Analytical Protein A Chromatography as a Quantitative Tool for the Screening of Methionine Oxidation in Monoclonal Antibodies

Loew, Caroline; Knoblich, Constanze; Fichtl, Jürgen; Alt, Nadja; Diepold, Katharina; Bulau, Patrick; Goldbach, Pierre; Adler, Michael; Mahler, Hanns‐Christian; Grauschopf, Ulla

doi:10.1002/jps.23286

Cited by 39 publications

(31 citation statements)

References 32 publications

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“…Only HC-Met-100c (located in the CDR 3), HC-Met-252, and HC-Met-428 displayed a demonstrative elevation in Met oxidation upon application of temperature stress, suggesting that HC-Met-100c represents a potential degradation site in the CDR of mAb2. These results are in agreement with previous studies on the evaluation of IgG1 conserved Met residues (Met-252 and Met-428) in recombinant antibodies 31 , 32 , 34 - 36 , 45 - 50 . No additional light or heavy chain Trp was found to be oxidized in the variable mAb2 region at detectable levels.…”

Section: Resultssupporting

confidence: 93%

Assessment of chemical modifications of sites in the CDRs of recombinant antibodies

Haberger

Bomans

Diepold

et al. 2014

mAbs

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134

165

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Modifications like asparagine deamidation, aspartate isomerization, methionine oxidation, and lysine glycation are typical degradations for recombinant antibodies. For the identification and functional evaluation of antibody critical quality attributes (CQAs) derived from chemical modifications in the complementary-determining regions (CDRs) and the conserved regions, an approach employing specific stress conditions, elevated temperatures, pH, oxidizing agents, and forced glycation with glucose incubation, was applied. The application of the specific stress conditions combined with ion exchange chromatography, proteolytic peptide mapping, quantitative liquid chromatography mass spectrometry, and functional evaluation by surface plasmon resonance analysis was adequate to identify and functionally assess chemical modification sites in the CDRs of a recombinant IgG1. LC-Met-4, LC-Asn-30/31, LC-Asn-92, HC-Met-100c, and HC Lys-33 were identified as potential CQAs. However, none of the assessed degradation products led to a complete loss of functionality if only one light or heavy chain of the native antibody was affected.

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Section: Resultssupporting

confidence: 93%

Assessment of chemical modifications of sites in the CDRs of recombinant antibodies

Haberger

Bomans

Diepold

et al. 2014

mAbs

Self Cite

134

165

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“…8,9,21,32 Attempts to oxidize one methionine position selectively while leaving the second position intact have all failed, since both positions are similarly prone to oxidation. The rate of oxidation of Met252 is approximately twice that of Met428, leading to a higher oxMet252 than oxMet428 content after forced oxidation or real-time storage.…”

Section: Structure-function Relationship-current Situationmentioning

confidence: 98%

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

Stracke¹,

Emrich²,

Rueger

et al. 2014

mAbs

102

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Preserving the chemical and structural integrity of therapeutic antibodies during manufacturing and storage is a major challenge during pharmaceutical development. Oxidation of Fc methionines Met252 and Met428 is frequently observed, which leads to reduced affinity to FcRn and faster plasma clearance if present at high levels. Because oxidation occurs in both positions simultaneously, their individual contribution to the concomitant changes in pharmacokinetic properties has not been clearly established. A novel pH-gradient FcRn affinity chromatography method was applied to isolate three antibody oxidation variants from an oxidized IgG1 preparation based on their FcRn binding properties. Physico-chemical characterization revealed that the three oxidation variants differed predominantly in the number of oxMet252 per IgG (0, 1, or 2), but not significantly in the content of oxMet428. Corresponding to the increase in oxMet252 content, stepwise reduction of FcRn affinity in vitro, as well as faster clearance and shorter terminal half-life, in huFcRn-transgenic mice were observed. A single Met252 oxidation per antibody had no significant effect on pharmacokinetics (PK) compared with unmodified IgG. Importantly, only molecules with both heavy chains oxidized at Met252 exhibited significantly faster clearance. In contrast, Met428 oxidation had no apparent negative effect on PK and even led to somewhat improved FcRn binding and slower clearance. This minor effect, however, seemed to be abrogated by the dominant effect of Met252 oxidation. The novel approach of functional chromatographic separation of IgG oxidation variants followed by physico-chemical and biological characterization has yielded the first experimentally-backed explanation for the unaltered PK properties of antibody preparations containing relatively high Met252 and Met428 oxidation levels.

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“…Oxidation is a common degradation pathway that may occur during the life cycle of proteins and peptides . The resulting chemical modifications may affect in vitro stability and in vivo biological functions . The position of oxidized amino acids in the protein sequence is crucial for their impact on protein structure, function, and biological response, especially in the case of monoclonal antibodies (mAbs) where binding properties are essential to reach target antigen.…”

Section: Introductionmentioning

confidence: 99%

Selective Oxidation of Methionine and Tryptophan Residues in a Therapeutic IgG1 Molecule

Folzer

Diepold

Bomans

et al. 2015

Journal of Pharmaceutical Sciences

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Analytical Protein A Chromatography as a Quantitative Tool for the Screening of Methionine Oxidation in Monoclonal Antibodies

Cited by 39 publications

References 32 publications

Assessment of chemical modifications of sites in the CDRs of recombinant antibodies

Assessment of chemical modifications of sites in the CDRs of recombinant antibodies

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

Selective Oxidation of Methionine and Tryptophan Residues in a Therapeutic IgG1 Molecule

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