Opalescence of an IgG1 Monoclonal Antibody Formulation is Mediated by Ionic Strength and Excipients - Can increase in ionic strength result in higher viscosity? - BioPharm International

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Opalescence of an IgG1 Monoclonal Antibody Formulation is Mediated by Ionic Strength and Excipients
Can increase in ionic strength result in higher viscosity?


BioPharm International
Volume 22, Issue 4

Effects of Viscosity

We also explored the effect concentration and NaCl had on the viscosity of MAb1 solutions. It was determined that the viscosity increased as the protein concentration increased. Similar results were obtained by Liu and colleagues for an IgG at a concentration range of 30 to 125 mg/mL in a formulation containing 16 mM histidine, 266 mM sucrose, and 0.03% PS-20, pH 6.22 The viscosity of MAb1 solutions also increased with NaCl concentration. The increase in viscosity of MAb1 solutions with increasing NaCl, however, was in contrast to the results obtained with an IgG by Liu and colleagues.22 In their studies, increasing the molarity of NaCl resulted in a decrease in viscosity.22 The differences in the viscosity increase or decrease in the presence of NaCl when comparing MAb1 to the IgG studied by Liu and colleagues may be caused by sequence-specific variability in the complementary determining regions (CDRs) in the heavy and light chains of the molecules.

Differential Scanning Calorimetry

Salts can affect proteins by several mechanisms and can either increase or decrease thermal stability.23 The dominant mechanism of the interaction of salts with proteins and the amplitude of the stabilizing or destabilizing effect depend on the type and concentration of the salt, but also on other solvent conditions.24

The thermal unfolding of MAb1 presents two main transitions. Based on the amplitude of these transitions, the first transition corresponds to the unfolding of the CH2 domain in the Fc fragment and the second transition corresponds to the unfolding of the Fab fragment and CH3 domain in the Fc fragment of the IgG molecule.25 The apparent melting temperature for both transitions is slightly decreased as the concentration of the salt is increased. In the absence of salt, the second transition presents a shoulder that may reflect a reduced overlap between CH3 and Fab unfolding. This observation suggests that the Fab fragment is more sensitive to the presence of salt than the CH3 domain.

Second Virial Coefficient

In the presence of NaCl, MAb1 had a negative B22. It is not known if the negative second virial coefficient of MAb1 was associated with the opalescence observed or was caused by the properties of the MAb. It could be hypothesized that net attractive interactions are associated with opalescence, however additional studies will be required to test the idea. It would also be interesting to evaluate MAb1 formulations lacking NaCl to determine if the second virial coefficient changes.

Studies with other proteins have shown that the second virial coefficient decreases as the concentration of NaCl increases.26 For example, it was demonstrated that the second virial coefficient of the peptide enfuviritide decreases with an increase in salt.26 In other studies, including one with an IgG, it was demonstrated that the second virial coefficient decreases as the concentration of NaCl increases.22,27

Stability of Opalescent IgG Formulations

It was determined that the ionic strength of the MAb1 formulation does not affect the stability at 4 C after four weeks. However, at elevated temperatures for four weeks in the presence of NaCl, irreversible dimer formation occurs, as demonstrated by SEC–HPLC. Interestingly, there was no significant change in high-order aggregate formation as the amount of NaCl was increased (data not shown).

The stability of an opalescent IgG1 formulation has been previously described by Sukumar and colleagues. In those studies, it was determined that the hydrodynamic size of formulations ranging from 0.5 to 50 mg/mL at 25 C remained unchanged up to 300 minutes.4

An example in which NaCl induced dimerization of an IgG occurred was with the monoclonal antibody rhuMAb (VEGF).28 In the presence of 1M NaCl, a decrease in the kD (the dissociation constant) for rhuMAb (VEGF) was observed, which resulted in the formation of dimers.28 In the same study, it was demonstrated that another salt, CaCl2, had the same effect as NaCl, in that the increased ionic strength resulted in dimerization of rhuMAb (VEGF).28 These results parallel the study described here, in which higher levels of NaCl result in dimer formation. In separate studies with an IgG, it was demonstrated that the percentages of IgG aggregates increased with increasing ionic strength.29

Overall, the studies reported here demonstrate that the ionic strength of a formulation as well as the excipients, play an important role in the opalescent appearance of an IgG1 monoclonal antibody. These factors may be relevant to the appearance and stability of other IgG formulations under stressed conditions.


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