -18 OCTakes about 7.5 min (Comparable to CRF)

-1.1 OC/minAlmost similar to CRF

Very Slow -0.4 OC/mincompared to CRF

* Fast freezing rate in phase I: biologically not optimal Long Total freezing time to -80C: 180 min (150- 260 min).

II

III

I

IV

Impact of freezing on product quality: Passive. F vs. CRF: unit post-thaw findings

% Recovery CRF (n = 25) Mechanical F (n= 12)

TB viable NC 78 + 9 84 + 5CFU-C 72 + 14 73 + 77AAD viable CD34+ cells

63 + 15 67 + 7

Recovery of viable NC, viable CD34+ cells and CFU-C after passive freezing are comparable to recoveries obtained after CRF

Conclusion: In vitro findings suggest passive freezing is valid alternative method for CRF

Vlaidation of segments : Units vs Segments (n=25) findings

Higher viability in units (78 + 9) compared to their attached segments (70 + 6)

Higher viability in units (78 + 9) compared to their attached segments (70 + 6)

Higher % recovery of CFU-C from units (72 + 14) compared to their attached segments (46 + 16)

Higher % recovery of CFU-C from units (72 + 14) compared to their attached segments (46 + 16)

Although unit and integral segments have different freezing dynamics (volumes and surface areas), viability and CFU-C recoveries from segments correlate well with recoveries from their corresponding units.Conclusions: Integral segments can be used for confirmation of frozen unit quality Unit quality is superior to seg quality: Good seg findings suggest very good unit quality

SLCBB

Approximately 25 000 units processed and frozen Freezing graphs are initialed for acceptance by lab techs, reviewed by lab director and investigated/approved by quality specialistLess than 5% of our frozen units exhibit abnormal freezing graphs/curve

Main cases of abnormal graphs

Mechanical: Stuck or uncontrolled valves Impurities in Liquid NitrogenSoftware issuesProbe placement and probe connection malfunction Residual air around overwrapped units

Abnormal graph/curve interpretation and decision depend on:

1. The type of abnormality found: for evaluation were classified abnormaities into 3 categories: Normal curve variation or simple common artifact Uncommon minor abnormality, or Major change in the freezing graph/curve

2. The abnormal freezing phase; injuries in earlier phases are expected to be more harmful 3. Whether one or all sample curves in the run are abnormal

4. Assesment of degree to which the abnormality in freezing would diminish unit quality. This critical assessment shall be concluded based in- site historical observations and post-thaw findings

SLCBB: Multiple products with separate probes and cooling curves

Normal run with normal curves

A print of independent sample curve can be obtained

Normal run, abnormal sample 2 yellow curve.

Sample 2 curve is almost completely overlapped with the target curve in all phases (Sample probe and cassette contact)

Category 1: Normal curve variation or simple common artifact: Example 1

Normal curve variation or simple common artifact: Example 2

Single sample curve with heat of fusion phase shift to the right

Normal curve variation or simple common artifact: Example 3

Single sample curve probe fluctuation

Normal curve variation or simple common artifactExample 4

Single sample probe error

Normal curve variation or simple common artifact Investigation: Segments and unit thaws studies indicated recovery of viable NC and CFU-C comparable (within mean +/- 1 SD) to control group (data not shown)

Conclusion : when normal curve variation or simple common artifact in single sample curve is found, no change of product quality is predicted

Recommendations: - The unit can be made available, segment CFU recovery/TB viability MUST be evaluated before export

In general: Simple common variation/artifact are considered serious (major) if all sample curves in the run are abnormal

Category 2: Uncommon minor abnormality: Cases explained by sample probe placement error, air in the

overwrap, or real transient sample warming up/cooling

Case 1

Unit Acceptable

Segments study: unit quality is acceptable if recovery of both viable NC and CFU-C within mean + 1SD of the control group

Uncommon minor abnormalities

Case 2: seg show acceptable recovery of CFU-C but not viable cells : unit for research

Uncommon minor abnormalities

Case 3Not studies

Case 4Not studies

Uncommon minor abnormalities

Case 5: curve up shift Seg study: Both viability and CFU-C recovery were Not acceptableUnit for research

Uncommon minor abnormalities

Conclusion: different uncommon minor abnormalities yields different impact on product quality

Recommendations:

Segment CFU recovery/TB viability to be evaluated before stored unit is made available

In general: uncommon minor abnormalities are considered serious (major) if all sample curves in the run are abnormal

Investigation if Minor abnormality/artifact (category 1 & 2) found in whole run

Pull segs on two units from the run and evaluate post-thaw CFU/Viability results

TB CFC TB CFC

Recommendation

A A A A 4 out 4 results acceptable, Units made available

A A N N If results of one seg are not acceptable, pull another seg from another unit and repeat investigation,

A N A A If 3 out 4 results are acceptable and CFC recoveries > 25%, units made available

N A N A If 1-2 out 4 results are acceptable OR CFC recovery < 25%, sacrifice 1 or 2 units (depends on run size)

If results of unit (s) are acceptable, units made available otherwise units made for research

Seg 1 of unit 1

Seg 2 of unit 2

Category 3: Major change in the freezing run

Case 1: 1 hr delay Case 2: 2 hrs delay

Delayed run

Test Case 1 (n= 2) Case 2 (n=2) Control group

TB viable NC 66, 83 50, 56 71 + 6

CFU-C 25, 22 34, 23 72 + 14

7AAD viable CD34+ cells 51, 38 31, 35 63 + 15

Units post-thaw recoveries: Significant loss of cell viability and/or potency (black). Units to research.

Major change in the freezing curve/run

Failure after phase II: Rapid cooling through phase III and IV. Units were at -15 to -20 C

Test Case 3 (n =4) Control group

TB viable NC 64, 63, 78, 59 71 + 6

CFU-C 87, 71, 30, 12 72 + 14

7AAD viable CD34+ cells 58, 51, 54, 47 63 + 15

Units post-thaw recoveries: In all cases, nucleated cells viability and/or CFU-C capacity is compromised (black) but not CD34+ viable cells . Units for research

Cooling rate in late phases was about - 10 C /min

Note: if rapid cooling occurs when units are colder than – 60 C (after phase III), no significant change predicted

Major change in the freezing curve/run Failure end of phase III: rapid warming is anticipated, samples transferred to Mechanical freezing

Units post-thaw recoveries: Warming up of frozen units would significantly diminished Nucleated cells viability and potency (black). Case 4 units required 2nd unit thaw, while case 5 units for research

Test Case 4 (n= 1) Case 5 (n=2) Control group

TB viable NC 75 55, 57 71 + 6

CFU-C 52 7, 7 72 + 14

7AAD viable CD34+ cells 62 42, 19 63 + 15

Case 4: transferred shortly before warming up

Case 5: samples were warmed up to -10C

Major change in the freezing curve/run

Case -6 :Constant cooling rate about -1 C/ min in all phases

Samples were transiently warmed up during heat of fusion phase

Units post-thaw recoveries: nucleated cells viability is compromised (black) but not stem cell potency (CFU-C, CD34+ viable cells) . Units for research

Test Case 6 (n =2) Control group

TB viable NC 59, 62 71 + 6

CFU-C 58, 60 72 + 14

7AAD viable CD34+ cells 57, 61 63 + 15

Case 7: chamber temp curve shows Phase II Shift to left

Major change in the freezing curve

Unexpected Change in chamber temperature

Units post-thaw recoveries: NC viability and/or CFU-C recovery were unacceptable (Data not shown). All run units to research

Major change in the freezing curve

Conclusion

Based on our findings, freezing curves with major abnormalities are associated with seriuos injuries that, most likely, will significantly diminish unit quality

Recommendations If one unit is affected, must investigate segment before making unit available. Available if seg viability and CFU-C recoveries are acceptable

If whole run is affected, at least one unit from the run should be thawed and investigated. If viability or CFU-C recovery is unacceptable, all units to research

Acknowledgement

St. Louis Cord Blood Bank Team

With a special thank for:

Christianna Henderson: Senior quality technologist

Kathy Fortune: Quality specialist

Donna Regan, Executive Director