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

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

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

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*

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

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)

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)

Sheep RBC in suspension

Sheep RBC upon initial freezing in the absence of protective agent

…and 1 minute later…

…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…

Human RBC in suspension

with buffer + protective agent

Human RBC suspension frozen

with buffer + protective agent

Thawed suspension with

buffer + protective agent

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

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

1

Non-

polymer 2

Polymer

Protectant concentration High Medium Low

Cooling rate High Medium Low

1 D shelf temperature High Medium Low

Rehydration solution Salt Buffer Polymer

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.

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

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-

polymer 1

Non-

polymer 2

Polymer

Protectant concentration High Medium Low

Cooling rate High Medium Low

1 D shelf temperature High Medium Low

Rehydration solution Salt Buffer Polymer

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

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

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

Biopharma House, Winnall Valley Road, Winchester SO23 0LD, UK

Tel: +44 (0)1962 841092 Web: www.btl-solutions.net