Elevated levels of soluble aggregates (high molecular weight species, HMW) were observed during the Cation-exchange chromatography for several different biologics including mAbs and Fc-fusion proteins. Factors such as high load pH, short protein/resin contact time, hydrophilic resin surface, and weak ionizable ligand were effective, to some extent, to reduce aggregate formation by improving the structural integrity of the bound protein. Differential scanning fluorimetry (DSF) using Sypro Orange dye confirmed that the bound protein exposed more hydrophobic area than the native molecule in free solution, especially in the pH 4–5 range for a Fc-fusion protein tested. The Sypro Orange dye study of resin surface property also demonstrated that the poly[styrene-divinylbenzene]-based Poros XS with polyhydroxyl surface coating is more hydrophobic compared to the agarose-based CM Sepharose FF and SP Sepharose FF. The hydrophobic property of Poros XS contributed to stronger interactions with the partially unfolded bound protein and consequently to the higher aggregate levels seen in Poros XS eluate. This work also investigates the aggregation reversibility in CEX eluate where up to 66% of the aggregates were observed to dissociate into native monomers over a period of 120 h, and links the aggregate stability to such conditions as resin surface properties and charged ligand type. Experimental data was correlated semi-quantitatively with theoretical protein charge and hydrophobicity calculations using homology modeling within the BIOVIA Discovery Studio software. Finally, an arginine-sulphopropyl (Arg-SP) agarose resin immobilized with multi-functional ligands was prepared to verify the proposed hypothesis and to eliminate the aggregate formation. The inconsistency between the protein stability property observed in solutions and its aggregation propensity in chromatography process emphasizes the importance of acquiring molecules’ downstream processibility information (i.e., this study) in designing manufacturing-friendly biologic molecules and in developing robust CEX polishing processes.

Aggregation phenomena in cation exchange chromatography and implications to process development for mAb and Fc-fusion proteins

253rd National Meeting (2017)

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