Large quantities of donated blood are discarded due to stability issues. Stocks of rare blood types rarely last more than a week. Therefore there is considerable value in achieving more stable material. This study was carried out in conjunction with the University of Cambridge and sponsored by the UK TSB.

1. Lyophilization of Red Blood Cells
Isobel Cook B.Sc. (Hons), MRSC
Principal Scientist
BTL, Winchester SO23 0LD, UK
Tel: +44 (0)1962 841092
E-mail: icook@biopharma.co.uk

2. Synopsis of Presentation
• Background and reasons for looking at
blood / red blood cells
• Aims of TSB-sponsored study
• Issues with freeze-drying of RBC
• Variables studied for human RBC
• Results 1 – RBC ‘survival’
• Results 2 – Haemoglobin oxidation
• Next steps

3. Why Blood?
• In the UK, large volumes of donated blood are
discarded due to stability issues
• NHSBT assigns donated blood a shelf life of 35
days at 4 2 C
• Stocks of rare blood types for transfusion rarely
exceed enough for more than 1 week
• Therefore there is considerable value in
achieving more stable material – stockpiling for
military use / disaster zones / emergencies
• Freeze-drying offers the opportunity of
stabilising whole blood / blood components

4. RBC/Blood in the body
• Blood 8% body weight
• RBC are concave discs,
6-8 µm diameter
• RBC (erythrocytes) account
for 45% of whole blood in the human body
• RBC circulate for about 120 days in the body
• *RBC carry haemoglobin*

5. Why Red Blood Cells (RBCs)?
• RBCs present a challenge – other factors
have successfully been lyophilised but no
reports of 100% success with RBC for
either freeze-thawing or freeze-drying
• RBC membrane not as robust as
nucleated cells – therefore, more cryo- /
lyo- protection is likely to be needed
• RBC lysis is relatively simple to quantify in
terms of haemoglobin leakage

6. Work to date
• Cryo-preservation – glycerol as a protectant
at ultra low temperatures – feasibility
• Intracellular loading – Lysis levels (20%)
• Lyophilisation with sugar solutions – high
moisture levels (~30%)
• Novel intracellular loading techniques (as
discussed in this presentation)

7. Aims of study (sponsored by the
UK Govt. Technology Strategy Board [TSB])
• To investigate a series of variables
(formulation and process) on the survival of
RBC after freeze-drying + rehydration
• To quantify RBC ‘survival’ by haemoglobin
release in supernatant compared with pellet
• To quantify level of haemoglobin oxidation
by UV-visible spectrophotometry
(comparing Hb, met-Hb, oxy-Hb)

8. Sheep RBC in suspension

9. Sheep RBC upon initial freezing
in the absence of protective agent

10. …and 1 minute later…

11. …and after rapid thawing…
Was it the freezing or the
thawing that caused lysis?
It’s difficult to tell using
conventional methods,
because the cells may have
been damaged during
freezing, yet fixed in position,
thereby making damage
impossible to identify…

12. Human RBC in suspension
with buffer + protective agent

13. Human RBC suspension frozen
with buffer + protective agent

14. Thawed suspension with
buffer + protective agent

15. Issues with RBC lyophilisation
• Freezing damage:
– from ice crystal growth
– from pH gradients
– from freeze-concentration / osmotic effects
• Drying damage:
– Physical action of ice removal on membrane
– Dehydration causing deformation of RBC
• Rehydration damage:
– Concentration and pH effects (e.g. localised
hypotonic / acidic / alkaline regions, causing lysis)
– Wetting issues exacerbating the above effects

16. Variables for Human RBC Study
Variable: Option 1 Option 2 Option 3
Buffer type Buffer 1 Buffer 2 Buffer 3
Buffer concentration High Medium Low
Protectant type Non-polymer Non- Polymer
1 polymer 2
Protectant concentration High Medium Low
Cooling rate High Medium Low
1 D shelf temperature High Medium Low
Rehydration solution Salt Buffer Polymer

17. Additionally…
• Our academic partner (Cambridge University) had
shown a novel biopolymer had the ability of providing a
mechanism of intracellular loading of protectant
• Therefore, a duplicate set of samples would also be
exposed to polymer
• BUT 2 x 37 sets of conditions plus controls would be
~4,400 samples, and…
– Triplicate runs would = 13,200 samples
– 3 UV/vis cuvettes per sample would = 39,600 cuvettes
– UV/vis readings at 4 wavelengths would = 158,400 UV readings!
• The project timeframe (6 months) did not allow for a DoE
approach. Therefore, the study was rationalised and
only selected combinations were studied.

18. Haemoglobin types
• Haemoglobin (metalloprotein enables O2
binding)
• Oxy-haemoglobin (saturated blood with
oxygen reversibly bound)
• De-oxyhaemoglobin (~75% saturated)
• Methoxy-haemoglobin (oxidised state III)
o 2-3% of RBC

19. RESULTS (1) – RBC ‘survival’
• Levels of RBC survival of 96% achieved for the combination below
• Surprisingly, this was achieved in the absence of biopolymer, which
implies that intracellular protectant may not be necessary
• Oxidation level of Hb was quite high (60%)
Variable: Option 1 Option 2 Option 3
Buffer type Buffer 1 Buffer 2 Buffer 3
Buffer concentration High Medium Low
Protectant type Non- Non- Polymer
polymer 1 polymer 2
Protectant concentration High Medium Low
Cooling rate High Medium Low
1 D shelf temperature High Medium Low
Rehydration solution Salt Buffer Polymer

20. RESULTS (2) – Hb oxidation
• While the biopolymer did not necessarily lead to
higher RBC survival (85%) under the conditions
employed here, it was noted that it was only in
samples containing the biopolymer that the
haemoglobin oxidation was reduced to below
detectible levels, and was typically below 10%
• Prevention of Hb oxidation may have been
attributable to the direct action of the polymer itself
and/or to the presence of intracellular protectant
facilitated by the presence of the polymer

21. What Next?
• Further studies, building on the data from this study:
– Using a DoE approach to cover all combinations of variables
– Extending the number of formulation and processing
variables
– Looking in detail at a novel method of iso-osmotic RBC
concentration / rehydration using specialist membranes
– Examining RBC deformability upon rehydration
– Identifying other significant RBC parameters to be studied
– Fine tuning novel biopolymer to match best non-polymer
survival rate
– Looking at long term stability in the freeze-dried state
• Application of the principles of this study to the freeze-
drying of nucleated cells

22. SUMMARY
• A large number of combinations of formulation
and process variables were tested in the freeze-
drying of RBC
• Best RBC survival rate was 96%, but with 60%
Hb oxidation
• Use of novel biopolymer led to Hb oxidation
levels below detectable limits, but maximum
survival was 85%
• Valuable lessons learned that will be applied to
further studies on RBC and nucleated cells

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