3. Effect of mobile phase additives on the separation of monoclonal antibodies

The use of organic solvents such as isopropyl alcohol (IPA) or salts can decrease the secondary interaction as reported by many scientists and mentioned earlier briefly in this chapter in Section 2.5. Peak resolution and retention time shift were not impacted with the use of 15% IPA as demonstrated in the two examples (Figure 12 and Figure 13). It is necessary to evaluate the individual SEC column regarding the impact of the additives since a stationary phase with minimum impact is always a favorable choice. After important mobile phase parameters have been set, such as pH, stationary phase, and ionic strength, significant improvements can, in fact, be made to separate mAb monomers from aggregates and fragments. There are no universal additives which can be applied for every mAb or protein purification. Other common additives are methanol and ethanol. Use of sodium perchlorate may also improve the separation and resolution. By switching from 0.2 mol/L sodium chloride to 0.2 mol/L of the more chaotropic sodium perchlorate salt, together with a twofold reduction in the buffer concentration, less peak tailing and distinct peaks for the dimer and trimer could be noticed [41]. Sodium dodecyl sulfate (SDS), urea, guanidine hydrochloride, etc., are sometimes used when the proteins need to be solubilized, leading to denaturation of the protein and breakage of the noncovalent bonds. The use of additives should be considered only when needed. In many cases, the performance of the column is irreversibly changed when a column is subjected to particular additives. Retention time may shift when the additive is added and the resolution may change, expectedly to a better resolution but these values should remain constant and the analysis should be reproducible. It is always better to dedicate the SEC column if the column is subjected to additives, since we still have no clear idea how the pore characteristic of the stationary phase may behave with and without additives. Analysts should check the operational and conditions (OCS) sheet for the column as provided by the vendor to make sure that additives are compatible with the stationary phase. If compatible the analyst should be aware of the percentage of organic solvent the column is compatible with. Generally, when the organic solvent is used, the column may need a slower ramping rate for proper equilibration of the column with the mobile phase containing the additive, generally by using gradual solvent changes using a shallow gradient at low flow rate.
