Affinity Bio separation Column Wuraola Akande School of Pharmacy and Biomolecular Sciences, University of Brighton, Brighton, UK
Cryogel preparation Cryogel was produced by free radical polymerisation at sub zero temperature from HEMA (hydroxyethyl methacrylate) and AGE (allyl glycidyl ether) monomers MBAA as a cross linker At the presence of APS/TEMED as initiator Cryogel in 5cm x 1cm glass tube Ice crystals & initial forming polymers Monomers & initators Macroporous gel
Characteristics of cryogel Cryogel , possess the following characteristics. • High mechanical and chemical stability • Low flow resistance • Good pore interconnection • Ease of functionalisation and attachment of biological ligands • Large surface area
Objectives were achieved by • Producing Macroporous Monolithic polymer cryogels with a range of interconnected pore sizes • Investigating the mechanical stability • Investigating haemocompatibility • Functionalising the surface of the matrix
8% PHEMA cryogel can withstand a maximum pressure of 180mmHg without deviation in linearity .
Creep test of 8% PHEMA cryogel, at approximately 8 minutes the cryogel had 100% strain recovery , while with 10% and 16% PHEMA cryogel there was 98% and 96% strain recovery
8% PHEMA had approximately 97% recovery after applying a constant load of 0.2N for one minute, cryogel was compressed to approximately 60% and left to recovery for two minutes in a repeated cycle five times . The difference in recovery in the first cycle compared to the fifth cycle is less than 1%.
After approximately 6ml of elution from the column 100% of the red blood cells were eluted . Heamolysis was calculated according to Blankey and Dinewoodie and no haemolysis was observed
There is no significant difference between the platelet loss in tube with adsorbent and without adsorbent, but there is a significant platelet protection in EDTA to control.( n= 3) *P> 0.05 as analysed by one way ANOVA
There is no significant difference between the platelet loss in tube with adsorbent and without adsorbent, but there is a significant platelet protection with EDTA compared to control.( n= 3) *P> 0.05 as analysed by one way ANOVA
There is no significant difference between the platelet activation in tube with adsorbent and without adsorbent in sigmacote tube, but there is a difference in platelet activation in plain tube with and without adsorbent. EDTA protected activation of PAC-1. ( n= 3) *P> 0.05 as analysed by one way ANOVA
There is no significant difference between the platelet activation in tube with adsorbent and without adsorbent in both plain tube and sigmacote tube, But there is a significant difference compared to control ( n= 3) *P> 0.05 as analysed by one way ANOVA
Summary of results • The PHEMA monolithic cryogel column consists from ~13% of polymer phase and ~87% of the interconnected pores within the range of 10-120 µm • Column can withstand a maximum pressure of 180mmHg without deviation in linearity • Compression release cyclic test showed approximately 97% strain recovery after been compressed to about 60% and thus cryogel mechanically stable • ~100% of Red Blood Cells passed through the column • No evidence of haemolysis was found in blood eluted
6. Results from Sysmex cell counter correlates with data obtained from the FACS analysis of free platelets 7. EDTA protect the loss of platelet in all surface 8. PHEMA cryogel is a material of low thrombogenicity. PHEMA cryogel can be an appropriate matrix for further functionalisation and use as affinity bio separation column.
Future work 1.Functionalisation of cryogel matrix surface 2. In vitro biocompatibility of cryogel matrix in an extracorporeal circulation
Acknowledgements The team at Krems Donau University • Prof .Dr. Dieter Falkenhagen • Dr Viktoria Weber • Ute Fichtinger • Ingrid Linsberger • Anita Schildberger • Stephan Harm The team at Polymerics • Dr Aneila Leistner • Andre Leistner • Anke Lehmann • Marina Stier
This work was funded by the FP7 MONACO EXTRA project and University of Brighton Acknowledgements • Dr Lyuba Mikhalovska • Dr Irina Savina • Dr Stuart James • Prof Sergey Mikhalovsky • Dr Lyuba Mikhalovska • Dr Irina Savina • Dr Stuart James • Prof Sergey Mikhalovsky