A novel approach to investigate the effect of methionine oxidation on pharmacokinetic properties of therapeutic antibodies

Stracke, Jan Olaf; Emrich, Thomas; Rueger, Petra; Schlothauer, Tilman; Kling, Lothar; Knaupp, Alexander; Hertenberger, Hubert; Wolfert, Andreas; Spick, Christian; Lau, Wilma; Drabner, Georg; Reiff, Ulrike; Koll, Hans; Papadimitriou, Apollon

doi:10.4161/mabs.29601

Cited by 95 publications

(113 citation statements)

References 49 publications

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“…We did not measure a significant decrease in affinity at pH 6 or in fraction bound at neutral pH on FcRn after methionine Ox, whereas other publications 20, 21, 22, 23 indicate that FcRn binding is reduced upon methionine Ox (only studied at pH 6). However, highest Ox levels that we induced were around 7%, whereas other groups report differences in FcRn binding at levels close to 80% of methionine Ox.…”

Section: Resultscontrasting

confidence: 73%

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Rapid screening of IgG quality attributes – effects on Fc receptor binding

Geuijen¹,

Oppers-Tiemissen

Egging

et al. 2017

FEBS Open Bio

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The interactions of therapeutic antibodies with fragment crystallizable γ (Fcγ) receptors and neonatal Fc receptors (FcRn) are measured in vitro as indicators of antibody functional performance. Antibodies are anchored to immune cells through the Fc tail, and these interactions are important for the efficacy and safety of therapeutic antibodies. High‐throughput binding studies on each of the human Fcγ receptor classes (FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb) as well as FcRn have been developed and performed with human IgG after stress‐induced modifications to identify potential impact in vivo. Interestingly, we found that asparagine deamidation (D‐N) reduced the binding of IgG to the low‐affinity Fcγ receptors (FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb), while FcγRI and FcRn binding was not impacted. Deglycosylation completely inhibited binding to all Fcγ receptors, but showed no impact on binding to FcRn. On the other hand, afucosylation only impacted binding to FcγRIIIa and FcγRIIIb. Methionine oxidation at levels below 7%, multiple freeze/thaw cycles and short‐term thermal/shake stress did not influence binding to any of the Fc receptors. The presence of high molecular weight species, or aggregates, disturbed measurements in these binding assays; up to 5% of aggregates in IgG samples changed the binding and kinetics to each of the Fc receptors. Overall, the screening assays described in this manuscript prove that rapid and multiplexed binding assays may be a valuable tool for lead optimization, process development, in‐process controls, and biosimilarity assessment of IgGs during development and manufacturing of therapeutic IgGs.

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Section: Resultscontrasting

confidence: 73%

“…Stracke et al . 21 found that only one of the two heavy chains is oxidized when Ox levels are around 50% or lower, and the other heavy chain of the antibody is still able to bind to FcRn. This agrees well with our results as no impact is measured at 7% Ox.…”

Section: Resultsmentioning

confidence: 99%

Rapid screening of IgG quality attributes – effects on Fc receptor binding

Geuijen¹,

Oppers-Tiemissen

Egging

et al. 2017

FEBS Open Bio

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“…Only molecules with both heavy chains oxidized at M252 show a significantly faster clearance whereas a single oxidized M252 per antibody exhibits no significant effect. [84] …”

Section: Methionine Oxidationmentioning

confidence: 99%

“…Later research, however, suggests that while oxidation of M252 has a considerable impact on binding toward FcRn, oxidation of M428 seems to play a less important role and might even counteract the effect of M252. [84] Additionally, it seems as if the number of oxidized M252 residues on the individual molecule is of decisive importance for the effect on FcRn binding and pharmacokinetics. Only molecules with both heavy chains oxidized at M252 show a significantly faster clearance whereas a single oxidized M252 per antibody exhibits no significant effect.…”

Section: Methionine Oxidationmentioning

confidence: 99%

Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact

Beyer

Schuster

Jungbauer

et al. 2017

Biotechnology Journal

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Antibodies are typical examples of biopharmaceuticals which are composed of numerous, almost infinite numbers of potential molecular entities called variants or isoforms, which constitute the microheterogeneity of these molecules. These variants are generated during biosynthesis by so‐called posttranslational modification, during purification or upon storage. The variants differ in biological properties such as pharmacodynamic properties, for example, Antibody Dependent Cellular Cytotoxicity, complement activation, and pharmacokinetic properties, for example, serum half‐life and safety. Recent progress in analytical technologies such as various modes of liquid chromatography and mass spectrometry has helped to elucidate the structure of a lot of these variants and their biological properties. In this review the most important modifications (glycosylation, terminal modifications, amino acid side chain modifications, glycation, disulfide bond variants and aggregation) are reviewed and an attempt is made to give an overview on the biological properties, for which the reports are often contradictory. Even though there is a deep understanding of cellular and molecular mechanism of antibody modification and their consequences, the clinical proof of the effects observed in vitro and in vivo is still not fully rendered. For some modifications such as core‐fucosylation of the N‐glycan and aggregation the effects are clear and should be monitored, but with others such as C‐terminal lysine clipping the reports are contradictory. As a consequence it seems too early to tell if any modification can be safely ignored.

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“…9,23,24 Recently, a clear effect of Met oxidation in the constant region of an IgG1 on the pharmacokinetics has been reported in 2 independent in vivo studies. 13,25 For large biomolecules such as recombinant antibodies, bottom-up liquid chromatography-mass spectrometry (LC-MS) of proteolytic peptides is often the method of choice for monitoring site-specific oxidation reactions. 2,5,[9][10][11][12][13]17,19,22,26 However, advances in native mass spectrometry has enabled the analysis of intact protein and protein complexes under more physiological conditions.…”

Section: Introductionmentioning

confidence: 99%

Functional assessment of antibody oxidation by native mass spectrometry

Haberger

Heidenreich

Schlothauer

et al. 2015

mAbs

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(2015) Functional assessment of antibody oxidation by native mass spectrometry, mAbs, 7:5, 891-900, DOI: 10.1080DOI: 10. /19420862.2015 To link to this article: https://doi.org/10. 1080/19420862.2015 Oxidation of methionine (Met) residues is one of several chemical degradation pathways for recombinant IgG1 antibodies. Studies using several methodologies have indicated that Met oxidation in the constant IgG1 domains affects in vitro interaction with human neonatal Fc (huFcRn) receptor, which is important for antibody half-life. Here, a completely new approach to investigating the effect of oxidative stress conditions has been applied. Quantitative ultraperformance liquid chromatography mass spectrometry (MS) peptide mapping, classical surface plasmon resonance and the recently developed FcRn column chromatography were combined with the new fast-growing approach of native MS as a near native state protein complex analysis in solution. Optimized mass spectrometric voltage and pressure conditions were applied to stabilize antibody/huFcRn receptor complexes in the gas phase for subsequent native MS experiments with oxidized IgG1 material. This approach demonstrated a linear correlation between quantitative native MS and IgG-FcRn functional analysis.In our study, oxidation of the heavy chain Met-265 resulted in a stepwise reduction of mAb3/huFcRn receptor complex formation. Remarkably, a quantitative effect of the heavy chain Met-265 oxidation on relative binding capacity was only detected for doubly oxidized IgG1, whereas IgG1 with only one oxidized heavy chain Met-265 was not found to significantly affect IgG1 binding to huFcRn. Thus, mono-oxidized IgG1 heavy chain Met-265 most likely does not represent a critical quality attribute for pharmacokinetics.

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A novel approach to investigate the effect of methionine oxidation on pharmacokinetic properties of therapeutic antibodies

Cited by 95 publications

References 49 publications

Rapid screening of IgG quality attributes – effects on Fc receptor binding

Rapid screening of IgG quality attributes – effects on Fc receptor binding

Microheterogeneity of Recombinant Antibodies: Analytics and Functional Impact

Functional assessment of antibody oxidation by native mass spectrometry

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