Implications for monoclonal antibody aggregation
In this endeavor, we observed differences in aggregation behavior between the IgG1 and IgG2 forms of the same antibody. To understand the aggregation mechanism, oligomer and aggregate species of the two antibodies were purified and characterized using a variety of approaches at a single (neutral) pH. Other studies from Amgen examining pH trends on IgG1 and IgG2 aggregation will be published at a later time.
One question that arises from all these studies is: why was the IgG2 form of the antibody more prone to aggregation compared with the IgG1 form? One possibility was the intrinsic heterogeneity of the IgG2 molecule because of the existence of disulfide-mediated isoforms that were recently discovered.17, 18 Potentially, the behavioral characteristics of these different isoforms are not identical and may have a role to play in this phenomenon. The existence and relative populations of these IgG2 isoforms of anti-streptavidin were not explored in this study. Another aspect to consider would be whether the recently discovered clip-mediated aggregation pathway26 was in some way more prevalent for IgG2 antibodies compared with IgG1. However, the IgG1 form tended to show greater fragmentation yet lower aggregation, so this clip-mediated mode of aggregation may be restricted to IgG2 antibodies for the most part.
Given the high degree of covalent character in the aggregates, our study suggested that the increase in aggregation of the IgG2 subclass was partly because of the 2.4-fold increase in free cysteines in the IgG2 form as detected by DTNB. Indeed, this theory is supported by a recent study with multiple antibodies,27 which showed a linear correlation between increased free thiol in the antibody and lower thermal stability (as monitored by thermal transition measurements). Although the IgG2 form aggregated to a greater extent in our work, the fraction of the total aggregate that was covalent was preserved between IgG1 and IgG2 forms. This suggested that the free thiols were responsible for increasing the rate of aggregation, although perhaps not the final covalent composition of the aggregate. One aspect to consider is that the relative contribution to covalent versus noncovalent aggregation would depend on the pH of the incubation. At neutral pH, it is expected to see a much greater contribution from disulfides than at pH 5. Indeed, the overall magnitude of the conformational changes was relatively small, consistent with previous observations, consistent with the recently proposed coagulation mechanism for antibody aggregation.5
Comparison of IgG1 and IgG2 aggregation characteristics: similarities and differences
Typically, differences between antibodies exist in their CDRs because of target specificity. These sequence differences often lead to different biochemical and biophysical behavior, which has been observed even within the same subclass class.28 IgG1 and IgG2 subclasses are structurally similar, consisting of 12 Ig-like domains with a conserved disulfide bond buried within each domain.29 The major difference between the subclasses originates from the highly flexible hinge region30; IgG1 and IgG2 have two and four disulfide bonds, respectively, and the IgG1 hinge is approximately two amino acids longer. The IgG1 and IgG2 anti-streptavidin antibodies used in this study had identical CDRs, but had 6% amino acid sequence differences in the hinge and heavy chain conserved regions. Increased hydrophobicity of a protein could potentially lead to higher aggregation rates.31 The variations in hydrophobicity between IgG1 and IgG2 anti-streptavidin are slight (grand average of hydropathicity index (GRAVY)32 scores of −0.396 for antiSA1 and −0.383 for antiSA2) indicating that changes in hydrophobicity were not a major determinant for the aggregation differences observed between the subclasses. It is unknown what directly leads to the aggregation differences, but it is presumed that this sequence variation and/or differences in amounts of free cysteine could potentially result in different aggregation rates and properties between the two subclasses as described in this work.
Similarities and differences in secondary and tertiary structural features were explored using a host of biophysical approaches. In the case of secondary structural features, both purified aggregate species were similar according to far-UV CD and Fourier transfer infrared spectroscopy (FTIR), with a few exceptions. The far-UV CD spectra of both antiSA1 and antiSA2 aggregates showed the same loss in ring stacking interactions and an increase in random structure as indicated by bands located at 230 and 198 nm, respectively, and a broadening of the β-sheet band at 217 nm. The FTIR spectra showed one noticeable difference between the aggregate species at 1610 cm−1, which was attributed to β-sheet: the IgG1 aggregate species had a complete loss in this band indicating heterogeneous β-sheet energies upon aggregation, whereas the IgG2 aggregate species had a shift in this band to a higher wavenumber indicating a shift in the β-sheet energy. This shift could be due to strong hydrogen bonding and the formation of distorted intermolecular β-sheets during aggregation.33 Distortion of existing secondary structures may point to a disruption in tertiary structure rather than global secondary structural rearrangements.
Near-UV CD spectroscopy revealed that the aggregates of both subclasses retained significant tertiary structure. The majority of the differences observed between subclasses originated from the tryptophan signals (290–295 nm). The near-UV CD spectra of the purified IgG1 aggregate species showed a change in local environments surrounding the tryptophan residues, whereas that of the IgG2 aggregate showed a more significant loss of structure in this region [Figs. 8(A,B)]. Interestingly, the tryptophan locations are conserved in both subclasses and the near-UV CD signal originating from the tryptophans for native IgG1 and IgG2 molecules are similar. Therefore, differences observed in near-UV CD signals during aggregation between the subclasses must arise from changes in the environments of the tryptophan residues, and not due to differences in tryptophan location. In addition, ANS binding data suggested that there may be a change in hydrophobic exposure in the purified aggregates compared with the monomer or oligomer samples, indicating an exposure of hydrophobic patches during the aggregation process, which may have been sequestered in the native molecule.
It has been established in the literature that proteins that contain disulfide bonds are more stable in their oxidized rather than their reduced forms.34, 35 Recently, however, the impact that a reduced disulfide bond can play in multidomain antibodies has recently come to light.27 Lacy et al. demonstrated a correlation between antibody thermal stability and the extent of free cysteines present in a series of antibodies, with greater free cysteines translating to lower thermal stability. In other studies, it has been observed that antibodies and antibody constituent domains can contain free cysteines that can have a deleterious effect on protein stability and cause an increase in aggregation propensity.34, 36, 37 It also has been noted that antibody free cysteines appear to be in buried regions of immunoglobulin domains and become solvent-exposed during agitation, thus forming covalent aggregates consisting of non-native, intermolecular disulfide bonds that can stabilize and act as a nucleus for further aggregate propagation.37 In this study, the anti-streptavidin IgG2 molecule contained 2.4 times the free cysteines when compared with the IgG1 subclass. The thermal stability by DSC of the subclasses showed measurable differences with the IgG2 molecule having a decrease of ∼0.6°C in its apparent thermal melting temperature, which is consistent with a previous report.27 It is worth noting that as one of the major differences between IgG1 and IgG2 antibodies is the number of disulfide bonds that comprise the hinge; therefore, it is plausible that the covalent aggregate could be dictated by free cysteine reactivity from this region. However, it has been previously shown that the level of free cysteine in an IgG2 was negligible in the native state indicating hinge cysteines are involved in disulfide bonding37 and, therefore, it is plausible that the aggregation increase observed with IgG2 was due to domain instability associated with buried unpaired cysteines.
Additionally, the IgG2 subclass aggregated 2.4 times faster than the IgG1 molecule in this study. These results agree well with previously reported results36 showing that the presence of free cysteines had a more negative effect on the IgG2 subclass compared with the IgG1. Strengthening the argument, ∼10 and 30% of the covalent aggregate observed by SDS dSE-HPLC (denatured size-exclusion chromatography) for IgG1 and IgG2, respectively, was reducible. This indicated that free cysteines were involved in the covalent interaction, further suggesting that increased amounts of free cysteines in IgG2 led to greater levels of covalent aggregate in the IgG2 version.
However, the observed covalent aggregate differences between the subclasses cannot be explained solely by the formation of non-native, intermolecular disulfide bonds. The nonreduced SDS dSE-HPLC data showed the presence of ∼62% covalently associated HMW species for both subclasses. Upon reduction, the majority of these HMW species persisted, indicating that the majority of the covalent bonds may not be disulfide-mediated. A possible cause for these nonreducible aggregates could be through beta elimination. At neutral to alkaline pH, crosslinking reactions between a charged or polar amino acid (e.g., lysine or tyrosine) and serine or cysteine residues that have undergone a beta elimination reaction to form dehydroalanine crosslinks can occur and form nonreducible aggregates.38, 39 It did not appear that these nonreducible aggregates were an artifact of sample preparation, as they were virtually nonexistent in nonstressed samples [Fig. 9(C)]. Interestingly, once the sample was stressed at neutral pH and 45°C for a period of time, all the isolated species contained nonreducible aggregate, increasing in order from monomer to small oligomer to aggregate. Further, the nonreducible species were always higher in the IgG1 samples. This may indicate real covalent differences that accompanied aggregation and differences between subclasses. The SDS dSE-HPLC data gave a deeper insight into the composition of the nonreducible aggregates. The SDS dSE-HPLC data were analyzed based on protein backbone detection at 215 nm; therefore, the theoretical percentages of heavy chain and light chain should be 66 and 33%, respectively, due to their molecular weights. However, the experimentally observed heavy chain percentage under reducing conditions for IgG1 and IgG2 was 23 and 30%, respectively, which was 66 and 55% lower than the theoretical percentages. A similar observation was noted for light chain, but to a lesser extent (45 and 25% reduction in light chain for IgG1 and IgG2, respectively, compared with their theoretical percentages). The significant loss in heavy chain indicated that the nonreducible covalent aggregate could consist of primarily of heavy chain. A detailed exploration of the mechanism of the nonreducible species would need further exploration.
In the future, a potential effort would be to determine the locations of free thiols in the antibody, and then distinguish areas of free thiols in IgG1 versus IgG2 subclasses. Another area of interest for the IgG2 antibodies would be to develop purification methods to effectively isolate the disulfide isoforms, characterize them, and directly compare their stability. Because of the intense interest in this area, we are likely to witness several important developments in the near future.