Evaluation of Hydrogen Exchange Mass Spectrometry as a Stability-Indicating Method for Formulation Excipient Screening for an IgG4 Monoclonal Antibody

Toth, Ronald; Pace, Samantha E.; Mills, Brittney J.; Joshi, Sangeeta B.; Esfandiary, Reza; Middaugh, C. Russell; Weis, David D.; Volkin, David B.

doi:10.1016/j.xphs.2017.12.009

Cited by 22 publications

(30 citation statements)

References 29 publications

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“…However, in related work in our laboratory, good correlations of enhanced local flexibility in the aggregation hot spot #1 (as measured by HX) with elevated levels of soluble aggregate and subvisible particle formation, as measured by size exclusion chromatography (SEC) and Microflow imaging (MFI) respectively, during thermal stress of a full length IgG4 mAb has been demonstrated. 51…”

Section: Discussionmentioning

confidence: 99%

Impact of Glycosylation on the Local Backbone Flexibility of Well-Defined IgG1-Fc Glycoforms Using Hydrogen Exchange-Mass Spectrometry

Toth

Okbazghi

et al. 2018

Journal of Pharmaceutical Sciences

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We have used hydrogen exchange-mass spectrometry to characterize local backbone flexibility of 4 well-defined IgG1-Fc glycoforms expressed and purified from Pichia pastoris, 2 of which were prepared using subsequent in vitro enzymatic treatments. Progressively decreasing the size of the N-linked N297 oligosaccharide from high mannose (Man8-Man12), to Man5, to GlcNAc, to nonglycosylated N297Q resulted in progressive increases in backbone flexibility. Comparison of these results with recently published physicochemical stability and Fcγ receptor binding data with the same set of glycoproteins provide improved insights into correlations between glycan structure and these pharmaceutical properties. Flexibility significantly increased upon glycan truncation in 2 potential aggregation-prone regions. In addition, a correlation was established between increased local backbone flexibility and increased deamidation at asparagine 315. Interestingly, the opposite trend was observed for oxidation of tryptophan 277 where faster oxidation correlated with decreased local backbone flexibility. Finally, a trend of increasing C'E glycopeptide loop flexibility with decreasing glycan size was observed that correlates with their FcγRIIIa receptor binding properties. These well-defined IgG1-Fc glycoforms serve as a useful model system to identify physicochemical stability and local backbone flexibility data sets potentially discriminating between various IgG glycoforms for potential applicability to future comparability or biosimilarity assessments.

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Section: Discussionmentioning

confidence: 99%

Impact of Glycosylation on the Local Backbone Flexibility of Well-Defined IgG1-Fc Glycoforms Using Hydrogen Exchange-Mass Spectrometry

Toth

Okbazghi

et al. 2018

Journal of Pharmaceutical Sciences

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“…HDX-MS localized the changes in the dynamic properties of the mAbs near the modifications to understand the consequences of the modifications. The other type of perturbation is the change in the environment of a mAb as a result of formulation, including salts, 17 excipients, 18,19 as well as the conditions the mAb is exposed to, such as thermal stress 20 and dimerization. 21 In these studies, the presence and the location of the changes in HDX-MS patterns are unknown prior to the analysis.…”

Section: Introductionmentioning

confidence: 99%

The effects of intramolecular and intermolecular electrostatic repulsions on the stability and aggregation of NISTmAb revealed by HDX‐MS, DSC, and nanoDSF

et al. 2021

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The stability and aggregation of NIST monoclonal antibody (NISTmAb) were investigated by hydrogen/deuterium exchange mass spectrometry (HDX-MS), differential scanning calorimetry (DSC), and nano-differential scanning fluorimetry (nanoDSF). NISTmAb was prepared in eight formulations at four different pHs (pH 5, 6, 7, and 8) in the presence and absence of 150 mM NaCl and analyzed by the three methods. The HDX-MS results showed that NISTmAb is more conformationally stable at a pH near its isoelectric point (pI) in the presence of NaCl than a pH far from its pI in the absence of NaCl. The stabilization effects were global and not localized. The midpoint temperature of protein thermal unfolding transition results also showed the C H 2 domain of the protein is more conformationally stable at a pH near its pI. On the other hand, the onset of aggregation temperature results showed that NISTmAb is less prone to aggregate at a pH far from its pI, particularly in the absence of NaCl. These seemingly contradicting results, higher conformational stability yet higher aggregation propensity near the pI than far away from the pI, can be explained by intramolecular and intermolecular electrostatic repulsion using Lumry-Eyring model, which separates folding/unfolding equilibrium and aggregation event. The further a pH from the pI, the higher the net charge of the protein. The higher net charge leads to greater intramolecular and intermolecular electrostatic repulsions. The greater intramolecular electrostatic repulsion destabilizes the protein and the greater intermolecular electrostatic repulsion prevents aggregation of the protein molecules at pH far from the pI.

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“…In addition to these conventional measurements, there is growing interest in temperature-dependent HDX–MS. The latter can provide a more comprehensive view of protein dynamics. − Of particular importance is the characterization of thermally stressed protein drugs (such as therapeutic antibodies) to assess their stability and aggregation propensity. ,, …”

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Analysis of Temperature-Dependent H/D Exchange Mass Spectrometry Experiments

Tajoddin

Konermann

2020

Anal. Chem.

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H/D exchange (HDX) mass spectrometry (MS) is a widely used technique for interrogating protein structure and dynamics. Backbone HDX is mediated by opening/closing (unfolding/refolding) fluctuations. In traditional HDX–MS, proteins are incubated in D2O as a function of time at constant temperature (T). There is an urgent need to complement this traditional approach with experiments that probe proteins in a T-dependent fashion, e.g., for assessing the stability of therapeutic antibodies. A key problem with such studies is the absence of strategies for interpreting HDX–MS data in the context of T-dependent protein dynamics. Specifically, it has not been possible thus far to separate T-induced changes of the chemical labeling step (k ch) from thermally enhanced protein fluctuations. Focusing on myoglobin, the current work solves this problem by dissecting T-dependent HDX–MS profiles into contributions from k ch(T), as well as local and global protein dynamics. Experimental profiles started off with surprisingly shallow slopes that seemed to defy the quasi-exponential k ch(T) dependence. Just below the melting temperature (T m) the profiles showed a sharp increase. Our analysis revealed that local dynamics dominate at low T, while global events become prevalent closer to T m. About half of the backbone NH sites exhibited a canonical scenario, where local opening/closing was associated with positive ΔH and ΔS. Many of the remaining sites had negative ΔH and ΔS, thereby accounting for the shallowness of the experimental HDX–MS profiles at low T. In summary, this work provides practitioners with the tools to analyze proteins over a wide temperature range, paving the way toward T-dependent high-throughput screening applications by HDX–MS.

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Evaluation of Hydrogen Exchange Mass Spectrometry as a Stability-Indicating Method for Formulation Excipient Screening for an IgG4 Monoclonal Antibody

Cited by 22 publications

References 29 publications

Impact of Glycosylation on the Local Backbone Flexibility of Well-Defined IgG1-Fc Glycoforms Using Hydrogen Exchange-Mass Spectrometry

Impact of Glycosylation on the Local Backbone Flexibility of Well-Defined IgG1-Fc Glycoforms Using Hydrogen Exchange-Mass Spectrometry

The effects of intramolecular and intermolecular electrostatic repulsions on the stability and aggregation of NISTmAb revealed by HDX‐MS, DSC, and nanoDSF

Analysis of Temperature-Dependent H/D Exchange Mass Spectrometry Experiments

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