Modelling of pH-dependence to develop a strategy for stabilising mAbs at acidic steps in production

Ooishi

2022

Preprint

Protein denaturation is a ubiquitous process that occurs both in vitro and in vivo. While the molecular understanding of the denatured structures of proteins is limited, it is commonly accepted that the loss of unique intramolecular contacts makes proteins larger. Herein, we report compaction of the immunoglobulin G1 (IgG1) protein upon acid denaturation. Small-angle X-ray scattering coupled with size-exclusion chromatography revealed that IgG1 radii of gyration at pH 2 were ~75% of those at neutral pH. Scattering profiles showed a globular shape with approximately triaxial ellipsoids, supported by analytical ultracentrifugation. A theoretical model indicated that the acid denaturation of proteins with size reduction was energetically costly, and thus acid-induced compaction required an attractive force for domain reorientation. Such intramolecular aggregation may be widespread in multidomain proteins as non-canonical structures. Herein, we discuss the potential biological significance of these non-canonical structures of immunoglobulins.

Section: Discussionmentioning

confidence: 99%

Getting Smaller by Denaturation: Acid-Induced Compaction of Antibodies

Ooishi

2022

Preprint

“…In addition, the precise prediction of antibody stability in an acidic environment requires the characterization of aciddenatured structures, including in the dimeric forms (Figure S5), which is an important aspect of the engineering process of antibodies. 65 The current knowledge-based principle of proteins getting larger by denaturation that has been established by many studies using single-domain proteins should be reconsidered. The new view should now include the potential for proteins to get smaller by denaturation, as we demonstrated for aciddenatured IgG1 proteins (mAb-A and mAb-B).…”

Section: The Journal Ofmentioning

confidence: 99%

“…It is unclear whether the molecular characteristics of the acid-denatured IgG1, such as intramolecular aggregation, decelerate or accelerate these reactions. In addition, the precise prediction of antibody stability in an acidic environment requires the characterization of acid-denatured structures, including in the dimeric forms (Figure S5), which is an important aspect of the engineering process of antibodies …”

mentioning

confidence: 99%

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Getting Smaller by Denaturation: Acid-Induced Compaction of Antibodies

Ooishi

2023

J. Phys. Chem. Lett.

Protein denaturation is a ubiquitous process that occurs both in vitro and in vivo. While our molecular understanding of the denatured structures of proteins is limited, it is commonly accepted that the loss of unique intramolecular contacts makes proteins larger. Herein, we report compaction of the immunoglobulin G1 (IgG1) protein upon acid denaturation. Small-angle X-ray scattering coupled with size exclusion chromatography revealed that IgG1 radii of gyration at pH 2 were ∼75% of those at a neutral pH. Scattering profiles showed a compact globular shape, supported by analytical ultracentrifugation. The acid denaturation of proteins with a decrease in size is energetically costly, and acid-induced compaction requires an attractive force for domain reorientation. Such intramolecular aggregation may be widespread in immunoglobulin proteins as noncanonical structures. Herein, we discuss the potential biological significance of these noncanonical structures of antibodies.

“…Although the aggregate evolution mechanism has been increasingly captured [8], the earlier processes preceding the aggregation remain. Identifying the structural details of the acid-denatured structure is also a necessity for antibody engineering [9].…”

Section: Introductionmentioning

confidence: 99%

Cue to Acid-Induced Long-Range Conformational Changes in an Antibody Preceding Aggregation: The Structural Origins of the Subpeaks in Kratky Plots of Small-Angle X-ray Scattering

2023

IJMS

Antibody aggregation, followed by acid denaturation and neutralization of pH, is one of the reasons why the production of therapeutic monoclonal antibodies (mAbs) is expensive. Determining the structural details of acid-denatured antibodies is important for understanding their aggregation mechanism and for antibody engineering. Recent research has shown that monoclonal antibodies of human/humanized immunoglobulin G1 (IgG1) become smaller globules at pH 2 compared to their native structure at pH 7. This acid-denatured species is unstable at pH 7 and prone to aggregation by neutralization of pH. Small-angle X-ray scattering (SAXS) data have revealed an acid-induced reduction in the subpeaks in Kratky plot, indicating conformational changes that can lead to aggregation. The subpeaks are well resolved at pH > 3 but less pronounced at pH ≤ 2. One of the weakened subpeaks indicates loosely organized inter-region (Fab-Fab and Fab-Fc) correlations due to acid denaturation. However, the structural origin of the other subpeak (called q3 peak in this study) has not been established because its q region could represent the various inter-region, inter-domain, and intra-domain correlations in IgG1. In this study, we aimed to untangle the effects of domain–domain correlations on Kratky’s q3 peak based on the computed SAXS of the crystal structure of IgG1. The q3 peak appeared in the static structure and was more prominent in the Fc region than in the Fab or isolated domains. Further brute-force analysis indicated that longer domain–domain correlations, including the inter-region, also positively contribute to Kratky’s q3 peak. Thus, the distortion of the Fc region and a longer inter-region correlation initiate acid denaturation and aggregation.