Characterization of Aggregation Propensity of a Human Fc-Fusion Protein Therapeutic by Hydrogen/Deuterium Exchange Mass Spectrometry

Huang, Richard Y.‐C.; Iacob, Roxana E.; Krystek, Stanley R.; Jin, Mi Sun; Wei, Hui; Tao, Liyuan; Das, Tapan K.; Tymiak, Adrienne A.; Engen, John R.; Chen, Guodong

doi:10.1007/s13361-016-1452-7

Cited by 23 publications

(16 citation statements)

References 52 publications

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“…These data suggest that the autophagy‐lysosome pathway, involved in the intracellular protein quality control by removing large misfolded proteins and proteins aggregates, is activated in liver cells expressing hFIX‐Fc fusion proteins . This hypothesis is in accordance with the propensity of Fc fusion proteins to form protein aggregates, which is mostly induced by hydrophobic interactions . Indeed, it has been shown that the coupling of the Fc region to the hFIX resulted in a difference in hydrogen atom exposition of residues 408‐414, which potentially favoured hydrophobic interactions and subsequent formation of hFIX‐Fc fusion protein aggregates.…”

Section: Discussionmentioning

confidence: 58%

“…Studies with NH 4 Cl, which inhibits the autophagic flux into lysosomes, led to hFIX‐Fc proteins be redirected from the lysosomal compartment to organelles such as endosome with a quicker degradation rate, or increased the level of misfolded hFIX‐Fc protein within the cell resulting in elevated endoplasmic reticulum stress and a higher apoptotic rate . These data suggest that the autophagy‐lysosome pathway, involved in the intracellular protein quality control by removing large misfolded proteins and proteins aggregates, is activated in liver cells expressing hFIX‐Fc fusion proteins . This hypothesis is in accordance with the propensity of Fc fusion proteins to form protein aggregates, which is mostly induced by hydrophobic interactions .…”

Section: Discussionmentioning

confidence: 96%

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Potential limits of AAV‐based gene therapy with the use of new transgenes expressing factor IX fusion proteins

et al. 2018

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Introduction The variety of treatment for haemophilia B (HB) has recently improved with the emergence of both AAV‐based gene therapy and bioengineered human factor IX (hFIX) molecules with prolonged half‐life due to fusion to either albumin (Alb) or immunoglobulin Fc fragment (Fc). Aim Adeno‐associated viral vectors (AAV) mediating expression of hFIX‐Alb and hFIX‐Fc fusion proteins was investigated for gene therapy of HB to explore if their extended half‐life translates to higher plasma levels of FIX. Methods Single‐stranded cross‐packaged AAV2/8 vectors expressing hFIX‐Alb, hFIX‐Fc and hFIX were evaluated in vitro, and in mice. Results Both hFIX‐Alb and hFIX‐Fc fusion proteins were synthesized and expressed as single chains of expected size following AAV‐mediated gene transfer in vitro and in vivo. The procoagulant properties of these hFIX‐fusion proteins were comparable to wild‐type hFIX. However, their expression levels were threefold lower than wild‐type hFIX in vivo most likely due to inefficient secretion. Conclusion This, the first, evaluation of hFIX‐fusion proteins in the context of AAV gene transfer suggests that the hFIX‐fusion proteins are secreted inefficiently from the liver, thus preventing their optimal use in gene therapy approaches.

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Section: Discussionmentioning

confidence: 58%

Section: Discussionmentioning

confidence: 96%

Potential limits of AAV‐based gene therapy with the use of new transgenes expressing factor IX fusion proteins

et al. 2018

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“…Structural modeling platforms, such as the Integrated Modelling Platform (IMP), rely on homology modeling in combination with structural restraints obtained from various techniques [106]. These hybrid modeling approaches incorporate information from a myriad of techniques such as electron microscopy, small angle X-ray scattering, crosslinking, chemical labeling, and hydroxyl footprinting [67, 107–109]. Amide accessibility obtained from HDX provides an additional level of information for structural modeling as it can differentiate regions of stable secondary structure from unfolded regions.…”

Section: Introductionmentioning

confidence: 99%

Tracking Higher Order Protein Structure by Hydrogen-Deuterium Exchange Mass Spectrometry

Benhaim

Lee

Guttman

2019

PPL

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Structural biology has provided a fundamental understanding of protein structure and mechanistic insight into their function. However, high-resolution structures alone are insufficient for a complete understanding of protein behavior. Higher energy conformations, conformational changes, and subtle structural fluctuations that underlie the proper function of proteins are often difficult to probe using traditional structural approaches. Solution state structural techniques are needed to interrogate the dynamic nature of proteins under native conditions. Hydrogen/deuterium exchange with mass spectrometry (HDX-MS) provides a means to probe the accessibility of backbone amide protons under native conditions, which reports on local structural dynamics of solution protein structure. HDX-MS also provides a tool for tracking complex structural rearrangements that occur in the course of a protein’s function. Here we review recent advances in HDX methodology and highlight some novel applications where HDX has shed light on particularly challenging systems.

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“…The deuterium uptake information can be improved to the single-residue level by using subtraction of deuterium uptake from overlapping peptides or the low-energy dissociation methods, , e.g., ETD or ECD. HDX-MS reports on the hydrogen bonding and solvent accessibility of the protein backbone amide hydrogens and is sensitive to protein structural changes caused by different perturbations. − Most importantly, key insights into the protein–protein interactions can be obtained by comparing HDX profiles to map the interfaces for the mAb reversible self-association , and pinpoint the regions with structural changes induced by aggregation. , Understanding protein aggregation at the molecular level could lead to rational design strategies for reducing mAb aggregation during product design, development, and manufacturing.…”

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confidence: 99%

“…26−28 Most importantly, key insights into the protein−protein interactions can be obtained by comparing HDX profiles to map the interfaces for the mAb reversible self-association 29,30 and pinpoint the regions with structural changes induced by aggregation. 31,32 Understanding protein aggregation at the molecular level could lead to rational design strategies for reducing mAb aggregation during product design, development, and manufacturing. In this study, our focus was on how dimers of an IgG2 mAb (mAb2) formed under two different conditions: (1) during long-term storage under the recommended storage condition and (2) during short-term exposure to a high-temperature thermal stress.…”

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confidence: 99%

Structural Changes and Aggregation Mechanisms of Two Different Dimers of an IgG2 Monoclonal Antibody

Zhang

Woods

et al. 2018

Biochemistry

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Protein therapeutics, monoclonal antibodies (mAbs) in particular, are large, structurally complex molecules that are prone to numerous modes of degradation during their production and long-term storage. Physical degradation via protein aggregation is a major concern when developing protein therapeutic candidates for clinical use. A dimer is perhaps the simplest element of protein aggregation, and thus, a better understanding of protein dimers in terms of their structures, intermolecular interactions, and chemical nature will help in the development of rational strategies for reducing aggregation propensity. In this study, two different mAb dimers were generated from an IgG2 monoclonal antibody solution, i.e., a native dimer generated under long-term storage and a thermal dimer from a thermal stress condition. Both IgG2 dimers were characterized in terms of their chemical and physical properties, bioactivity, and conformational dynamics. The native IgG2 dimer was formed mainly through noncovalent association. It displayed minimal differences in biophysical properties and higher-order structure compared to the monomer yet showed compromised in vitro potency, likely because of steric hindrance. In contrast, the thermal IgG2 dimer was mainly disulfide-linked, but even so, no new non-native disulfide bonds were detected by peptide mapping. Two regions within the Fc-C2 domain of the thermal IgG2 dimer exhibited significantly increased flexibility as measured by hydrogen-deuterium exchange mass spectrometry, and notably, these regions are connected by an intrachain disulfide bond under natively folded conditions. These findings provide a better understanding of dimer formation under long-term storage and thermal stress conditions for this IgG2 mAb, and possible aggregation mechanisms are discussed.

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Characterization of Aggregation Propensity of a Human Fc-Fusion Protein Therapeutic by Hydrogen/Deuterium Exchange Mass Spectrometry

Cited by 23 publications

References 52 publications

Potential limits of AAV‐based gene therapy with the use of new transgenes expressing factor IX fusion proteins

Potential limits of AAV‐based gene therapy with the use of new transgenes expressing factor IX fusion proteins

Tracking Higher Order Protein Structure by Hydrogen-Deuterium Exchange Mass Spectrometry

Structural Changes and Aggregation Mechanisms of Two Different Dimers of an IgG2 Monoclonal Antibody

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