A systematic multitechnique approach for detection and characterization of reversible self-association during formulation development of therapeutic antibodies

Abstract

In addition to controlling typical instabilities such as physical and chemical degradations, understanding monoclonal antibodies' (mAbs) solution behavior is a key step in designing and developing process and formulation controls during their development. Reversible self-association (RSA), a unique solution property in which native, reversible oligomeric species are formed as a result of the noncovalent intermolecular interactions has been recognized as a developability risk with the potential to negatively impact manufacturing, storage stability, and delivery of mAbs. Therefore, its identification, characterization, and mitigation are key requirements during formulation development. Considering the large number of available analytical methods, choice of the employed technique is an important contributing factor for successful investigation of RSA. Herein, a multitechnique (dynamic light scattering, multiangle static light scattering, and analytical ultracentrifugation) approach is employed to comprehensively characterize the self-association of a model immunoglobulin G1 molecule. Studies herein discuss an effective approach for detection and characterization of RSA during biopharmaceutical development based on the capabilities of each technique, their complementarity, and more importantly their suitability for the stage of development in which RSA is investigated.

Hydrogen exchange mass spectrometry reveals protein interfaces and distant dynamic coupling effects during the reversible self-association of an IgG1 monoclonal antibody

“…10 For example, Pathak et al demonstrated that the presence of reversibly associated clusters at high protein concentrations contributed to an increase in solution viscosity. 52 Similar trends in viscosity in response to changes in solution conditions that we describe here for mAb-C have been reported for other IgG1 mAbs, where the extent of viscosity increased in a concentration-dependent manner with increasing ionic strength 11,18,30,53,54 and solution pH, 11,55 related to elevated levels of protein RSA due to charge shielding effects. [56][57][58] The isoelectric point (pI) range of mAb-C is basic (pI»9.1-9.4), 45 and therefore, the overall surface charge of mAb-C is expected to be positive at neutral pH.…”

“…These results are consistent with trends previously reported for the RSA of mAb-C at low protein concentrations by DLS, SV-AUC and CG-MALS. 11 Many of these same trends were apparent at higher protein concentrations, as revealed by changes in viscosity, where an exponential increase in viscosity was observed ranging from »1 to »75 mPa¢s depending on the protein concentration and solution conditions (Fig. 3).…”

“…These DLS results are consistent with those reported previously by Esfandiary et al for the same antibody molecule formulated under similar solution conditions. 11,45 To further assess the effect of salt on reversible self-association (RSA) of mAb-C, we used chemical cross-linking to measure the effect of sulfate on the extent of RSA at pH 7. In the presence of 300 mM sodium sulfate, higher molecular weight bands at »300 kDa and »450 kDa were observed (Fig.…”

“…These analytical limitations were encountered with DLS and SV-AUC measurements of mAb-C under different solution conditions as described in this work and as reported previously. 11,45 In the present study, we correlate solution effects on the RSA of mAb-C, as determined by DLS and chemical crosslinking at lower mAb-C concentrations, to solution viscosity measurements at higher protein concentrations. We then directly mapped the local regions of mAb-C involved at the interface of RSA at the higher protein concentration using HX-MS, an analytical tool that has been widely used to map interfaces of protein-protein interactions.…”

“…[5][6][7] The RSA of mAbs presents various pharmaceutical challenges, including the formation of protein aggregation precursors that can lead to the irreversible formation of oligomers. 8,9 In addition, the RSA of mAbs gives rise to a network of associated higher-order species that can affect the viscoelastic properties of the solution, 10 resulting in increased viscosity, [5][6][7]11 solution turbidity, 12 and, under certain conditions, even phase transitions. 13 The increase in solution viscosity also imposes manufacturing challenges including high back-pressure and clogging of membranes, 2 as well as elevated levels of shear stress during pumping.…”