Adsorption Kinetics of Proteins for Increased Understanding of Immobilisation Behaviour
S. Hedberg*, J.Y.Y Heng*, D.R. Williams* and J. Liddell**
*Surface and Particle Engineering Laboratory **Research and Development
Department of Chemical Engineering Fujifilm Diosynth Biotechnologies
Imperial College London, SW7 2BY, Billingham, TS23 1LH, United Kingdom
Immobilisation of proteins and enzymes onto a solid phase has shown to be very important process for both commercial and research uses. A key application for the immobilising of a protein with a solid phase is that it allows the study of protein-protein interactions between a ‘free’ protein and an immobilised protein.
Protein-protein molecular interactions are known to be involved in protein solution aggregation behaviour which can lead to better understanding of the mechanisms behind protein aggregation and aggregation kinetics. This work includes a number of experimental studies that includes both model proteins and therapeutic mAbs, of different sizes being immobilised onto different standard affinity resins of with various active surface groups.
The maximum amount of protein that can be adsorbed onto the resin and the kinetics of the immobilisation procedure have been investigated. Also the amount of protein has been reduced and the resin type changed to observe the difference in kinetics observed. After the immobilisation procedure has finished the resins with immobilised proteins are washed a number of times with different solution conditions, including high salt buffers, to evaluate the strength of the covalent immobilisation linkage of the proteins. This study links the experimental results to the theory of the kinetics of adsorption. It will also be able to determine the precise amount of protein needed for a certain surface coverage of the resin when there is a shortage of proteins available to improve the economics of the process. These results are useful for the application of Self-Interaction Chromatography that has become an increasingly popular method of determining the osmotic second virial coefficient of proteins as well as the cross virial coefficient between two proteins.
The results have shown that many proteins with amine surface groups used have very high rate of adsorption with at least around 75% of protein immobilised on the resin with aldehyde surface groups with continuous mixing. The amount will differ for individual proteins, with almost 80% immobilised for the therapeutic mAb. However, resins with different surface groups and different solution conditions can easily and significantly affect the extent of immobilisation.
In summary these results obtained will contribute better understanding of adsorption kinetics and equilibria of a number of model and therapeutic proteins.
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2. Roberts, C.J., Das, T.K., and Sahin, E. (2011) Predicting Solution Aggregation Rates for Therapeutic Proteins: Approaches and Challenges. International Journal of Pharmaceutics. 418(2): p. 318-333.