Table 1.

Recent reports on the impact of major factors on intrinsic aggregation of mAbs and Fc-fusion proteins.

Factor	Cofactors	Type of Protein	Mode of instability	Reported effects	References
pH		mAbs	conformational	Low pH (2.7) increases IgG hydrophobicity and induces aggregation.	[29]
pH		mAbs	conformational	pH-shift from 2 to 7 induces irreversible aggregates.	[30]
pH		mAbs	conformational	IgG subclasses respond differently to pH-induced aggregation.	[31]
pH		mAbs	conformational	Nivolumab is susceptible to aggregation (30%) at pH 3.5. IgG4 aggregation is dependent on the Fc region. Propensity to acid-induced aggregation: IgG4 > IgG2 > aglyco-IgG1 > IgG1.	[32]
pH	Temp	Fc fusion protein	conformational	Reducing pH 7, 30°C to pH 6.7, 27°C significantly decreases aggregation.	[33]
Temp	pH	Fc fusion protein mAb	conformational	Low temperature and/or low pH in a mammalian cell culture reduce misfolded and aggregated proteins.	[34]
pH	Excipient	mAbs	colloidal	Isoelectric precipitation of plasma proteins leads to dextrose-mediated mAbs aggregation.	[35]
Freeze-thaw	pH	mAbs Fc fusion protein	conformational	Adjusting protein solution (frozen at pH 3) in a higher pH buffer (pH 7) during thawing increases aggregation.	[36]
Freeze-thaw		mAbs	conformational	Slow thawing induces highest aggregate and subvisible particle levels. Fast freeze and fast thaw are better for mAbs.	[37]
Chemical modification	pH	mAbs	colloidal	Deamination increases aggregation at low pH 3.8.	[38]
Chemical modification	pH/Temp	mAbs Fc fusion proteins	conformational colloidal	Met oxidation induces aggregation at low pH and high temperature. Trp oxidation and Asn deamidation induces covalent crosslinked and attractive colloidal interactions.	[39]
Chemical modification		Biotherapeutic protein	conformational	Asn deamidation causes less unfolding at the aggregation hotspots identified with NMR compared to methionine oxidation.	[40]
Buffer	Ion	bispecific Ab	colloidal	His + Glu at low ionic strength stabilize antibody and reduce aggregation.	[41]
Ion	pH	mAbs	colloidal	Accumulation of citrate ions at the protein surface decreases protein-protein repulsion (compared to acetate buffer).	[42]
Ion		mAbs	conformational colloidal	Sodium citrate buffer reduces protein stability compared to sodium acetate buffer.	[43]