Chromatographic Tools to Predict the Stability of mAbs for Faster Identification of Therapeutic Candidates
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
Protein-protein molecular interactions are known to be involved in protein solution aggregation behaviour and are a common issue for the manufacturing of therapeutic proteins such as mAbs. Much effort has been employed to gain a better understanding of aggregation, however the mechanisms leading to protein aggregation are still not fully understood. The osmotic second virial coefficient (B22) is a fundamental physiochemical property that describes protein-protein interactions solution, which can be a useful tool to predict aggregation propensity of proteins.
One way of predicting aggregation propensity is self-interaction chromatography (SIC), which recently have shown to be a promising tool for better understanding of phase behaviour of proteins. Another technique, cross-interaction chromatography (CIC), has shown to be an even more high-throughput technique than its predecessor with possibly the same capabilities. This work consists of two experimental studies with therapeutic mAbs to improve SIC and CIC as a tool to predict protein aggregation.
The first part includes a 10 times scale-down study of therapeutic mAbs from laboratory scale macro-columns to micro-scale columns, which will enable the determination of B22 for the individual protein as well as the cross-virial coefficient, B23, between two proteins. Micro SIC and CIC uses only a few milligrams of mAb in order to obtain a complete formulation study. The results from the first part of the study proved to give good comparable results between the micro and macro scales enabling the use of micro SIC for B22 determinations.
The second part of this work presents an extensive formulation study of mAbs, varying pH and salt, as well as the presence of different stabilisers as well as different external factors known to induce aggregation. The B22 and B23 values determined from the formulation study are then correlated with aggregation data obtained from size-exclusion chromatography. It was shown that over all test conditions, good correlations could especially be found between B22 and aggregation rate.
In summary this work hopes to lead to a better insight into the mechanisms of aggregation by looking at the extent of tailing of the peaks in the chromatograms and comparison with conformational studies.
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